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6 Kobayasi et al. - Effects of Temperature, Solar Radiation, and Vapor Pressure Deficit on Flower Opening

7 Time in Rice

8 Effects of Temperature, Solar Radiation, and Vapor Pressure Deficit on the Flower Opening Time in

9 Rice

10 Kazuhiro Kobayasi1, Tsutomu Matsui2, Mayumi Yoshimoto3 and Toshihiro Hasegawa3

11 (1 Faculty of Life and Environmental Science, Shimane University, 1060 Nishi-Kawatsu-Cho, Matsue, 690-

12 8504 Japan;

13 2
Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan;

14 3
National Institute for Agro-Environmental Sciences, 3-1-3, Kannondai, Tsukuba, Ibaraki 305-8604, Japan;)

15 Footnotes:

16 Corresponding author: K. Kobayasi (kobayasi@life.shimane-u.ac.jp, fax +81-852-32-6507).

17 Abbreviations: FOT, flower opening time; GLM, general linear model; Rs, solar radiation; Ta, air

18 temperature; VPD, vapor pressure deficit.

19 Abstract: Flower opening in the early morning is an important characteristic that helps to avoid sterility of

20 rice plants caused by heat stress at anthesis. Although flower opening time (FOT) is under strong genetic

21 control, it is also strongly affected by weather, and particularly by air temperature (Ta), but how variations in

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22 Ta, solar radiation (Rs) and vapor pressure deficit (VPD) influence FOT of various genotypes remains

23 unclear, particularly under field conditions; this limits our ability to predict FOT. We therefore attempted to

24 determine the relationship between FOT and Ta, Rs, and VPD at various periods before anthesis under field

25 conditions. By photographing spikelets at 10-min intervals, we determined the FOT of five cultivars. To

26 evaluate single effects of cultivars, Ta, Rs, and VPD on FOT, general linear models (GLMs) were used. In

27 this analysis, Ta, Rs, and VPD averaged every three hour from 0000 to 1200 were calculated. From GLMs,

28 air temperature, Rs, and VPD averaged during 0600 and 0900 most strongly affected FOT (adjusted R square

29 = 0.399; p<0.001). Standardized partial regression coefficients of Ta and Rs were negative and that of VPD

30 was positive. These facts mean that higher Ta, higher Rs, and/or lower VPD in the early morning will be

31 effective to cause FOT to occur earlier. However, multiple regression analysis suggested that the most

32 sensitive period when Ta, Rs, and VPD determine FOT and the ratio among the contributions of the three

33 weather factors were different according to the cultivars.

34

35 Key words: Flower opening in the early morning, Flower opening time, Heat-induced sterility, Rice, Solar

36 Radiation, Temperature, Vapor pressure deficit.

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37 Global climate change will pose a serious challenge to crop production around the world. An anticipated high

38 temperature > 34 °C at the time of flowering may induce floret sterility and decrease rice yield even in

39 temperate regions such as southern Japan if the cropping seasons do not change (Kim et al., 1996; Horie et

40 al., 1996; Nakagawa et al., 2003). Using crop simulation models, Horie et al. (1996) suggested that the yield

41 of current rice varieties in southern Japan would be reduced by up to 40% under future climate scenarios. In

42 the summer of 2003, the Yangtze River rice cropping region in China suffered from an extremely high

43 temperature above 39 °C, leading to a large reduction in rice production due to a low seed-set rate (30 to

44 70%; Wang et al., 2004).

45 Opening of rice flowers in the early morning is an important adaptation that can avoid the sterility caused by

46 heat stress at anthesis. Spikelet sensitivity to high temperatures decreases during the 1-hr period after

47 flowering (Satake and Yoshida, 1978). Thus, even if flower opening occurs only 1 hr earlier, this may still

48 significantly reduce sterility, because air temperature can rise at a rate of 3 °C hr-1 or more by around 1000

49 (Nishiyama and Blanco, 1980). An experiment under controlled environments revealed that the flowers of

50 Milyang 23 which opened earlier in the morning and at a lower temperature had higher seed-set rates than

51 those which opened later (near midday) and at higher temperatures (Imaki et al., 1987).

52 Selection for cultivars with early flower opening is thus an important method for reducing heat-induced

53 sterility (Nishiyama and Blanco, 1980; Jagadish et al., 2008). For example, the flowers of Oryza glaberrima

54 Steud. open earlier than those of Oryza sativa L. (Nishiyama and Blanco, 1980; Jagadish et al., 2008), and

55 the flowers of interspecific hybrids between O. glaberrima and O. sativa also open earlier than those of O.

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56 sativa (Nishiyama and Satake, 1981). Among the cultivars of O. sativa, Imaki et al. (1983; 1987) found

57 cultivars whose flowers opened 1 or 2 hr earlier than those of standard Japanese cultivar ‘Koshihikari’.

58 Under controlled conditions, Milyang 23 exhibited heat tolerance due to the earlier opening of its flowers

59 (Imaki et al., 1983).

60 Although flower opening time (FOT) is under strong genetic control, it is also strongly affected by weather,

61 and particularly by temperature (Imaki et al., 1983; Nishiyama and Satake, 1981; Hoshikawa, 1989; Jagadish

62 et al., 2007; 2008; Nakagawa and Nagata, 2007). The response of flower opening to high temperature differs

63 among cultivars. For example, high temperature influences FOT in Milyang 23 and Nipponbare, but the

64 response is different. In Milyang 23, FOT occurs earlier under high temperatures, but FOT is delayed in

65 Nipponbare (Imaki et al., 1983). In a study of indica cultivars, FOT occurred about 45 min earlier at higher

66 temperatures (Jagadish et al., 2007). However, most studies of FOT (Imaki et al., 1983; Nishiyama and

67 Blanco, 1981; Jagadish et al., 2007; 2008) have been conducted under controlled environments, and rice

68 plants in a glass house or growth chamber tend to open their flowers 1 or 2 hr later than those grown

69 outdoors (Imaki et al., 1982).

70 Other weather factors such as solar radiation (Rs) and humidity may affect FOT. Light also influences

71 FOT. Under a continuously illuminated or dark condition, anthesis persisted over a long period (Hoshikawa,

72 1989). Solar radiation from 0400 to 0800 influenced FOT in Koshihikari, but not in EG0. Strong winds

73 (Tsuboi, 1961), low atmospheric pressure, and rain (Hoshikawa, 1981) also change FOT.

74 How variations in temperature, Rs, and humidity influence FOT of various genotypes remains unclear,

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75 particularly under field conditions, and this limits our ability to predict FOT. We therefore attempted to

76 determine the effects of temperature, Rs, and humidity at various periods before anthesis on FOT under field

77 conditions. First, we analyzed correlation between FOT and air temperature (Ta), Rs, or vapor pressure

78 deficit (VPD) averaged every hour from 0000 to 1000. Second, using general linear models (GLMs), we

79 evaluated effects of cultivars, Ta, Rs, and VPD separately. Third, for each cultivar we applied multiple

80 regressions and evaluate the contribution of Ta, Rs, and VPD to determining FOT because the sensitive

81 period and the degrees of contribution of Ta, Rs, and VPD may be different according to cultivars.

82

83 Materials and Methods

84 1. Field, plant materials, and culture methods

85 Experiment was conducted at the experimental field of Shimane University in Matsue, Shimane Prefecture,

86 Japan (35°29’N, 133°04’E, altitude 4 m asl) in 2007. We used five cultivars in this study: the indica cultivars

87 ‘IR72’, and the japonica cultivars ‘Hanaechizen’, ‘Fujihikari’, ‘Shinriki’, and ‘Asahi’. Hanaechizen and

88 Fujihikari were used to obtain flowering plants over a long period of the cropping season because they have

89 a weak photoperiodic sensitivity and by shifting transplanting dates, we can easily obtain flowering plants

90 over a long period. Shinriki and Asahi were used to obtain flowering plants in September, when temperatures

91 are decreasing day by day and we can obtain a wide range of weather data. The soil was alluvial and sandy

92 clay. Thirty-day-old seedlings grown in nursery boxes were manually transplanted into the field several times

93 to obtain plants flowering under different weather conditions. The planting density was 22.2 hills m -2 (one

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94 seedling per hill; hill spacing was 15 cm and row spacing was 30 cm). At basal dressing, 5 g/m 2 of nitrogen,

95 5.6 g/m2 of phosphate, and 4.4 g/m2 of potash were applied. Topdressing was not done. Culture methods such

96 as irrigation and chemical use followed the standard local practices for rice production in Shimane

97 Prefecture.

98 2. Measurements of flower opening time and weather

99 Physical stimuli such as touching sometimes promote the opening of the flowers of rice plants (Tsuboi,

100 1961). To avoid this phenomenon, we used digital photographs of the panicles instead of physical

101 inspections. By photographing the panicles at 10-min intervals with a digital camera (Optio W30, Pentax,

102 Tokyo, Japan), we determined the FOT of the five cultivars. We defined FOT as the time when 50% of the

103 flowers opened on a given day although the flowering pattern is also affected by temperature (Jagadish et al.,

104 2007).

105 Air temperature, relative humidity, and Rs were measured every 5 min; we used a wireless weather station

106 (Wireless Vantage Pro, Davis Instruments, Hayward, CA, U.S.). The data of the weather station were

107 obtained at the site of Shimane University (http://www.ipc.shimane-u.ac.jp/weather/station/index.html).

108 Vapor pressure deficits were calculated from Ta and relative humidity (Buck, 1981).

109

110 3. Statistical analysis

111 We analyzed our data through GLMs and multiple regression procedures using SPSS (Version 14J for

112 Windows, SPSS Japan Inc., Tokyo, Japan). Correlation analysis was used to identify relationships between

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113 weather factors (Ta, Rs, and VPD) and FOT. The correlation coefficients between FOT and Ta, Rs, and VPD

114 averaged every hour during 0000 and 1000 were calculated.

115 There are usually relatively high correlations between Ta, Rs, and VPD. To evaluate single effects of

116 cultivars, Ta, Rs, and VPD on FOT, GLMs were used. In this analysis, Ta, Rs, and VPD averaged every three

117 hour (from 0000 to 0300, from 0300 to 0600, from 0600 to 0900, from 0900 to 1200) were calculated.

118 It is probable that the sensitive period and the degrees of contribution of Ta, Rs, and VPD in determining

119 FOT are different according to cultivars. For each cultivar, multiple regressions were applied to analyze the

120 relative contribution of weather components to FOT according to their standardized coefficients, with the

121 overall strength of this relationship quantified with the multiple correlation coefficients.

122 Results

123 1. Correlation analysis between weather factors and FOT

124 Because we transplanted seedlings several times, the measurement periods were longer than 17 days for all

125 cultivars (Table 1). The daily mean, maximum, and minimum temperatures during the FOT observation

126 period across the cultivars ranged from 22.1 to 28.7 °C, from 22.9 to 34.6 °C, and from 17.3 to 25.2 °C,

127 respectively. Daily Rs and daily mean VPD during the FOT observation period across the cultivars ranged

128 from 2.3 to 27.4 MJ/m2 and from 1.2 to 13.4 hPa, respectively. IR72 opened flowers earlier than other four

129 cultivars. The range of FOT was more than 2.5 hours in all cultivars.

130 IR72 had the negative, high correlations between FOT and Ta averaged every hour during 0000 and 0800

131 (Fig. 1). We did not detect correlation significant at 5% level between FOT and Ta averaged every hour

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132 during 0000 and 0700 in other four cultivars. Shinriki had the negative correlation significant at 5% level

133 between FOT and Ta averaged every hour during 0700 and 0900. Fujihikari and Asahi had the negative

134 correlation significant at 5% level between FOT and mean Ta during 0800 and 0900.

135 Fujihikari had the negative correlations between FOT and Rs averaged every hour during 0500 and 1000

136 (Fig. 2). On the other hand, IR72 had the positive correlation significant at 5% level between FOT and Rs

137 averaged every hour during 0500 and 0700. Shinriki had the negative correlation significant at 5% level

138 between FOT and Rs averaged every hour during 0800 and 0900. Asahi had the correlation significant at 5%

139 level between FOT and Rs averaged every hour during 0800 and 1000.

140 In most cases, there were not correlations significant at 5% level between FOT and VPD averaged every

141 hour (Fig. 3). We only detected correlations significant at 5% level between FOT and VPD in IR72 (from

142 0500 to 0900), Hanaechizen (from 0400 to 0700), and Asahi (from 0800 to 0900). The correlation

143 coefficients in IR72 were positive, and on the other hand, those in Hanaechizen and Asahi were negative.

144 2. Evaluation of effects of cultivars, Ta, Rs, and VPD on FOT using GLMs

145 Adjusted R squares analyzed in GLMs was highest (adjusted R square=0.399, p<0.001) for the periods from

146 0600 to 0900 (Table 2). In this period, all four factors (cultivars, Ta, Rs, and VPD) were significant at 0.1%

147 level. Standardized partial regression coefficient of Ta was -0.488 and negative, this result means that higher

148 Ta caused FOT to occur earlier. Standardized partial regression coefficient of Rs was -0.666 and negative,

149 this result means that higher Rs caused FOT to occur earlier. Standardized partial regression coefficient of

150 VPD was 0.475 and positive, this means that lower VPD (more humid condition) caused FOT to occur

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151 earlier. The contributions of these three weather factors to determining FOT were not so different, comparing

152 standard partial regression coefficients.

153 3. Evaluation of effects in each cultivar of Ta, Rs, and VPD on FOT using multiple regression analysis

154 In IR72, highest adjusted R square was obtained at the period between 0600 and 0900 (Table 3.). In this case,

155 standardized partial regression coefficient of Ta only was significant at 5% level. In Hanaechizen, highest

156 adjusted R square was obtained at the period between 0300 and 0600, and standardized partial regression

157 coefficient of VPD only was significant at 5% level. In Fujihikari, highest adjusted R square was obtained at

158 the period between 0900 and 1200, and all the three standardized partial regression coefficients were

159 significant at 5% level. We did not obtained adjusted R squares significant at 5% in Shinriki. In Asahi,

160 highest adjusted R square was obtained at the period between 0600 and 0900, and standardized partial

161 regression coefficient of Rs only was significant at 5% level.

162 Discussion

163 Air temperature, Rs, and VPD averaged during 0600 and 0900 affected FOT (adjusted R square is 0.399;

164 p<0.001; Table 2.). From considering the standardized partial regression coefficients, the contributions of Ta,

165 Rs, and VPD to FOT were not so different. However, simple correlation analysis showed that few

166 correlations between FOT and Ta (Fig. 1.), Rs (Fig. 2.), and VPD (Fig. 3.) were detected, and sometimes

167 conflicting results were obtained; Correlation coefficients between FOT and Rs in Fujihikari were negative,

168 but those in IR72 were positive. Correlation coefficients between FOT and VPD in IR72 were negative, but

169 those in Hanaechizen were positive. These facts showed that it is necessary to take the correlations between

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170 Ta, Rs, and VPD into consideration in analyzing weather effects on FOT.

171 Temperature had negative standardized partial regression coefficients, so higher Ta caused FOT to occur

172 earlier (Table 2.). Imaki et al. (1983) reported that high Ta caused FOT to occur earlier in Milyang 23, a

173 japonica–indica hybrid. Similarly, Jagadish et al. (2007; 2008) found that rice plants including indica,

174 japonica, and hybrid varieties open their flowers earlier at higher temperatures to avoid high midday

175 temperatures. These results suggest that rice plants might be able to open their flowers under cooler

176 conditions in the early morning by detecting and responding to high night temperatures. In mid-August, Ta at

177 0900 is more than 2 °C lower than that at 1100 in Matsue, Japan. Combining genetic improvement of FOT

178 with the ability for FOT to occur earlier under high night temperatures would allow flower opening in the

179 early morning to mitigate potential heat-induced sterility in rice.

180 How high night temperatures can lead to earlier FOT is not clear. In the day before flowering, development

181 of the embryo sac is completed, and the egg cells become physiologically capable of being fertilized

182 (Hoshikawa, 1989). Pollen grains continue developing until flowering occurs, and fill with starch 1 day

183 before anthesis. In pollen grains, starch is rapidly digested after the end of starch engorgement at the end of

184 the grain opposite to the germ poles, 3 to 4 hr before anther dehiscence, and more than 70% of pollen grains

185 became sugar-type grains by the time of anther dehiscence (Koike and Satake, 1987). These results suggest

186 that high night temperatures may promote the digestion of starch in the pollen grains, thereby causing FOT

187 to occur earlier.

188 Solar radiation also had negative standardized partial regression coefficients, so higher Rs caused FOT to

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189 occur earlier (Table 2.).Other aspects of light conditions may influence FOT. Rice plants in a glass house or

190 growth chamber tend to open their flowers 1 or 2 hr later than those grown outdoors (Imaki et al., 1982).

191 Artificial dark or light treatments can affect FOT (Nishiyama and Blanco, 1981). Levels of Rs in the early

192 morning affects FOT, and cultivars differ in their FOT response to levels of Rs (Nakagawa and Nagata,

193 2007). Higher level of Rs in the early morning caused FOT to occur earlier in ‘Koshihikari’, but not in

194 ‘EG0’. Under filed conditions, the synergistic effects of temperature and light on FOT will thus be important.

195 In the present study, the contribution of VPD to FOT was detected and lower VPD caused FOT to occur

196 earlier. In a rainy day, flowers, however, usually open in the afternoon (Hoshikawa, 1989). Transpirational

197 cooling by higher VPD (Matsui et al., 2007) in the early morning might decrease panicle tissue temperature

198 and cause FOT to occur later. It is possible that Ta, Rs, and VPD affect FOT through panicle tissue

199 temperature.

200 There were cultivar differences in the most sensitive period when Ta, Rs, and VPD determine FOT (Table 3).

201 The adjusted R square in Hanaechizen was highest in the period from 0300 to 0600. Highest adjusted R

202 square was obtained in Fujihikari in the period from 0900 to 1200. Flower opening times in Hanaechizen and

203 Fujihikari were not so different. In the field conditions, where Ta, Rs, and VPD change gradually, it is

204 difficult to detect the most sensitive period in determining FOT precisely. Experiments under controlled

205 weather conditions in a growth cabinet will be needed.

206 Judging from standardized partial regression coefficients, the contribution of Ta to FOT was large in IR72

207 (Table 3.). On the other hand, the contribution of VPD was little in IR72. T he contributions of VPD,

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208 however, were large in Hanaechizen and Fujihikari. The ratio among the contributions of Ta, Rs, and VPD

209 would be different among cultivars. Imaki et al. (1983) found that high Ta before flowering delayed flower

210 opening in japonica cultivars, but enhanced in indica and indica-japonica cultivars. The roles of temperature

211 and VPD might be different in indica (IR72) and japonica (Hanaechizen and Fujihikari).

212 In the conclusion, higher Ta, higher Rs, and/or lower VPD in the early morning will be effective to cause

213 FOT to occur earlier. However, the most sensitive period when Ta, Rs, and VPD determine FOT and the ratio

214 among the contributions of the three weather factors would be different according to the cultivars. To predict

215 FOT under field conditions, more information under field conditions and experiments under controlled

216 conditions will be needed about the effects of temperature, light, humidity, and other weather factors.

217 Acknowledgements

218 We thank Dr. Fumihiko Adachi for scientific support of making use of the weather data at the site of

219 Shimane University (http://www.ipc.shimane-u.ac.jp/weather/station/index.html).

220 References

221 Buck ,A. L. 1981. New equations for computing vapor pressure and enhancement factor. J. Appl. Meteorol.

222 20:1527-1532.

223 Horie, T., Matsui, T., Nakagawa, H. and Omasa, K. 1996. Effects of elevated CO2 and global climate change

224 on rice yield in Japan. In K. Omasa, K. Kai, H. Taoda, Z. Uchijima and M. Yoshino eds., Climate Change

225 and Plants in East Asia. Springer-Verlag, Tokyo. 39-56.

226 Hoshikawa, K. 1989. The Growing Rice Plant – An Anatomical Monograph. Nosan Gyoson Bunka Kyokai,

227 Tokyo. 1-310.

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228 Imaki, T, Jyokei, K. and Hara, K. 1982. Flower opening under the controlled environments in rice plants.

229 Bull. Fac. Agric. Shimane Univ. 16:1-7**.

230 Imaki, T., Jyokei, K. and Yamada, I. 1983. Sterility caused by high temperature at flowering in rice plants.

231 Bull. Fac. Agric. Shimane Univ. 17:1-7**.

232 Imaki, T., Tokunaga, S. and Obara, N. 1987. High temperature sterility of rice spikelets at flowering in

233 relation to flowering time. Jpn. J. Crop Sci. 56(Extra issue 2):209-210***.

234 Jagadish, S.V.K., Craufurd, P.Q. and Wheeler, T.R. 2007. High temperature stress and spikelet fertility in rice

235 (Oryza sativa L.). J. Exp. Bot. 58:1627-1635.

236 Jagadish, S.V.K., Craufurd, P.Q. and Wheeler, T.R. 2008. Phenotyping parents of mapping populations of

237 rice for heat tolerance during anthesis. Crop Sci. 48:1140-1146.

238 Kim, H.Y., Horie, T., Nakagawa, H. and Wada, K. 1996. Effect of elevated CO2 concentration and high

239 temperature on growth and yield of rice. II. The effect on yield and its components of Akihikari rice. Jpn. J.

240 Crop Sci. 65:644-651*.

241 Koike, S. and Satake, T. 1987. Sterility Caused by Cooling Treatment at the Flowering Stage in Rice Plants.

242 II. The abnormal digestion of starch in pollen grain and metabolic changes in anthers following cooling

243 treatment. Jpn. J. Crop Sci. 56:666-672.

244 Matsui, T., Kobayasi, K., Yoshimoto, M. and Hasegawa, T. 2007. Stability of rice pollination in the field

245 under hot and dry conditions in the Riverina Region of New South Wales, Australia. Plant Prod. Sci. 10:57-

246 63.

247 Nakagawa, H., Horie, T. and Matsui, T. 2003. Effects of climate change on rice production and adaptive

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248 technologies. In T. W. Mew, D. S. Brar, S. Peng, D. Dawe and B. Hardy eds., Rice Science: Innovations and

249 Impact for Livelihood. International Rice Research Institute, Laguna, the Philippines. 635-658.

250 Nakagawa, H. and Nagata, A. 2007. Internal and environmental factors affecting the time of flower-opening

251 in rice. Jpn. J. Crop Sci. 76(Extra issue 2):280-281***.

252 Nishiyama, I. and Blanco, L. 1980. Avoidance of high temperature sterility by flower opening in the early

253 morning. Jpn. Agric. Res. Quarterly 14:116-117.

254 Nishiyama, I. and Blanco, L. 1981. Artificial control of flower opening time during the day in rice plants. I.

255 Preliminary experiments. Jpn. J. Crop Sci. 50:59-66.

256 Nishiyama, I. and Satake, T. 1981. High temperature damages in rice plants. Jpn. J. Trop. Agric. 25:14-

257 19***.

258 Satake, T. and Yoshida, S. 1978. High temperature-induced sterility in indica rices at flowering. Jpn. J. Crop

259 Sci. 47:6-17.

260 Tsuboi, Y. 1961. Ecological studies on rice plants with regard to damages caused by wind. Bull. Natl. Inst.

261 Agric. Sci. A 8:1-156**.

262 Wang, C., Yang, J., Wa, J. and Cai, Q. 2004 Influence of high and low temperature stress on fertility and

263 yield of rice (Oryza sativa L.): Case study with the Yangtze River rice cropping region in China. Abstract,

264 World Rice Research Conference 2004, Tsukuba, Japan. 97.

265 * In Japanese with English abstract.

266 ** In Japanese with English summary.

267 *** In Japanese.

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268 Table and figure captions

269 Table 1. Measurement periods, numbers of measurements, variations (highest – lowest) in air temperature

270 (Ta, °C), solar radiation (Rs, MJ/m2), vapor pressure deficit (VPD, hPa), and flowering opening time (FOT) for

271 the five cultivars.

272 We did not measure FOT when heavy rain, strong wind, or digital camera disorder, so the number of

273 measurements was not corresponding to the measurement period.

274 Table 2. The results of multiple regression analysis to explain FOT across cultivars using GLMs.

275 ns indicates not significant. *, **, and *** indicate significant at 0.05, 0.01, and 0.001 probability levels,

276 respectively.

277 Table 3. Adjusted R squares and standardized partial regression coefficients in multiple regression analysis

278 for prediction of FOT of each cultivar using Ta, Rs, and VPD averaged every three hours from 0000 to 1200

279 as independent variables.

280 ns indicates not significant. *, **, and *** indicate significant at 0.05, 0.01, and 0.001 probability levels,

281 respectively.

282 Fig. 1. Time courses in five cultivars of correlation coefficients between FOT and Ta averaged every hour

283 from 0000 to 1000.

284 Symbols of cultivars are shown in the figure. Closed and open symbols represent that the correlation

285 coefficients are significant and not significant at the 5% level, respectively.

286 Fig. 2. Time courses of correlation coefficients between FOT and Rs averaged every hour from 0400 to 1000

287 in Fujihikari and from 0500 to 1000 in other four cultivars.

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288 Symbols are the same as in Fig. 1.

289 Fig. 3. Time courses in five cultivars of correlation coefficients between FOT and VPD averaged every hour

290 from 0000 to 1000.

291 Symbols are the same as in Fig. 1.

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292 Table 1. Measurement periods, numbers of measurements, variations (highest – lowest) in air temperature (Ta, °C), solar radiation (Rs, MJ/m 2), vapor

293 pressure deficit (VPD, hPa), and flowering opening time (FOT) for the five cultivars.

294

Measurement Numbers of
Maximum
Cultivars periods measurement Mean Ta Minimum Ta Rs VPD FOT
Ta
s
IR72 Sep 5 - Sep 12 23.6-28.3 26.6-34.4 18.4-25.2 5.2-22.4 4.7-11.3 0913-1200
22
Hanaechize Jul 20 - Sep 6 9 22.3-28.7 23.1-34.6 17.3-25.1 3.5-27.4 1.7-13.4 1041-1404
n
Fujihikari Jul 9 - Aug 1 24 22.1-27.6 22.9-34.2 17.3-24.9 3.1-27.4 1.2-11.2 1020-1606
Shinriki Sep 5 - Sep 17 23.6-28.3 26.6-34.4 18.4-25.2 5.2-22.4 4.7-11.3 1107-1346
23
Asahi Sep 1 - Sep 23 20 23.6-28.3 26.6-34.4 18.4-25.2 2.3-22.4 4.7-11.3 1120-1406
295
296 We did not measure FOT when heavy rain, strong wind, or digital camera disorder, so the number of measurements was not corresponding to the measurement

297 period.

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298 Table 2. The results of multiple regression analysis to explain FOT across cultivars using GLMs.

299
Adjusted R F values in GLMs Standardized partial regression coefficients
Periods
squares Cultivars Ta Rs VPD Ta Rs VPD
0000-0300 0.160* 4.156** 1.529ns 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
300
301 ns indicates not significant. *, **, and *** indicate significant at 0.05, 0.01, and 0.001 probability levels, respectively.

302

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303 Table 3. Adjusted R squares and standardized partial regression coefficients in multiple

304 regression analysis for prediction of FOT of each cultivar using Ta, Rs, and VPD averaged

305 every three hours from 0000 to 1200 as independent variables.

Adjusted R Standardized partial regression coefficients

Cultivars Periods
squares Ta Rs VPD
IR72 0000-0300 0.505* -0.851* 0.367ns
0300-0600 0.636* -0.562ns 0.412ns -0.067ns
0600-0900 0.672* -0.827* -0.102ns -0.058ns
0900-1200 0.036ns -0.462ns 0.496ns 0.319ns
Hanaechizen 0000-0300 0.123ns -0.646ns 0.378ns
0300-0600 0.854** -0.492ns 0.277ns 0.941**
0600-0900 0.747* -0.883* -0.237ns 0.953*
0900-1200 0.491ns -1.050* -0.635ns 0.712ns
Fujihikari 0000-0300 0.008ns 0.307ns -0.010ns
0300-0600 0.393** -0.539ns -0.864*** 0.548*
0600-0900 0.196ns -0.500ns -0.589** 0.564ns
0900-1200 0.549*** -1.394** -1.404*** 2.085**
Shinriki 0000-0300 0.228ns -0.467ns 0.658*
0300-0600 0.091ns -0.638ns -0.303ns 0.461ns
0600-0900 0.282ns -0.467ns -0.592* 0.279ns
0900-1200 -0.038ns -0.712ns -0.272ns 0.655ns
Asahi 0000-0300 -0.015ns -0.091ns 0.390ns
0300-0600 -0.110ns -0.191ns -0.259ns 0.221ns
0600-0900 0.346* -0.485ns -0.733** 0.397ns
0900-1200 0.033ns -0.496ns -0.180ns 0.146ns
306

307 ns indicates not significant. *, **, and *** indicate significant at 0.05, 0.01, and 0.001

308 probability levels, respectively.

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309
310

311 1 1 1

0.8
IR72

ハナエチゼン

フジヒカリ IR72 IR72 Fig. 1. Time courses in

神力
ハナエチゼン
Hanaechizen
0.6
朝日

312 0.8 0.8 0.4

0.2
フジヒカリ five cultivars of

313
0

Fujihikari
神力
correlation
0.6 0.6
-0.2

-0.4

Shinriki朝日
Correlation coefficient

314 coefficients between

-0.6

0.4 Asahi
-0.8

0.4 -1

315
0 1 2 3 4 5 6 7 8 9

FOT and Ta averaged

316 0.2 0.2

every hour from 0000

317 0
0 to 1000.

318 - 0.2 Symbols of cultivars

-0.2
319 are shown in the
- 0.4

320 -0.4 figure. Closed and

- 0.6
321 -0.6 open symbols
- 0.8
322 represent that the
-0.8
323 -1 correlation
-1 0 1 2 3 4 5 6 7 8 9
324 coefficients are
0 1 2 3 4 5 6 7 8 9
325 significant and not
Hour
326 significant at the 5%

327 level, respectively.

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328

329

330 Fig. 2. Time courses of

1 1 IR72

331 ハナエチゼン correlation coefficients

332 0.8 0.8 フジヒカリ

between FOT and Rs
神力
333 0.6 0.6
朝日 averaged every hour
Correlation coefficient

334 from 0400 to 1000 in

0.4 0.4

335 Fujihikari and from

336
0.2 0.2

0500 to 1000 in other

0
337 0 four cultivars.

338 - 0.2 Symbols are the same

-0.2
339 - 0.4 as in Fig. 1.

340
-0.4
- 0.6
-0.6
- 0.8
-0.8
-1
-1 0 1 2 3 4 5 6 7 8 9

0 1 2 3 4 5 6 7 8 9
Hour

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341

1 1 IR72

ハナエチゼン

0.8 0.8 フジヒカリ

神力

0.6 0.6
朝日
Correlation coefficient

0.4 0.4

0.2
0.2
0
0
- 0.2
-0.2
- 0.4
-0.4
- 0.6
-0.6
- 0.8
-0.8
-1
-1 0 1 2 3 4 5 6 7 8 9

0 1 2 3 4 5 6 7 8 9
342
Hour
343

344 Fig. 3. Time courses in five cultivars of correlation coefficients between FOT and VPD averaged every hour from 0000

345 to 1000.

346 Symbols are the same as in Fig. 1.

347

348

349

350

351 The information contained in this document is confidential, privileged and only for the information of the intended recipient and may not
352 be used, published or redistributed without the prior written consent of Crimson Interactive Inc.

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Proofreading Test
353

354

355 Proofread the following test keeping American English conventions in mind.

356 Abstract

357 Fasting hypertriglyceridemia is positively associated with the morbidity of coronary heart disease (CHD),
358 postprandial (non-fasting) hypertriglyceridemia also correlates with the risk status for CHD, which is related to the
359 increase in chylomicron (CM) remnant lipoproteins produced from the intestine. CM remnant particles, as well as
360 oxidized low density lipoprotein (LDL) or very low density lipoprotein (VLDL) remnants, are highly atherogenic and act
361 by enhancing systemic inflammation, platelet activation, coagulation, thrombus formation, and macrophage foam cell
362 formation. Cholesterol levels of remnant lipoproteins significantly correlate with small, dense LDL, impaired glucose
363 tolerance (IGT) and CHD prevalence. We have developed an assay of apolipoprotein(apo)B-48 levels to evaluate the
364 accumulation of CM remnants. Fasting apoB-48 levels correlate with the morbidity of postprandial
365 hypertriglyceridemia, obesity, type III hyperlipoproteinemia, the metabolic syndrome, hypothyroidism, chronic kidney
366 disease, and IGT. Fasting apoB-48 levels also correlate with carotid intima-media thickening and CHD prevalence, and
367 high apoB-48 level is a significant predictor of CHD risk independent of the fasting TG level. Diet interventions, such as
368 dietary fibers, polyphenols, medium-chain fatty acids, diacylglycerol, and long-chain n-3 polyunsaturated fatty acids
369 (PUFA), ameliorate postprandial hypertriglyceridemia; drugs for dyslipidemia (n-3 PUFA, statins, fibrates or ezetimibe)
370 and drugs for diabetes concerning incretins (dipeptidyl-peptidase IV inhibitor or glucagon like peptide-1 analogue) may
371 improve postprandial hypertriglyceridemia. Since the accumulation of CM remnants correlates to impaired lipid and
372 glucose metabolism and atherosclerotic cardiovascular events, further studies are required to investigate the
373 characteristics, physiological activities, and functions of CM remnants for the development of new interventions to
374 reduce atherogenicity.

375 1. Fasting and Postprandial Hypertriglyceridemia

376 In Japan, the morbidity and mortality of atherosclerotic cardiovascular diseases (ASCVD), including coronary
377 heart disease (CHD) and stroke have gradually increased for recent decades. An intensive intervention against
378 hypercholesterolemia or hyper low-density lipoprotein (LDL)-cholesterolemia using statins has improved the primary
379 and secondary prevention of CHD events; however, the complete suppression of CHD events has not yet been
380 accomplished. Recently, the importance of controlling “residual risk factors” for CHD has been emphasized;
381 hypertriglyceridemia (≧150 mg/dL) and hypo high-density lipoprotein(HDL)-cholesterolemia (<40 mg/dL) have both
382 been investigated in basic and clinical research settings to determine a possible method for the prevention of ASCVD
383 (1,2). As fasting triglyceride (TG) levels at the registration increased (<100, 100-149, 150-199, 200-499, and ≥500 mg/dL)
384 the age- and sex-adjusted hazard ratio (HR) for adjusted all-cause mortality worsened (1.06, 1.16, 1.29, and 1.28
385 compared with <100 mg/dL, respectively)(3). A systematic review and meta-analysis of 35 observational studies
386 revealed that fasting hypertriglyceridemia was significantly associated with cardiovascular death (odds ratios (OR) 1.80;
387 95% confidence interval (CI) 1.31-2.49), cardiovascular events (OR, 1.37; 95% CI, 1.23-1.53) and myocardial infarction
388 (OR, 1.31; 95% CI, 1.15-1.49)(4). Moreover, on a background of statin treatment after ACS, fasting triglycerides were
389 related to risk of CHD death, nonfatal myocardial infarction, stroke, and unstable angina in models adjusted for classic
390 CHD risk factors (5). The Japan Atherosclerosis Society Guidelines 2012 suggested that if a subject with
391 hypertriglyceridemia (fasting TG level ≥150 mg/dL) is defined as high risk for ASCVD (especially CHD) by an annual
392 medical checkup, he or she should be encouraged to receive secondary checkups and medical intervention (6).
393 However, fasting TG levels may be varied by the lipid content and the consumption time of the patient’s meal, fasting
394 TG level is not always positively correlated with the atherogenicity. Slightly elevated TG levels that are observed in
395 patients with impaired glucose tolerance (IGT) or the metabolic syndrome (MetS) are highly atherogenic while the
396 severely high TG levels that are observed in patients with primary chylomicronemia or lipoprotein lipase (LPL)

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397 deficiency are rarely atherogenic. Therefore, the measurement of the fasting TG level is insufficient for evaluating
398 individual ASCVD risks; the exact analysis of impaired lipoprotein metabolism as the background has been requested.

399 In contrast, many studies have revealed that postprandial (non-fasting) hypertriglyceridemia is likely to reflect the risk
400 for ASCVD. Iso et al. showed the positive correlation between the incidence of CHD (myocardial infarctions, angina
401 pectoris events, and sudden cardiac deaths) and non-fasting TG levels in a 15.5-year prospective observation; the
402 multivariate relative risk of CHD associated with a 1 mmol/L increase in TG level was 1.29 (95% CI: 1.09-1.53; p <0.01)
403 for men and 1.42 (1.15-1.75; p <0.01) for women independent of total cholesterol levels (7). Nordestgaard et al. also
404 showed that non-fasting TG levels correlated with the morbidity of CHD (8) and ischemic stroke (9) in a prospective
405 cohort study (Copenhagen City Heart Study). However, there has been no standardized reference value for
406 postprandial TG levels to define postprandial hypertriglyceridemia as a risk factor for ASCVD events. In 2016, the
407 European Atherosclerosis Society and European Federation of Clinical Chemistry and Laboratory Medicine published a
408 joint consensus statement recommending the routine use of non-fasting blood samples for assessing plasma lipid
409 profiles (10), based on the epidemiological data that there was no clinically significant difference between LDL-C and
410 non-HDL-C levels in both the fasting and the postprandial state. As maximal mean changes in TG levels at 1-6 h after
411 habitual meals were stable (+26 mg/dL), they suggested that the cut-off for abnormal postprandial TG levels should be
412 >2 mmol/L (175 mg/dL) and pointed out the usefulness of measuring non-fasting lipid levels in usual clinical settings
413 (10). For the future, the cut-off value of non-fasting TG level based upon the prospective study in a larger population
414 is strongly recommended for the purpose of evaluating ASCVD risks with high sensitivity.

415

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