such as "Introduction", "Conclusion"..etc
Increasing the heat tolerance of rice at flowering, one of the most sensitive stages of development to stress, is a vital adaptation strategy for variable and warmer climates (Horie et al., 1996). Tolerance classically comprises elements of escape, i.e. the timing of panicle emergence and spikelet/floret opening relative to the occurrence of the stress, and the absolute tolerance to stress of key processes, such as anther dehiscence. It is essential for phenotyping and modelling that these two mechanisms, as well as the effects of temperature on the rate of spikelet anthesis, are clearly differentiated.
Flowering and anthesis in most O. sativa genotypes of rice occurs over a 5 d period, with most spikelets reaching anthesis between 10.00 h and 12.00 h (Fig. 1; Nishiyama and Blanco, 1980; Prasad et al., 2006). Although indica spp and japonica spp have a similar pattern of flowering, it is worth noting that O. glaberrima genotypes flower much earlier in the day, with >90% spikelets nearing anthesis by 09.00 h (Nishiyama and Blanco, 1980; Prasad et al., 2006). This is a potentially useful escape mechanism that should be incorporated into O. sativa now that interspecific crosses can be made (Jones et al., 1997). For phenotyping for heat tolerance, it is clearly essential that high temperature (or other stresses) are timed to coincide with this peak and that escape is taken into account, as discussed later.
In both IR64 and Azucena, the peak of flowering occurred about 30–45 min earlier at 36.2 °C, compared with 29.6 °C, presumably reflecting a simple thermal response of overall rate of development (progress towards anthesis) to spikelet (nb. not ambient) temperature whose optimum is apparently 36.2 °C. In IR64, rates of spikelet opening were apparently also increased by temperature, and over 3 d about 20% more spikelets reached anthesis at 36.2 °C. By contrast, high temperature reduced the number of spikelets opening by 36% in Azucena, the effect increasing in severity over the three days. So although the overall pattern or timing of flowering was not adversely affected by temperature in Azucena, the number of potential seed sites was greatly reduced. Matsui et al. (2000) have shown that the sterility of japonica spp increases with increasing duration (days) of exposure during flowering, in contrast to indica spp where fertility was similar throughout the flowering period. These results agree with this, but suggest this is due as much to effects of temperature on the number of spikelets reaching anthesis as fertility, with increased rates of spikelet anthesis in the indica IR64 compensating for reduced fertility. The effects of high temperature on spikelet anthesis, both pattern and rate, are clearly important factors to be considered in phenotyping for heat tolerance, and not just fertility per se.
One hour at a spikelet tissue temperature of 33.7 °C was sufficient to cause sterility if this coincided with anthesis in both IR64 and Azucena, in agreement with previous studies (Satake and Yoshida, 1978). Sterility in rice is invariably associated with low numbers of pollen or germinated pollen on the stigma (Matsui et al., 2000, 2001; Prasad et al., 2006; Farrell et al., 2006). Anther dehiscence is acutely sensitive to high temperature, both prior to and during anthesis (Matsui et al., 2001). Rice florets open for about 30 min (Ekanyake et al., 1989), exposing anthers and pollen to ambient temperature and humidity. Pollen is also acutely sensitive and loses its viability within 10 min (Song et al., 2001). After pollination it takes about 30 min for the pollen tube to reach the embryo sac (Cho, 1956) and it is very likely that very high temperatures will also influence pollen germination and pollen tube growth (cf Kakani et al., 2002, in peanut). Nonetheless, poor anther dehiscence is apparently the main cause of sterility at high temperature in rice.
Although 1 h at 33.7 °C was sufficient to induce sterility, it is apparent that shorter periods than this may also affect spikelet fertility. From the marking studies, spikelets that opened in the hour before, and to a lesser extent the hour after, exposure to high temperature were affected as well. This probably reflects the fact that spikelets opening 30 min before high temperature, for example, will also experience 30 min at high temperature, seemingly enough to reduce spikelet fertility to some extent. The timing of anthesis is clearly important for phenotyping for heat tolerance in terms of spikelet fertility or sterility; even with spikelet marking, short periods of high temperature will still include an element of escape. The small effect of duration on spikelet fertility in IR64 (Fig. 3) was interpreted to be due to more spikelets escaping heat stress in shorter than longer duration treatments (Fig. 4), giving a higher apparent fertility.
In these experiments, plants were transferred to growth cabinets at the target temperature and spikelets were therefore given no opportunity to acclimate. However, given the very short period of exposure required to induce sterility, it is unlikely that acclimation would occur. Furthermore, under natural conditions, where temperature increases gradually during the morning, escape rather than acclimation is likely to be is more significant.
IR64 and Azucena responded differently to varying temperatures and durations of temperature. In Azucena, which was clearly very susceptible to temperatures >29.6 °C, there was a significant temperaturexduration interaction, which could be quantified by a cumulative temperature response above a threshold temperature of 33 °C. Vara Prasad et al. (1999) reported a similar response in groundnut. Although the relationship was strong (r2=0.86), the two temperature cohorts (33.7 °C and 36.2 °C) only overlap slightly and further data that create a wider range of accumulated temperatures are needed to confirm this response. By contrast, there was no interaction in IR64 such that a temperature stress of 1 h reduced spikelet fertility by 9% °C–1. Yoshida et al. (1981) previously described the effect of varying duration and temperature on seed-set in several genotypes, including N22 and IR747B, both indica spp. These data were re-analysed and no significant temperaturexduration interactions were found (data not presented). However, given that Matsui and Omasa (2002) have shown that a japonica sp., Akitokomachi is also highly tolerant, it is unlikely that the difference observed between IR64 and Azucena is subspecies-dependent. Although heat stress primarily affects anther dehiscence (Matsui et al., 2000, 2001; Prasad et al., 2006), it is possible that, in susceptible genotypes such as Azucena, that a number of other processes before fertilization are affected. For example, pollen swelling, anther pore size (Matsui and Kagata, 2003), and pollen stickiness may all be affected, as might pollen germination and the rate of pollen tube growth (Kakani et al., 2002). Farrell et al. (2006) have also shown that cold tolerance in rice is related to stigma size, as well as anther size. It is unlikely that processes after fertilization are affected as spikelets exposed to high temperature 1 h after opening are fertile and set seed in Azucena as well as IR64. Some of these factors are being investigated.
In conclusion, this study has shown that 1 h of exposure to high temperature is sufficient to induce sterility in rice (O. sativa). IR64 and Azucena exhibited different sensitivities and responses to temperature of spikelet fertility. Nonetheless, phenotyping of RILs and breeding lines of tolerant and susceptible types can be effectively carried out by exposing plants to high temperature (>33.7 °C) for 4 h, centred around the hours of maximum anthesis. This will ensure that >90% of spikelets opening experience high temperature, effectively removing ‘escape’ as a potential confounding factor. This study has also shown that high temperature affects the pattern of flowering and the number of spikelets that reach anthesis, and phenotyping needs to include these factors as well.
Acknowledgements We thank the Felix Scholarship for funding and The University of Reading for supporting the PhD of K Jagadish. We also thank Mr K Chivers for excellent engineering support and Ms C Hadley, Mr L Hansen, and Ms P Ling for technical assistance. IRRI, Philippines and WARDA, Cote d'Ivoire are thanked for supplying seed.
Enter the code exactly as it appears. All letters are case insensitive, there is no zero.