Two experiments were undertaken between May and October in 2003 and in 2004, using controlled environment facilities at the Plant Environment Laboratory, Department of Agriculture, The University of Reading, UK (51°27' N, 00°56 'W). Plants were grown in a greenhouse under optimum temperature and photoperiod conditions, and transferred at anthesis to growth cabinets to impose high temperature treatments.
Greenhouse
Plants were grown in a naturally-lit greenhouse with day and light-proof night compartments. Pots were placed on automated mobile trolleys (2.85 mx0.96 m) that were drawn out and in to night compartments (3.57 mx1.74 m) at 08.00 h and 19.00 h BST, respectively, giving a short, inductive photoperiod of 11 h. Temperature in the greenhouse was maintained at 30/24 °C day/night through a combination of heating (16 kW) and venting during the day, and heating at night. Fans in both the day and night compartments circulated air continuously. Aspirated air temperature was measured by copper-constantan thermocouples at canopy height and recorded every 30 min using data loggers (Delta T Devices, Burwell, Cambridge, UK).
Crop husbandry
Plants were grown in a soil-less medium. Steam-sterilized sand and gravel were mixed with peat compost and vermiculite in the proportion of 2:4:1:4 by vol., respectively. Pots of 12.5 cm diameter were filled with the potting medium and placed on the mobile trolleys in the greenhouse. Pots were soaked overnight and four to six seeds of IR64 and Azucena were sown per pot at a depth of 2–2.5 cm and then thinned down to one per pot at the three leaf stage. All the plants were de-tillered to leave three shoots to reduce overcrowding. After thinning, plants were irrigated automatically through a drip-irrigation system (greenhouse), or watered by hand (cabinet), with a complete nutrient solution containing 100 mg l–1 inorganic nitrogen. The nutrient solution was acidified to pH 5 to avoid Fe-deficiency (Yoshida et al., 1976). Plants were also sprayed with a foliar feed (Miracle-Gro®, The Scotts Company UK Ltd) at 3.75 g l–1 at panicle emergence. Azucena plants were sprayed with Torque (50 w/w fenbutatin oxide) at 0.5 g l–1 of water to control red spider mites 82 d after sowing (DAS). There were no other major pest or disease problems.
Growth cabinets and temperature treatments
At anthesis, plants were transferred to modified Sacxil growth cabinets (internal size 1.4x1.4x1.5 m). Cabinets were maintained at 360 µmol mol–1 CO2 of air. The photosynthetic photon flux density (PPFD) at the base of the cabinet was maintained at 650 µmol m–2 s–1 by a combination of cool white fluorescent tubes and incandescent lamps. A centrally placed fan circulated heat and air uniformly. Screened and aspirated air temperature and RH were measured every 10 s using copper constantan thermocouples and recorded in a data logger (Delta T Devices, Burwell, Cambridge, UK) and averaged over 10 min for the entire period of high temperature exposure.
In 2003 (Experiment 1), growth cabinets were maintained at air temperatures of 30 (control), 35, and 38 °C between 08.00 h and 19.00 h BST. Growth cabinets were not replicated. Vapour pressure deficit (VPD) was maintained at 1.2 kPa in all temperature regimes and therefore RH ranged from 70% to 85%. VPD/RH was controlled either by adding moisture to air passing through glycol maintained at a set temperature or by removing the excess humidity by condensation.
In 2004 (Experiments 2 and 3), growth cabinets were again maintained at 30, 35, and 38 °C during the day. In contrast to Experiment 1, growth cabinets were replicated in Experiment 2. RH was also held constant at 60% during the day in 2004 to maximize anther dehiscence (Matsui et al., 1999a, b, 2001; Matsui and Omasa, 2002) and therefore VPD ranged from 1.7 to 2.3 kPa. In addition, spikelet tissue temperature was measured by placing copper constantan thermocouples in spikelets of four different plants per cabinet. Temperatures were recorded by a data logger (Delta T Devices, Burwell, Cambridge, UK) every 10 s and averaged over 10 min for the entire period of high temperature exposure.
Effect of high temperature on spikelet fertility
The effect of temperature and the duration of temperature on spikelet fertility in IR64 and Azucena was examined in Experiments 1 and 2.
In Experiment 1 (2003), on the day after the first anthers were observed, individual plants of IR64 and Azucena were transferred at 08.00 h from the greenhouse to a growth cabinet kept at 30 °C. As IR64 and Azucena flowered at different times, transfers therefore occurred on different days. Transfers were at 10.00 h, to coincide with the period of peak flowering, plants were then transferred to adjacent cabinets at air temperatures of 30 (control), 35, and 38 °C for a 2 h period. A square wave heat treatment was applied to overcome the potentially confounding effects of gradually increasing temperature. At the end of this period plants were returned to the greenhouse. The previous day (after midday) anthers from spikelets at anthesis were carefully removed.
To identify spikelets exposed to high temperature, spikelets that opened (i.e. with a visible anther) during the 2 h temperature treatment were marked with red acrylic paint at the end of the temperature treatment. Most opened spikelets were towards the top of the panicle. Ten to 12 d after the temperature treatments, marked spikelets were scored for spikelet fertility. There were 10 replicate pots in each temperature (growth cabinet) treatment.
In Experiment 2 (2004), plants were again subjected to temperature treatments of 30, 35, and 38 °C in growth cabinets, using the same transfer and spikelet marking system as in Experiment 1. On the day that anthesis was first observed, plants were moved to the control cabinet (30/24 °C). On the following day, plants were subjected to 30, 35, and 38 °C for 1, 2, 4, or 6 h, centred on the peak flowering time of 11.00 to 11.30 h, i.e. plants were transferred at different times of the morning. At the end of the respective temperature treatments, plants were transferred back to the control cabinet, and from there to the greenhouse the following morning. Spikelets reaching anthesis during the high temperature treatments were marked with different coloured acrylic paints for different durations, either at the end of the treatment (1 and 2 h) or in the middle and at the end (4 and 6 h). Plants were removed from the growth cabinets for marking to ensure temperature and RH conditions were maintained at specified levels during the temperature treatments.
Spikelet fertility (seed-set) of the painted spikelets was scored 10–12 d after anthesis. In this experiment temperature treatments (growth cabinets) were replicated twice, and there were five replicate plants per cabinet.
The total numbers of spikelets marked at each temperature by duration treatment for each genotype in Experiments 1 and 2 are given in Table 1.
Time of spikelet anthesisThe effect of the time of spikelet anthesis relative to the imposition of a high temperature stress was also examined in Experiments 1 and 2. In Experiment 1, spikelets that reached anthesis prior to the 2 h temperature treatment, i.e. between 08.00 h and 10.00 h, were marked with blue paint to distinguish them from those that reached anthesis during the temperature treatment (see above). In Experiment 2, spikelets opening 1 h before and 1 h after the temperature treatment were marked with blue and yellow paint, respectively. Seed-set of these spikelets was scored 10–12 d after anthesis.
Flowering patterns
The effect of high temperature on flowering patterns was examined in Experiment 3 in 2004. Three replicate plants of each of IR64 and Azucena were transferred from the greenhouse immediately before anthesis to a growth cabinet at either 30/24 °C or 38/24 °C. For three consecutive days, from the start of anthesis, the number of spikelets at anthesis (i.e. anthers extruding or spikelets gaping and anthers visible), were counted every 30 min between 09.00 and 15.00 h BST.
Statistical analysis
Spikelet fertility (Experiments 1 and 2) was treated as binomial data, there being only two states possible; fertile/seed-set (1) and infertile/no seed-set (0). These binomial data were analysed as logits [log(p/(100–p)) of the percentages p(0 p Genstat version 7.1) using plant as a random factor and the spikelet as the unit of treatment. Hence, the results are expressed taking into account each individual spikelet marked as a data point from all the treatments. The logit results (A) can be back-transformed to the original scale using 1/(1+EXP(–A)), giving predicted probabilities or Odds Ratio of success (OR).
To examine the interaction between temperature and duration of exposure, a threshold temperature response (Vara Prasad et al., 1999) was used, wherein spikelet fertility was reduced in proportion to accumulated temperature above a threshold value. The threshold value was set at 33 °C based on visual observation of the data and following Nakagawa et al. (2002). Accumulated temperature or thermal time (TT) above this threshold was then calculated from day temperature (T) and duration of treatment (t) by:
using values from 2003 and 2004. Logit spikelet fertility was then regressed against
TT.
The total number of anthesing spikelets per day (Experiment 3) was analysed by ANOVA as a completely randomized experiment with three replicates.
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