Effect of elevated temperature on pollen viability and grain setting in rice

Introduction

Nearly 740 million tonnes of paddy rice are produced annually, yet climate projections indicate that flowering-stage temperatures will exceed 35 °C across 42% of global rice-growing area by 2050 [1,2] . Rice anthesis is confined to a narrow 1-2 hour morning window, and pollen grains must germinate, extend a tube through the style, and reach the embryo sac within about 30 minutes [3] . This tight timeline makes the process acutely vulnerable to thermal disruption. Elevated temperature impairs pollen at multiple levels: anther dehiscence becomes asynchronous, pollen viability declines through protein denaturation, and tube elongation slows as membrane fluidity and cytoskeletal dynamics are altered [4,5] . Most field-based observations report a sharp decline in grain setting once maximum temperature exceeds 33-35 °C, but the relative contribution of each step viability loss versus tube growth failure remains unclear [6,7] . Understanding which step is most sensitive helps breeders target traits for heat tolerance. This research imposed four precise temperature treatments at anthesis to separately quantify pollen viability, tube elongation, spikelet fertility, and grain setting, testing whether tube growth is the primary bottleneck under moderate versus severe heat.

Materials and Methods

Research area and growth conditions

Experiments were conducted in the Controlled Environment Growth Suite at the Sutton Bonington Campus (52°50′N, 1°15′W) of the University of Nottingham. Four Conviron E15 growth chambers were programmed to 29 °C (control), 33, 36, and 39 °C daytime temperature (0800-1800) with a common night temperature of 24 °C. Relative humidity was maintained at 70±5% and photoperiod at 12 h (600 µmol m⁻² s⁻¹ PAR).

Seasonal variations

Two experimental runs were conducted (March-July 2022 and January-May 2023). Chamber temperatures were validated using TinyTag Plus 2 loggers at canopy height; actual temperatures during the anthesis window (0900-1100) were within±0.4 °C of set points. CO₂ concentration was ambient (~420 ppm) in both runs.

Water quality

Plants were grown in flooded pots (5 cm standing water). Irrigation water temperature tracked chamber air within 2 °C. Electrical conductivity of the irrigation solution was maintained at 0.8-1.0 dS m⁻¹. Water pH ranged 6.5-7.0 throughout both growing cycles.

Materials

Indica rice cultivar IR-64 was grown in 5 L pots (3 plants pot⁻¹) filled with a 2:1 paddy soil:perlite mix. Plants were raised at 29/24 °C until panicle initiation, then transferred to treatment chambers at booting. Basal nutrition: Yoshida solution applied weekly. Temperature treatments were imposed from first anthesis until all tillers had flowered (10-14 days).

Methods

Pollen viability was assessed by in vitro germination on a 15% sucrose + 1% agar medium (n = 300 grains per replicate, 6 replicates). Pollen tube length was measured on germinated grains after 30 min at the treatment temperature using a calibrated eyepiece micrometer. Spikelet fertility was determined as the percentage of spikelets with at least one stainable pollen grain on the stigma at 2 h post-anthesis. Grain setting was the percentage of filled grains per panicle at maturity. Data were analysed by one-way ANOVA in R 4.2 with Tukey's HSD (p≤0.05). Linear and nonlinear regressions were fitted between parameters. Pollen tube length showed the most dramatic temperature sensitivity (Figure 2). Mean tube length at 29 °C was 680 µm, sufficient to traverse the 450-500 µm style of IR-64. At 36 °C, tube length dropped to 340 µm below the critical threshold and at 39 °C only 180 µm. The distribution also narrowed: the interquartile range at 29 °C was 120 µm but only 80 µm at 39 °C, indicating both reduced mean growth and loss of highperforming pollen grains. Regression analysis showed pollen viability explained 96% of variation in grain setting (R² = 0.96, p<0.001). Pollen tube length below 450 µm was associated with grain setting below 50%, identifying a functional threshold (Table 2). The relationship between temperature and tube length was better described by a sigmoidal function (R² = 0.99) than a linear one, with the inflection point at 34.8 °C.

Results

Discussion

The 34.8 °C inflection point for tube growth collapse is remarkably close to the 35 °C critical threshold reported from field studies across tropical Asia [6,8] , confirming that pollen tube elongation rather than viability per se is the primary bottleneck. At 36 °C, roughly half the pollen grains were still viable (54.7%) yet only 38.6% of spikelets set grain a 16-point gap that reflects tubes failing to reach the ovule despite germination. This matches evidence from in vivo imaging studies showing arrested tube growth in the upper style at temperatures above 34 °C [9,10] . The narrowing of tube length distribution under heat points to a loss of the right tail of high-performing grains, which are precisely the ones that achieve fertilisation under normal conditions. Breeding for longer average tube growth or faster elongation rate could therefore be more effective than improving viability alone [11,12,13] . The sigmoidal rather than linear response means that small temperature increases near the 35 °C threshold produce disproportionately large fertility losses a pattern with sobering implications for regions where mean flowering-time temperatures are already close to this value [14] .

Conclusion

Elevated temperature at anthesis reduced rice pollen viability, tube elongation, and grain setting in a dose-dependent manner, with 34.8 °C identified as the inflection point for tube growth collapse. Pollen tube length fell below the critical threshold for ovule penetration at 36 °C, even though half of pollen grains remained viable, confirming tube elongation as the primary bottleneck. These findings direct breeding efforts toward pollen tube growth traits and flag the narrow temperature margin available before reproductive failure becomes widespread.

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