Fig. 7.

Flavonols regulate pollen viability, tube length, and ROS levels during heat stress. (A) Quantification of pollen viability in VF36 and the are mutant grown in two different greenhouse temperature regimes. (A–C) Data are shown as the mean ± SEM. Asterisks indicate significant differences between VF36 and are and pound signs indicate differences between temperature treatments, according to two-way ANOVA followed by a Fisher’s LSD test with P < 0.05. (B) Quantification of in vitro tube growth in VF36 and the are mutant at two different temperature regimes. Pollen tubes were grown in germination medium for 90 min at 28 °C and then for 150 min at 34 °C or kept at 28 °C for another 150 min. n = 166–168 tubes measured across three independent experiments. (C) Quantification of DCF fluorescence in pollen tubes of VF36 and are grown in vitro at the two different temperature regimes described above. n = 97–123 tubes measured across four independent experiments. (D) Quantification of pollen tube length in VF36 and the are mutant. Pollen tubes of VF36 and the are mutant were grown for 90 min at 28 °C and then transferred for 150 min to 34 °C (red symbols) or kept for another 150 min at 28 °C (blue symbols). Supplementation of the pollen germination medium with the nonenzymatic antioxidant ascorbic acid or the NADPH oxidase inhibitor DPI occurred after 90 min of growth at 28 °C. Data represent mean ± SEM. n = 80 tubes measured across two independent experiments. Gray, dotted lines with asterisks indicate significant differences between control and treatment within the same genotype and temperature regime. Pound signs indicate significant differences in pollen tube length between 28 °C and 34 °C within the same genotype and treatment. (E) Model for the role of heat stress and flavonols in pollen viability, germination, and tube growth. Flavonols scavenge ROS produced in pollen at normal and increased temperatures, thereby regulating pollen viability, germination, and tube growth. Solid arrows depict relationships described in our study, whereas dashed arrows highlight relationships reported for vegetative tissues in the literature. Green dots represent viable pollen grains, while magenta dots are dead grains.