To test whether BR is involved in Fe deficiency-induced changes in physiological processes in rice plants, the effect of exogenous EBR at varying concentrations (0–500 nM) on cholorophyll concentrations of rice seedlings grown in Fe-sufficient and Fe-deficient media was studied. Apparent chlorosis was observed in young leaves of rice seedlings grown in Fe-deficient medium for 2 weeks (Fig. 1A), leading to a decrease in cholorophyll concentration (Fig. 1B). Furthermore, the chlorosis became more evident by application of EBR to Fe-deficient seedlings. In contrast, application of EBR had no apparent effect on leaf cholorosis in Fe-sufficient seedlings (Fig. 1A). Accordingly, treatments with EBR led to a significant decrease in chlorophyll concentration of Fe-deficient plants, while the same treatment had no effect on chlorophyll concentration in Fe-sufficient seedlings (Fig. 1B). The reduction in chlorophyll concentration in Fe-deficient seedlings by EBR occurred at a concentration of 1 nM, and no further reduction in the chlorophyll concentration was observed when EBR concentration was increased up
to 500 nM, suggesting that the effect of EBR on the chlorophyll concentration is independent of EBR dose, and that BR may be an important signal during Fe deficiency. Although 1 nM EBR can have the negative effect, at >100 nM it can partly restore the phenotype of the d2-1 mutant. Therefore, an EBR concentration of 100 nM was used hroughout this study, which can partly restore growth of d2-1 and has no effect on growth of WT plants. Since rice can take up both Fe2+- and Fe3+-MA, in addition to Fe-EDTA, we also investigated the effects of EBR on plants with different forms of Fe present in the medium (FeSO4, Fe3+EDTA, and FeCl3). Similar to FeEDTA, EBR-induced leaf chlorosis was also detected under conditions of deficiency in FeSO4, Fe3+EDTA, or FeCl3 in the growth medium (Supplementary Figure S1).These results indicate that the effect of BR on Fe nutrition is independent of Fe species in the growth medium. We therefore
focused on the effect of BR on rice plants with FeEDTA in the medium throughout our study.
The involvement of BR in the mediation of Fe deficiencyinduced changes in physiological processes was further evaluated using a BR-deficient mutant d2-1. Similar to WT plants, Fe deficiency also decreased leaf chlorophyll in the d2-1 mutant, and exogenous application of EBR enhanced Fe-deficiency-induced leaf chlorosis in young leaves of d2- 1 seedlings (Fig. 2). However, the d2-1 mutant had a higher chlorophyll concentration than the WT plant, and the magnitude of decrease in chlorophyll concentration by EBR was much greater in WT plants than in d2-1 mutants under edeficient
conditions (Fig. 2), indicating that the WT is more sensitive to EBR than d2-1 mutant plants under Fe-deficient conditions. In contrast, application of EBR had no apparent
effect on foliar chlorophyll concentration in Fe-sufficient d2-1 mutant seedlings (Fig. 2).