Fig. 7: ILR3 and RH24 are in condensates and BTS facilitates the condensate formation.

a RH24 interacts with ILR3 and were co-localized with BTS in the nucleus. The proteins were transiently expressed in N. benthamiana leaves. YFP fluorescence was assigned with pseudocolor green. Bar = 10 μm. b ILR3 and RH24 are in phase-separated condensates. The upper panel shows the FRAP of ILR3-nYFP and cYFP-RH24 that were transiently co-expressed in N. benthamiana leaf epidermal cells. Red arrows indicate the nuclear bodies that are bleached. The images at the bottom show the fusion of the nuclear bodies. White arrows indicate the nuclear bodies that undergo fusion. Bar = 2 μm. Images are representative of three independent experiments. c BTS and cold treatments facilitate condensate formation. ILR3-nYFP, cYFP-RH24, GUS-FLAG, and BTS-FLAG were transiently expressed in N. benthamiana leaves at 22 °C for 2 days and then transferred to 22 °C or 4 °C for another 1 h. Bar = 7.5 μm. The percentage of ILR3-RH24 condensations were presented. Images are representative of three independent experiments. d ILR3-mediated chilling sensitivity depends on RH24. 2-4# and 3-9# are two independent 35S:ILR3-ox transgenic lines. The plants were grown on ½ MS at 22 °C for 1 week or at 22 °C for 1 week and treated at 8 °C for another 4 weeks. The concentration of chlorophyll (μg/mg FW) was presented. Data presented as means ± SD (n = 3 biological replicates) by two-factor ANOVA with Tukey’s HSD test (P ≤ 0.05). e Iron concentration in ILR3-ox and ILR3-ox/rh24 plants. The leaves from plants grown in soil under 22 °C for 4 weeks were used for Fe measurement. Data presented as means ± SD (n = 4 biological replicates) by one-way ANOVA with Tukey’s HSD test (P ≤ 0.05). f The working model: RH24 unwinds ILR3 mRNA to facilitate the accumulation of ILR3. Under chilling stress, RH24 is accumulated, which sequesters ILR3 in phase-separated bodies by forming RH24-ILR3 condensates in the nucleus. BTS and cold stress facilitate RH24-ILR3 condensate formation. As a result, the ILR3-regulated iron uptake is reduced under chilling stress, which consequently attenuates the iron-overload caused damage. Under standard growth conditions, BTS finetunes free ILR3 to maintain iron homeostasis.