In rice (Oryza sativa) and other plants, plant architecture and seed size are closely related to yield. Brassinosteroid (BR) signaling and the mitogen-activated protein kinase (MAPK) pathway (MAPK kinase kinase 10 [MAPKKK10]–MAPK kinase 4 [MAPKK4]–MAPK6) are two major regulatory pathways that control rice architecture and seed size. However, their possible relationship and crosstalk remain elusive. Here, we show that WRKY53 mediated the crosstalk between BR signaling and the MAPK pathway. Biochemical and genetic assays demonstrated that glycogen synthase kinase-2 (GSK2) phosphorylates WRKY53 and lowers its stability, indicating that WRKY53 is a substrate of GSK2 in BR signaling. WRKY53 interacted with BRASSINAZOLE-RESISTANT 1(BZR1); they function synergistically to regulate BR-related developmental processes. We also provide genetic evidence showing that WRKY53 functions in a common pathway with the MAPKKK10–MAPKK4–MAPK6 cascade in leaf angle and seed size control, suggesting that WRKY53 is a direct substrate of this pathway. Moreover, GSK2 phosphorylated MAPKK4 to suppress MAPK6 activity, suggesting that GSK2-mediated BR signaling might also regulated MAPK pathway. Together, our results revealed a critical role for WRKY53 and uncovered sophisticated levels of interplay between BR signaling and the MAPK pathway in regulating rice architecture and seed size.

Cold tolerance at the booting (CTB) stage is a major factor limiting rice (Oryza sativa L.) productivity and geographical distribution. A few cold-tolerance genes have been identified, but they either need to be overexpressed to result in CTB or cause yield penalties, limiting their utility for breeding. Here, we characterize the function of the cold-induced transcription factor WRKY53 in rice. The wrky53 mutant displays increased CTB, as determined by higher seed setting. Low temperature is associated with lower gibberellin (GA) contents in anthers in the wild type but not in the wrky53 mutant, which accumulates slightly more GA in its anthers. WRKY53 directly binds to the promoters of GA biosynthesis genes and transcriptionally represses them in anthers. In addition, we uncover a possible mechanism by which GA regulates male fertility: SLENDER RICE1 (SLR1) interacts with and sequesters two critical transcription factors for tapetum development, UNDEVELOPED TAPETUM1 (UDT1), and TAPETUM DEGENERATION RETARDATION (TDR), and GA alleviates the sequestration by SLR1, thus allowing UDT1 and TDR to activate transcription. Finally, knocking out WRKY53 in diverse varieties increases cold tolerance without a yield penalty, leading to a higher yield in rice subjected to cold stress. Together, these findings provide a target for improving CTB in rice.

Cold stress at the booting stage leads to a lower seed setting rate and seriously threatens the production of rice (Oryza sativa L.), which has become a major yield-limiting factor in higher-altitude and -latitude regions. Because cold tolerance at the booting stage (CTB) is a complex trait and is controlled by multiple loci, only a few genes have been reported so far. In this study, a function of OsMKKK70 (Mitogen Activated Protein Kinase Kinase Kinase 70) in response to CTB was characterized. OsMKKK70 expression was rapidly induced by cold stress at the booting stage. OsMKKK70 overexpression (OsMKKK70-OE) plants were more sensitive to cold stress at the booting stage with a lower seed setting and pollen fertility, but there was no significant difference between the osmkkk70 mutant and WT. Considering the effect of functional redundancy, we further tested the CTB response of osmkkk62/70 and osmkkk55/62/70, the double and triple mutants of OsMKKK70 with its closest homologs OsMKKK62 and OsMKKK55, and found that osmkkk62/70 and osmkkk55/62/70 displayed significantly increased CTB with a higher seed setting and pollen fertility, indicating that OsMKKK70 negatively regulates rice CTB. Moreover, under the low-temperature (LT) condition, the osmkkk62/70 mutant had slightly higher Gibberellin (GA) contents, increased expression of GA biosynthesis genes, and lower protein level of OsSLR1 in anthers than those in WT. By contrast, OsMKKK70-OE anther had a lower GA biosynthesis than that of WT. Together, these findings suggest that OsMKKK70 negatively regulates rice CTB by fine-tuning GA levels in anthers.