WRKY53 negatively regulates rice cold tolerance at the booting stage by fine-tuning anther gibberellin levels.

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.

WRKY53 integrates classic brassinosteroid signaling and the mitogen-activated protein kinase pathway to regulate rice architecture and seed size.

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.

OsWRKY78 regulates panicle exsertion via gibberellin signaling pathway in rice.

Panicle exsertion is one of the crucial agronomic traits in rice (Oryza sativa). Shortening of panicle exsertion often leads to panicle enclosure and severely reduces seed production. Gibberellin (GA) plays important roles in regulating panicle exsertion. However, the underlying mechanism and the relative regulatory network remain elusive. Here, we characterized the oswrky78 mutant showing severe panicle enclosure, and found that the defect of oswrky78 is caused by decreased bioactive GA contents. Biochemical analysis demonstrates that OsWRKY78 can directly activate GA biosynthesis and indirectly suppress GA metabolism. Moreover, we found OsWRKY78 can interact with and be phosphorylated by mitogen-activated protein kinase (MAPK) kinase OsMAPK6, and this phosphorylation can enhance OsWRKY78 stability and is necessary for its biological function. Taken together, these results not only reveal the critical function of OsWRKY78, but also reveal its mechanism via mediating crosstalk between MAPK and the GA signaling pathway in regulating panicle exsertion.

Functions of OsWRKY24, OsWRKY70 and OsWRKY53 in regulating grain size in rice.

MAIN CONCLUSION: OsWRKY24 functions redundantly with OsWRKY53, while OsWRKY70 functions differently from OsWRKY53 in regulating grain size. Grain size is a key agronomic trait that affects grain yield and quality in rice (Oryza sativa L.). The transcription factor OsWRKY53 positively regulates grain size through brassinosteroid (BR) signaling and Mitogen-Activated Protein Kinase (MAPK) cascades. However, whether the OsWRKY53 homologs OsWRKY24 and OsWRKY70 also contribute to grain size which remains unknown. Here, we report that grain size in OsWRKY24 overexpression lines and oswrky24 mutants is similar to that of the wild type. However, the oswrky24 oswrky53 double mutant produced smaller grains than the oswrky53 single mutant, indicating functional redundancy between OsWRKY24 and OsWRKY53. In addition, OsWRKY70 overexpression lines displayed an enlarged leaf angle, reduced plant height, longer grains, and higher BR sensitivity, phenotypes similar to those of OsWRKY53 overexpression lines. Importantly, a systematic characterization of seed length in the oswrky70 single, the oswrky53 oswrky70 double and the oswrky24 oswrky53 oswrky70 triple mutant indicated that loss of OsWRKY70 also leads to increased seed length, suggesting that OsWRKY70 might play a role distinct from that of OsWRKY53 in regulating grain size. Taken together, these findings suggest that OsWRKY24 and OsWRKY70 regulate rice grain size redundantly and independently from OsWRKY53.

OsMKKK70 Negatively Regulates Cold Tolerance at Booting Stage 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.

bZIP71 delays flowering by suppressing Ehd1 expression in rice.

Flowering time is a fundamental factor determining the global distribution and final yield of rice (Oryza sativa). Although diverse flowering time genes have been reported in this crop, the transcriptional regulation of its key flowering genes are poorly understood. Here, we report that a basic leucine zipper transcription factor, bZIP71, functions as a flowering repressor. The overexpression of bZIP71 delays flowering, while the bzip71 mutant flowers early in both long-day and short-day conditions. A genetic analysis showed that the regulation of flowering by bZIP71 might be independent of Heading date 2 (Hd2), Hd4, and Hd5. Importantly, bZIP71 directly associates with the Early heading date 1 (Ehd1) promoter and represses its transcription, and genetically the function of bZIP71 is impaired in the ehd1 mutant. Moreover, bZIP71 interacts with major components of polycomb repressive complex 2 (PRC2), SET domain group protein 711 (SDG711), and Fertilization independent endosperm 2 (FIE2), through which bZIP71 regulates the H3K27me3 level of Ehd1. Taken together, we present a transcriptional regulatory mechanism in which bZIP71 enhances the H3K27me3 level of Ehd1 and transcriptionally represses its expression, which not only offers a novel insight into a flowering pathway, but also provides a valuable putative target for the genetic engineering and breeding of elite rice cultivars.

OsMKKK70 regulates grain size and leaf angle in rice through the OsMKK4-OsMAPK6-OsWRKY53 signaling pathway.

Grain size and leaf angle are key agronomic traits that determine final yields in rice. However, the underlying molecular mechanisms are not well understood. Here we demonstrate that the Oryza sativa Mitogen Activated Protein Kinase Kinase Kinase OsMKKK70 regulates grain size and leaf angle in rice. Overexpressing OsMKKK70 caused plants to produce longer seeds. The osmkkk62/70 double mutant and the osmkkk55/62/70 triple mutant displayed significantly smaller seeds and a more erect leaf angle compared to the wild type, indicating that OsMKKK70 functions redundantly with its homologs OsMKKK62 and OsMKKK55. Biochemical analysis demonstrated that OsMKKK70 is an active kinase and that OsMKKK70 interacts with OsMKK4 and promotes OsMAPK6 phosphorylation. In addition, the osmkkk62/70 double mutant showed reduced sensitivity to Brassinosteroids (BRs). Finally, overexpressing constitutively active OsMKK4, OsMAPK6, and OsWRKY53 can partially complement the smaller seed size, erect leaf, and BR hyposensitivity of the osmkkk62/70 double mutant. Taken together, these findings suggest that OsMKKK70 might regulate grain size and leaf angle in rice by activating OsMAPK6 and that OsMKKK70, OsMKK4, OsMAPK6, and OsWRKY53 function in a common signaling pathway that controls grain shape and leaf angle.

Oryza sativa mediator subunit OsMED25 interacts with OsBZR1 to regulate brassinosteroid signaling and plant architecture in rice.

Brassinosteroids (BRs) are plant-specific steroid hormones which regulate plant growth, development, and adaptation. Transcriptional regulation plays key roles in plant hormone signaling. A mediator can serve as a bridge between gene-specific transcription factors and the RNA polymerase machinery, functioning as an essential component in regulating the transcriptional process. However, whether a mediator is involved in BR signaling is unknown. Here, we discovered that Oryza sativa mediator subunit 25 (OsMED25) is an important regulator of rice BR signaling. Phenotypic analyses showed that the OsMED25-RNAi and osmed25 mutant presented erect leaves, as observed in BR-deficient mutants. In addition, the OsMED25-RNAi and osmed25 mutant exhibited decreased BR sensitivity. Genetic analysis indicated that OsMED25-RNAi could suppress the enhanced BR signaling phenotype of Osbzr1-D. Further biochemical analysis showed that OsMED25 interacts with OsBZR1 in vivo, and OsMED25 is enriched on the promoter of OsBZR1 target genes. RNA sequencing analysis indicated that OsMED25 affects the expression of approximately 45% of OsBZR1-regulated genes and mainly functions as a corepressor of OsBZR1. Together, these findings revealed that OsMED25 regulates rice BR signaling by interacting with OsBZR1 and modulating the expression of OsBZR1 target genes, thus expanding our understanding of the roles of mediators in plant hormone signaling.