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Plant hormone cytokinin signals through histidine-aspartic acid (H-D) phosphorelay to regulate plant growth and development. While it is well known that the phosphorelay involves histidine kinases, histidine phosphotransfer proteins (HPs), and response regulators (RRs), how this process is regulated by external components remains unknown. Here we demonstrate that protein phosphatase with kelch-like domains (PPKL1), known as a signaling component of steroid hormone brassinosteroid, is actually a cryptic inhibitor of cytokinin phosphorelay in rice (Oryza sativa). Mutation at a specific amino acid D364 of PPKL1 activates cytokinin response and thus enlarges grain size in a semi-dominant mutant named s48. Overexpression of PPKL1 containing D364, either with the deletion of the phosphatase domain or not, rescues the s48 mutant phenotype. PPKL1 interacts with OsAHP2, one of authentic HPs, and D364 resides in a region resembling the receiver domain of RRs. Accordingly, PPKL1 can utilize D364 to suppress OsAHP2-to-RR phosphorelay, whereas mutation of D364 abolishes the effect. This function of PPKL1 is independent of the phosphatase domain that is required for brassinosteroid signaling. Importantly, editing of the D364-residential region produces a diversity of semi-dominant mutations associated with variously increased grain sizes. Further screening of the edited plants enables the identification of two genotypes that confer significantly improved grain yield. Collectively, our study uncovers a noncanonical cytokinin signaling suppressor and provides a robust tool for seed rational design. This study reveals that PPKL1, known as a signaling component of steroid hormone brassinosteroid, is actually a cryptic inhibitor of cytokinin phosphorelay in rice. PPKL1 utilizes the D364 site to interfere with AHP2-to-response regulator phosphorelay, and the D364 residential region could be a promising target for seed rational design. [ABSTRACT FROM AUTHOR]

Summary: Brassinosteroids (BRs) are a class of phytohormones that modulate several important agronomic traits in rice (Oryza sativa). GSK2 is one of the critical suppressors of BR signalling and targets transcription factors such as OsBZR1 and DLT to regulate BR responses. Here, we identified OFP3 (OVATE FAMILY PROTEIN 3) as an interactor of both GSK2 and DLT by yeast‐two‐hybrid screening and demonstrated that OFP3 plays a distinctly negative role in BR responses. While knockout of OFP3 promoted rice seedling growth, overexpression of OFP3 led to strong BR insensitivity, which resulted in reduced plant height, leaf angle, and grain size. Interestingly, both BR biosynthetic and signalling genes had decreased expression in the overexpression plants. OFP3 overexpression also enhanced the phenotypes of BR‐deficient mutants, but largely suppressed those of BR‐enhanced plants. Moreover, treatment with either BR or bikinin, a GSK3‐like kinase inhibitor, induced OFP3 depletion, whereas GSK2 or brassinazole, a BR synthesis inhibitor, promoted OFP3 accumulation. Furthermore, OFP3 exhibited transcription repressor activity and was able to interact with itself as well as additional BR‐related components, including OFP1, OSH1, OSH15, OsBZR1, and GF14c. Importantly, GSK2 can phosphorylate OFP3 and enhance these interactions. We propose that OFP3, as a suppressor of both BR synthesis and signalling but stabilized by GSK2, incorporates into a transcription factor complex to facilitate BR signalling control, which is critical for the proper development of various tissues. Significance Statement: Brassinosteroid (BR) modulates various important agronomic traits in rice, but how the diverse hormone responses are integrated remains largely unclear. This study identified an OVATE family protein, OFP3, as a critical integrator of a BR signalling transcription factor complex, that interacts with a number of known components of BR signalling to suppress BR responses. As OFP3 forms the negative regulatory loops with BR signalling and biosynthesis, OFP3 could function as a stabilizer of BR signalling to moderate the signal magnitude. [ABSTRACT FROM AUTHOR]

Grain size is an important agronomic trait affecting grain yield, but the underlying molecular mechanisms remain to be elucidated. Here, we isolated a dominant mutant, big grain3 (bg3‐D), which exhibits a remarkable increase of grain size caused by activation of the PURINE PERMEASE gene, OsPUP4. BG3/OsPUP4 is predominantly expressed in vascular tissues and is specifically suppressed by exogenous cytokinin application. Hormone profiling revealed that the distribution of different cytokinin forms, in roots and shoots of the bg3‐D mutant, is altered. Quantitative reverse transcription‐PCR (qRT‐PCR) analysis indicated that expression of rice cytokinin type‐A RESPONSE REGULATOR (OsRR) genes is enhanced in the roots of the bg3‐D mutant. These results suggest that OsPUP4 might contribute to the long‐distance transport of cytokinin, by reinforcing cytokinin loading into vascular bundle cells. Furthermore, plants overexpressing OsPUP7, the closest homolog of OsPUP4, also exhibited a similar phenotype to the bg3‐D mutant. Interestingly, subcellular localization demonstrated that OsPUP4 was localized on the plasma membrane, whereas OsPUP7 was localized to the endoplasmic reticulum. Based on these findings, we propose that OsPUP4 and OsPUP7 function in a linear pathway to direct cytokinin cell‐to‐cell transport, affecting both its long‐distance movement and local allocation. [ABSTRACT FROM AUTHOR]

Grain size is one of the most important determinants of crop yield in cereals. Here, we identified a dominant mutant, big grain2 ( bg2- D) from our enhancer-trapping population. Genetic analysis and Site Finding PCR (polymerase chain reaction) revealed that BG 2 encodes a cytochrome P450, OsCYP78 A13. Sequence search revealed that CYP 78 A 13 has a paralogue G rain L ength 3.2 ( GL 3.2, LOC _ Os 03 g 30420) in rice with distinct expression patterns, analysis of transgenic plants harbouring either CYP 78 A 13 or GL 3.2 showed that both can promote grain growth. Sequence polymorphism analysis with 1529 rice varieties showed that the nucleotide diversity at CYP 78 A 13 gene body and the 20 kb flanking region in the indica varieties were markedly higher than those in japonica varieties. Further, comparison of the genomic sequence of CYP 78 A 13 in the japonica cultivar Nipponbare and the indica cultivar 9311 showed that there were three In Dels in the promoter region and eight SNPs (single nucleotide polymorphism) in its coding sequence. Detailed examination of the transgenic plants with chimaeric constructs suggested that variation in CYP 78 A 13 coding region is responsible for the variation of grain yield. Taken together, our results suggest that the variations in CYP 78 A 13 in the indica varieties hold potential in rice breeding for application of grain yield improvement. [ABSTRACT FROM AUTHOR]