Fine-Tuning of MiR528 Accumulation Modulates Flowering Time in Rice

In plants,microRNA (miRNA) functions in the post-transcriptional repression of target mRNAs have been well explored.However,the mechanisms regulating the accumulation of miRNAs remain poorly under.stood.Here,we report that distinct mechanisms regulate accumulation of a monocot-specific miRNA,rice (Oryza sativa) miR528.At the transcriptional level,miR528 accumulated to higher levels in older plants than in young seedlings and exhibited aging-modulated gradual accumulation and diurnal rhythms in leaves;at the post-transcriptional level,aging also modulated miR528 levels by enhancing pri-miR528 alter.native splicing.We found that miR528 promotes rice flowering under long-day conditions by targeting RED AND FAR-RED INSENSITIVE2 (OsRFI2).Moreover,natural variations in the MIR528 promoter region caused differences in miR528 expression among rice varieties,which are correlated with their different binding affinities with the transcription factor OsSPL9 that activates the expression of miR528.Taken together,our findings reveal rice plants have evolved sophisticated modes fine-tuning miR528 levels and provide insight into the mechanisms that regulate MIRNA expression in plants.

A cryptic inhibitor of cytokinin phosphorelay controls rice grain size

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.

Modulation of nitrate-induced phosphate response by the MYB transcription factor RLI1/HINGE1 in the nucleus

The coordinated utilization of nitrogen(N)and phosphorus(P)is vital for plants to maintain nutrient balance and achieve optimal growth.Previously,we revealed a mechanism by which nitrate induces genes for phosphate utilization;this mechanism depends on NRT1.1B-facilitated degradation of cytoplasmic SPX4,which in turn promotes cytoplasmic-nuclear shuttling of PHR2,the central transcription factor of phosphate signaling,and triggers the nitrate-induced phosphate response(NIPR)and N-P coordinated utilization in rice.In this study,we unveiled a fine-tuning mechanism of NIPR in the nucleus regulated by Highly Induced by Nitrate Gene 1(HINGE1,also known as RLI1),a MYB-transcription factor closely related to PHR2.RLI1/HINGE1,which is transcriptionally activated by PHR2 under nitrate induction,can directly activate the expression of phosphate starvation-induced genes.More importantly,RLI1/HINGE1 competes with PHR2 for binding to its repressor proteins in the nucleus(SPX proteins),and consequently releases PHR2 to further enhance phosphate response.Therefore,RLI1/HINGE1 amplifies the phosphate response in the nucleus downstream of the cytoplasmic SPX4-PHR2 cascade,thereby enabling fine-tuning of N-P balance when nitrate supply is sufficient.

Molecular analysis of rice plants harboring a multi-functional T-DNA tagging system

About 25,000 rice T-DNA insertional mutant lines were generated using the vector pCAS04 which has both promoter-trapping and activation-tagging function. Southern blot analysis revealed that about 40% of these mutants were single copy integration and the average T-DNA insertion number was 2.28. By extensive phenotyping in the field, quite a number of agronomically important mutants were obtained. Histochemical GUS assay with 4,310 primary mutants revealed that the GUS-staining frequency was higher than that of the previous reports in various tissues and especially high in flowers. The T-DNA flanking sequences of some mutants were isolated and the T-DNA insertion sites were mapped to the rice genome. The flanking sequence analysis demonstrated the different integration pattern of the right border and left border into rice genome. Compared with Arabidopsis and poplar, it is much varied in the T-DNA border junctions in rice.