The transcriptional repressor OsPRR73 links circadian clock and photoperiod pathway to control heading date in rice.

The phase transition from vegetative to reproductive growth is triggered by internal and external signals that participate in circadian clock in plants. We identified a rice floral inhibitor OsPRR73 encoding a CONSTANS protein. Overexpression of OsPRR73 resulted in late heading under both long-day (LD) and short-day (SD) conditions. Knockout mutants led to early heading under LD conditions but no change under SD. OsPRR73 mRNA accumulated at noon and exhibited a robust oscillation under constant light (LL) and constant darkness (DD) conditions. OsPRR73 overexpression exerted negative feedback on endogenous OsPRR73 expression and altered diurnal expressions of key flowering genes and circadian clock genes. OsPRR73 bound to the promoters of the floral gene Ehd1 and the circadian gene OsLHY, and significantly suppressed their expression at dawn. In LL and DD, the oscillatory patterns of the circadian genes OsLHY, OsTOC1, OsGI and OsELF3 were varied in OsPRR73OX and osprr73 mutants. OsPRR73 expression was decreased in osphyb mutants, and overexpression of OsPRR73 complemented the early heading date phenotype of osphyb, indicating OsPRR73 works downstream of OsPhyB. Therefore, OsPRR73 is involved in a feedback loop of the rice clock and connects the photoperiod flowering pathway by binding to the Ehd1 promoter in rice.

Wide Grain 7 increases grain width by enhancing H3K4me3 enrichment in the OsMADS1 promoter in rice (Oryza sativa L.).

Grain size is a major determinant of grain weight, a key component of grain yield of rice. Here, we identified the grain size gene WIDE GRAIN 7 (WG7) from a T-DNA insertion mutant. The grain size of WG7 knockout mutants and WG7 overexpression lines indicated that WG7 is a positive regulator of grain size. WG7 encodes a cysteine-tryptophan (CW) domain-containing transcriptional activator. EMSAs and ChIP-qPCR assay confirmed that WG7 directly bound to the promoter of OsMADS1, a grain size gene, and thereby significantly activated its expression. Point mutations showed that the cis-element CATTTC motif in the promoter was the binding site of WG7. Compared with the wild-type, deletion mutants of the cis-element motif exhibited lower expression of OsMADS1 and produced narrower grains, implicating the requirement of this motif for WG7 function. ChIP-qPCR assays showed that WG7 enhanced histone H3K4me3 enrichment in the promoter of OsMADS1. WG7 underwent directional selection due to the poor fertility of the non-functional mutant. These findings demonstrated that WG7 upregulated OsMADS1 expression by directly binding to its promoter, enhanced histone H3K4me3 enrichment in the promoter and ultimately increased grain width. This study will enrich the knowledge concerning the regulatory network of grain size formation in rice.

Minor Effects of 11 Dof Family Genes Contribute to the Missing Heritability of Heading Date in Rice (Oryza sativa L.).

DNA binding with one finger (Dof) proteins are plant-specific transcription factors with important and diverse functions in seed germination, flowering time, and biotic and abiotic stresses. In this study, haplotype-based association analysis was conducted between heading date and 30 Dof family genes in a worldwide germplasm collection. Of these, 22 Dof genes were associated with heading date. Multiple comparisons among haplotypes revealed their diverse functions in promoting and suppressing heading date under short-day (SD) and long-day (LD) conditions. They cumulatively made a considerable contribution to the missing heritability of heading date. A set of knockout mutants of 30 Dof genes generated by CRISPR/Cas9-mediated genome editing technology showed that 11 and 9 Dof genes regulated heading date under LD and SD, respectively. Phenotype measurement of mutants showed that these 11 and 9 Dof genes slightly regulated heading with effects of 2-5 days under LD and SD, respectively. Both mutant and natural variation assays indicated functional redundancy in regulating heading date among Dof family genes. Nucleotide diversity analysis suggested that most Dof genes have been subjected to selection during domestication and improvement. Beyond heading date, this set of mutants is also a good resource for evaluating the function of Dof genes in regulating stress tolerance and seed germination.