Flowering time is an important agronomic trait which determines the yield and regional adaptability of rice, but the underlining molecular regulatory mechanism need further study. FCS-like Zinc finger proteins (FLZs) are a class of plant specific regulatory proteins which play essential roles in plant growth and stress response, but their functions in regulating flowering time have not been reported. This study aims to investigate the potential function of FLZ proteins in rice flowering time control. The finding will broaden our understanding on the molecular regulatory mechanism of rice flowering time .and provide new theoretical basis and gene resource for rice breeding.

Based on the target sequences published in RGAP database, OsFLZ18 overexpression vector and CRISPR-Cas9 vector were generated and introduced into Japonica variety Nipponbare by Agrobacterium tumefaciens-mediated genetic transformation assay. Homozygous CRISPR knockout mutants were screened by PCR and sequencing analyses. The quantitative real-time PCR (qRT-PCR) assay was used to examine the spatial-temporal expression and diurnal rhythmic expression of OsFLZ18, as well as the effects of OsFLZ18 on the transcription of several known flowering time-related genes. Yeast two-hybrid assay (Y2H) was used to test the interaction between OsFLZ18 and the flowering time-related regulatory proteins.

OsFLZ18 was ubiquitously expressed in various rice tissues, with the highest expression level in 14 day-old seedling, followed by leaf sheaths and leaf blades at the tillering stage, and stem and young panicles at reproductive stages. The OsFLZ18-CRISPR vector was constructed and transformed into Nipponbare. Two independent homozygous OE lines (OE-2, OE-3) with higher OsFLZ18 expression level and two homozygous mutants (CRISPR-21, CRISPR-25) were selected for further study. Phenotypic observation showed that the OE lines flowered later than the wild-type plants under both natural long-day and short-day conditions in Guangzhou, while the CRISPR lines had no obvious differences in heading date when compared to the wild-type plants. The expression levels of Ehd1, Hd3a and RFT1 were significantly decreased in OE-2 plants compared with those in the wild-type plants under artificial short-day conditions, but no significant difference in the expression level of Hd1 was observed between them. The results of Y2H experiment showed that OsFLZ18 interacted with OsMADS51, a positive regulator of rice flowering time. Furthermore, OsFLZ18 exhibits a diurnal rhythmic expression profile, showing lower expression levels in the daytime and higher expression levels at night with a peak at midnight.

Overexpression of OsFLZ18 delays rice flowering time.