Map-Based Cloning And Characterization Of A Novel And Oryza-Genus Specific Regulator, Ehd44, In Photoperiodic Control Of Flowering In Rice

Flowering time （or heading date in crops） is not only a profound transition from vegetative to reproductive development but also a critical determining factor of crop yields that determines cropping seasons and regional adaptability. Plants have the ability to measure day-length and use it as an indicator to initiate flowering, referred to as photoperiod control of flowering. Rice is a major staple crop worldwide and known as a short day plant that flowers more rapidly in short-day conditions （SDs） but delays under long-day conditions （LDs）. However, cultivated rice （Oryza sativa） is now grown widely in Asia, with a northern limit of nearly 53°N in northern Asia （where natural day length during rice cultivation is nearly 15 hours light）, whereas Oryza rufipogon, a wild rice that is the most relative ancestor of O. sativa, is mainly distributed at tropical latitudes with a northern limit about 28°N. The northward expansion of cultivated rice into higher latitudes must be accompanied by human selection of the flowering time trait during rice domestication and breeding, to secure a harvest before cold weather approaches. Accumulating evidence is suggesting that the regulation of photoperiodic flowering is more complex in rice than in Arabidopsis. The conserved OsGI （GI）-Hd1 （CO）-Hd3a （FT） regulatory module has a dual function in promoting flowering under SDs and suppressing flowering under LDs in rice. In addition, there is a "LD-activation" pathway composed of OsMADS50, Ehd2/RID1/OsID, Ehd3, and Ehdl in rice. However, the genetic components and the signaling mechanisms regulating flowering under LDs are still relatively poorly-studied in rice.In this study, we showed that loss-of-function mutation in Ehd4 resulted in a never-flowering phenotype under natural long-day conditions. Map-based cloning revealed that Ehd4 encodes a novel CCCH-type zinc finger protein, which is localized to the nucleus and is able to bind to nucleic acids in vitro and transactivate transcription in yeast, suggesting that it likely functions as a transcription factor. Ehd4 expression is most active in young leaves with a diurnal expression pattern similar to that of Ehdl, accumulating after dusk, reaching a peak at dawn, and damping rapidly thereafter under both SDs and LDs. We show that Ehd4 up-regulates the expression of the florigen genes Hd3a and RFTl through Ehdl, but acts independently of other known Ehdl regulators. Strikingly, Ehd4 is highly conserved in the Oryza genus including wild and cultivated rice, but has no homologs in other species, suggesting that Ehd4 is originated along with the diversification of the rice genus Oryza from the grass family during evolution. We found that Ehd4 has two major haplotypes, Hap₂ is the major haplotype in indica accessions mostly distributed in lower latitude and elevation zones, whereas Hap₃ is the major haplotype in japonica accessions mostly distributed in higher latitudes and elevation zones. Genetic studies showed that Hap₃ is functionally more potent in promoting flowering under NLDs, implying that EHD4 may have contributed to the northward expansion and regional adaptability of cultivated rice into higher latitudes. We conclude that Ehd4 is a novel, Oryza genus specific regulator of Ehdl and it plays an essential role in photoperiodic control of flowering time in rice.