Identification of a CONSTANS homologous gene with distinct diurnal expression patterns in varied photoperiods in ramie (Boehmeria nivea L. Gaud)

Ramie is an important natural fiber and forage crop in China. Breeding of late- or non-flowering varieties, with higher vegetative yields, is an important goal in ramie improvement. However, the ramie genes involved in flowering regulation have not previously been identified. In model plants, such as rice and Arabidopsis, the CONSTANS (CO) and CONSTANS-like (COL) genes play key roles in flowering regulation. In the present study, six ramie COL genes (BnCOL1-BnCOL6) with a full-length open reading frame (ORF) were identified. Sequence alignment revealed that all six BnCOL proteins contained conserved CCT (CO, COL, TOC1) and B-box I domains, but that only four of these proteins contained the B-box II domain. Expression pattern analysis showed that BnCOL1-BnCOL6 were mainly expressed in the stem xylem, flowers, and leaves. Phylogenetic analysis classified the six newly identified BnCOL proteins, and also 16 COL proteins with known functions in other species, into three groups. The BnCOL2-encoded protein was assigned to the same group as the CO- and Hd1-encoded proteins, suggesting that this BnCOL2-encoded protein showed the highest level of homology with the CO/Hd1-encoded proteins. Photoperiodic experiments showed that BnCOL2 exhibited a diurnal expression pattern under long- and short-day conditions. Subcellular localization examination revealed that the BnCOL2 protein fused with YFP was localized in the nucleus. Because the homologous sequence and similar expression pattern between BnCOL2 and CO/Hd1, the BnCOL2 possibly has a role in flowering modulation, and can be used as a candidate gene for research in the flowering regulation of ramie.