Isolation and functional characterization of a circadian-regulated CONSTANS homolog (GbCO) from Ginkgo biloba.

KEY MESSAGE: This is the first report to clone and functionally characterize a flowering time gene GbCO in perennial gymnosperm Ginkgo biloba. GbCO complements the co mutant of Arabidopsis, restoring normal early flowering. CONSTANS (CO) is a central regulator of photoperiod pathway, which channels inputs from light, day length, and circadian clock to promote the floral transition. In order to understand the role of CO in gymnosperm Ginkgo biloba, which has a long juvenile phase (15-20 years), a CO homolog (GbCO) was isolated and characterized from G. biloba. GbCO encodes a 1741-bp gene with a predicted protein of 400 amino acids with two zinc finger domains (B-box I and B-box II) and a CCT domain. Phylogenic analysis classified GbCO into the group 1a clade of CO families in accordance with the grouping scheme for Arabidopsis CO (AtCO). Southern blot analysis indicated that GbCO belongs to a multigene family in G. biloba. Real-time PCR analysis showed that GbCO was expressed in aerial parts of Ginkgo, with the highest transcript level of GbCO being observed in shoot apexes. GbCO transcript level exhibited a strong diurnal rhythm under flowering-inductive long days and peaked during early morning, suggesting that GbCO is tightly coupled to the floral inductive long-day signal. In addition, an increasing trend of GbCO transcript level was observed both in shoot tips and leaves as the shoot growth under long-day condition, whereas GbCO transcript level decreased in both tissues under short-day condition prior to growth cessation of shoot in G. biloba. GbCO complemented the Arabidopsis co-2 mutant, restoring normal early flowering. All the evidence being taken together, our findings suggested that GbCO served as a potential inducer of flowering in G. biloba.