Molecular and Physiological Characterization of the Flowering Time Control Protein, HvFCA and its Role in ABA Signalling and Seed Germination

Abstract

The RNA binding protein Flowering Time Control Locus A (FCA) regulates flowering in rice and Arabidopsis. The abscisic acid binding protein ABAP1 shares high sequence homology to FCA and was considered the FCA homologue in barley. The current study investigates the existence of ABAP1 as an independent gene product and also the cloning, characterization and functional significance of the gamma (γ) isoform of FCA from barley. Barley FCA protein showed higher sequence similarity to wheat and rice FCA compared to Arabidopsis FCA. It contains two RNA recognition motifs (RRMs), a glycine rich region at the N-terminal end, the WW domain and a poly-glutamine region immediately downstream of WW domain at the C-terminal. In developing barley embryos, FCA transcripts could be detected from 2 days after pollination (DAP) up to late maturity without any detectable change within these stages. FCA transcript levels declined as germination progressed in barley embryos and the FCA transcripts were retained for longer duration when germination was reduced with application of ABA. FCA also showed up-regulation by ABA and abiotic stresses in barley germinating seeds and seedlings. Transient co-expression of barley FCA or a truncated FCA (lacking RRM) with a maize VP1 promoter-GUS construct or a wheat Em gene promoter-GUS construct in barley aleurone layer protoplasts led to increased GUS activity in both cases. Adding ABA during the incubation enhanced the observed increase due to FCA expression. Similar effects of transient over-expression of FCA in barley embryos affected VP1. Barley FCA localized to the nucleus. This nuclear localization was due to the nuclear localization signal within the protein and not due to the RNA recognition motifs (RRMs) as the truncated FCA lacking RRMs also localized to the nucleus. Barley FCA did not restore the flowering phenotype in an Arabidopsis fca-1 mutant. In conclusion, I have shown that barley FCA is up-regulated by ABA and stress in embryos and affects ABA signalling in barley caryopses. The properties of FCA appear to have diverged between dicot and monocot systems.