Circadian Clock in Arabidopsis thaliana Determines Flower Opening Time Early in the Morning and Dominantly Closes Early in the Afternoon.

Many plant species exhibit diurnal flower opening and closing, which is an adaptation influenced by the lifestyle of pollinators and herbivores. However, it remains unclear how these temporal floral movements are modulated. To clarify the role of the circadian clock in flower movement, we examined temporal floral movements in Arabidopsis thaliana. Wild-type (accessions; Col-0, Ler-0 and Ws-4) flowers opened between 0.7 and 1.4 h in a 16-h light period and closed between 7.5 and 8.3 h in a diurnal light period. In the arrhythmic mutants pcl1-1 and prr975, the former flowers closed slowly and imperfectly and the latter ones never closed. Under continuous light conditions, new flowers emerged and opened within a 23-26 h window in the wild-type, but the flowers in pcl1-1 and prr975 developed straight petals, whose curvatures were extremely small. Anti-phasic circadian gene expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYLE (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) occurred in wild-type flowers, but non-rhythmic expression was observed in pcl1-1 and prr975 mutants. Focusing on excised petals, bioluminescence monitoring revealed rhythmic promoter activities of genes expressed (CCA1, LHY and PHYTOCLOCK 1/LUX ARRHYTHMO, PCL1/LUX) in the morning and evening. These results suggest that the clock induces flower opening redundantly with unknown light-sensing pathways. By contrast, flower closing is completely dependent on clock control. These findings will lead to further exploration of the molecular mechanisms and evolutionary diversity of timing in flower opening and closing.

Design and site-directed immobilization of single-chain Fv antibody to polystyrene latex beads via material-binding peptides and application to latex turbidimetric assay.

In this study, immobilization of single-chain Fv (scFv) antibodies on the surfaces of polystyrene (PS) latex beads via material-binding peptides was investigated for sensitive immuno-turbidimetric assay of C-reactive protein (CRP). Anti-CRP scFvs fused with polystyrene-binding peptide (PS-tag) and poly(methylmethacrylate)-binding peptide (PMMA-tag) were over-expressed in Escherichia coli cells and recovered in the active form following refolding. The beads with PMMA-tag-fused scFv (scFv-PM) were successfully suspended with sufficient dispersion at pH 8.0. Three types of alternative scFv-PMs with a penta-asparatic acid tag (D5-tag) introduced at different positions were then designed. All of the D5-tagged scFv-PMs were successfully immobilized on the surfaces of beads with no significant change in the diameter of the latex beads at pH levels ranging from 6.0 to 8.0. According to the results of turbidimetric assay for the detection of CRP, 13 ng/ml of CRP was detectable using beads with D5-tagged scFv-PMs at 400 ng/cm3, and no turbidity change was observed in the absence of antigen. When the density of scFv-PM was 250 ng/cm2, which was 63% of the maximum density, the beads were dispersed well and reactive with the antigen at a concentration range comparable to those with D5-tagged scFv-PMs. These results indicate that controlling charge density on the surface of beads after site-directed immobilization is definitely important in order to maintain high levels of dispersion and reactivity. Thus, the usefulness of the scFv-PM as well as D5-tagged scFv-PMs developed in the present study should be significant when used as ligand antibodies in the preparation of immuno-latex beads.