Application of Green Fluorescent Protein in Immunoassays
Immunomodulation is a means to modulate an organism’s function by antibody production to capture either endogenous or exogenous antigens. We have recently succeeded in obtaining gibberellin (GA)-deficient phenotypes in Arabidopsis thaliana by using anti-bioactive GA antibodies. In this study, a single-chain antibody (scFv) against GA24, a precursor GA, was utilized to repress the biosynthesis of bioactive gibberellins. Stable accumulation of the scFv in endoplasmic reticulum (ER) was achieved by being produced as a fusion with GFP as well as KDEL ER-retention signal. The transgenic plants showed GFP fluorescence in the reticulate cortical ER network in epidermal cells. The GFP-scFv fusion produced in plants maintained its binding activity. The transgenic plants showed GA-deficient phenotypes, including reduced rosette leaf development, delayed flower induction and reduced stem elongation of the main culm, especially in the early stage of inflorescence growth. Contrarily, stem elongation of the main culm at a later stage, or that of lateral shoots was much less affected by scFv production. These phenotypes were different from anti-bioactive GA scFv-producing lines, whose stem elongation was continuously repressed throughout the inflorescence development. The GA-deficient phenotypes were recovered by treatment with GA24 and bioactive GA4, the latter being more effective. The transgenic lines contained conspicuously higher endogenous GA24 and clearly less GA4 than wild-type plants. The expression of GA 20-oxidase and GA 3-oxidase genes, which are feedback-regulated by GA signaling, were up-regulated in those plants. These results demonstrate that the scFv trapped GA24 in ER and inhibited metabolism of GA24 to bioactive GA4.