Expression of Oat-phyA-cDNA in a Suspension Cell Culture of Transgenic Tobacco: A Single-Cell System for the Study of Phytochrome Function

Abstract

A culture of callus cells has been developed from a transgenic line of tobacco which contains an introduced phyA-cDNA encoding phytochrome A. Suspension cultures of the cells were shown to accumulate a significant immunodetectable level of the heterologous phytochrome, but not of the native phyA-gene product. The red-irradiated form (Pfr) of the heterologous phytochrome was specifically degraded in vivo, and the red-irradiated (Pfr) and far-red-irradiated (Pr) forms demonstrated different patterns of in vitro proteolytic cleavage. These results strongly suggested that the phytochrome apoprotein was associated with a chromophore moiety which mediated red/far-red sensitive conformational changes of the molecule. Exogenous application of 4-amino-5-hexynoic acid (AHA) to the transgenic suspension cultures resulted in the accumulation of a population of phytochrome which was stable under red light and gave identical patterns of in vitro digestion in the red and far-red irradiated forms, i.e. the spectral activity of phytochrome was inhibited. Application of exogenous 5-aminolevulinic acid (ALA) or biliverdin overcame the inhibitory effects of AHA to restore spectral sensitivity of the phytochrome pool. These results are consistent with the proposed pathway of phytochrome chromophore biosynthesis in intact plant systems. Thus, the transgenic suspension cultures provided a single-cell system in which spectrally-active phytochrome, apparently indistinguishable from the native phytochrome synthesized in etiolated seedlings, was accumulated. Photoregulation of expression of the genes encoding the small subunit of ribulosc-1,5-tephosphate carboxylase and chlorophyll aib binding proteins demonstrated that the heterologous phytochrome population mediated rapid changes in gene expression in the de-differentiated cells. It is therefore proposed that such a suspension culture of transgenic cells offers a model system for the study of phytochrome function.