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

A culture of callus cells has been developed from a transgenicline of tobacco which contains an introduced phyA-cDNA encodingphytochrome A. Suspension cultures of the cells were shown toaccumulate a significant immunodetectable level of the heterologousphytochrome, but not of the native phyA-gene product. The red-irradiatedform (P_fr) of the heterologous phytochrome was specificallydegraded in vivo, and the red-irradiated (P_fr) and far-red-irradiated(P_r) forms demonstrated different patterns of in vitro proteolyticcleavage. These results strongly suggested that the phytochromeapoprotein was associated with a chromophore moiety which mediatedred/far-red sensitive conformational changes of the molecule.Exogenous application of 4-amino-5-hexynoic acid (AHA) to thetransgenic suspension cultures resulted in the accumulationof a population of phytochrome which was stable under red lightand gave identical patterns of in vitro digestion in the redand far-red irradiated forms, i.e. the spectral activity ofphytochrome was inhibited. Application of exogenous 5-aminolevulinicacid (ALA) or biliverdin overcame the inhibitory effects ofAHA to restore spectral sensitivity of the phytochrome pool.These results are consistent with the proposed pathway of phytochromechromophore biosynthesis in intact plant systems. Thus, thetransgenic suspension cultures provided a single-cell systemin which spectrally-active phytochrome, apparently indistinguishablefrom the native phytochrome synthesized in etiolated seedlings,was accumulated. Photoregulation of expression of the genesencoding the small subunit of ribulose-1,5-bisphosphate carboxylaseand chlorophyll a/b binding proteins demonstrated that the heterologousphytochrome population mediated rapid changes in gene expressionin the de-differentiated cells. It is therefore proposed thatsuch a suspension culture of transgenic cells offers a modelsystem for the study of phytochrome function. Key words: Cell cultures, transgenic tobacco, phytochrome, oat-phy A-cDNA, gene expression