Comparison of the effects of exogenous native phytochrome and in-vivo irradiation on in-vitro transcription in isolated nuclei from barley (Hordeum vulgare).

In barley seedlings the transcription of genes coding for the light-harvesting chlorophyll a/b protein (LHCP) is stimulated and the transcription of genes coding for the NADPH-protochlorophyllide oxidoreductase (reductase) is repressed by light working via the phytochrome system. This phytochrome-mediated control of gene expression has been studied by monitoring in-vitro transcription in isolated nuclei. Two different experimental approaches have been used to elucidate the function of phytochrome (Pfr) during the transduction of the light signal. Concentrations of phytochrome were varied experimentally either by illuminating intact plants or macerated plant material prior to the isolation of nuclei or by adding purified phytochrome (Pfr) in its native 124-kDa form to the isolated nuclei. Our results indicate that there are at least two different steps involved in the phytochrome control of specific gene expression. (i) There is a rapid and transient change in the transcription rate which is saturated by very low levels of Pfr. (ii) There is a change in the duration and the maximum range of the transient change; this step requires relatively high Pfr concentrations and thus reacts very sensitively and rapidly to changes in Pfr levels as induced by secondary irradiations. This second step, but not the first one, could be triggered by the addition of purified oat phytochrome to a reconstituted nuclear system. This effect of purified phytochrome could only be shown if nuclei isolated from red-light (R)-irradiated seedlings were used. It was thus possible to simulate the effect of an in-vivo-applied second R pulse by the addition of Pfr to nuclei isolated from R-preirradiated plants.