Pea nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor that specifically interacts with regulatory DNA sequences upstream of the pea rbcS‐3A‐gene. This factor, designated GT‐1, binds to two short sequences (boxes II and III) in the −150 region that are known to function as light‐responsive elements (LREs) in transgenic tobacco. Binding of GT‐1 to homologous sequences further upstream (boxes II and III in the −220 region) indicates that these boxes comprise the redundant LRE that functions in vivo when boxes II and III are deleted. In both box II and box II, methylation interference experiments demonstrate that two adjacent G residues are critical for GT‐binding. Single Gs present in boxes III and III are also important. Since GT‐1 is present in nuclear extracts from leaves of light‐grown and dark‐adapted pea plants, its regulatory role does not depend on de novo synthesis. Thus if GT‐1 binds differentially in vivo it must be postranslationally modified or sterically blocked from binding by another factor in response to light.
We have investigated the effects of protein synthesis inhibitors on light-induced expression of two plant nuclear genes, Cab and rbcS, in wheat, pea and transgenic tobacco. Light activation of these two genes is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis but not to chloramphenicol, an inhibitor of organeliar protein synthesis. Studies with chimeric gene constructs in transgenic tobacco seedlings show that cycloheximide exerts its effect at the transcriptional level. As a control, we show that the expression of the cauliflower mosaic virus (CaMV) 35S promoter is enhanced by cycloheximide treatment, irrespective of the coding sequence used. Escape-time analyses with green wheat seedlings show that the cycloheximide block for Cab gene expression is after the primary signal transduction step linked to phytochrome photoconversion. Our results suggest that phytochrome activation of Cab and rbcS is mediated by a labile protein factor(s) synthesized on cytoplasmic ribosomes.
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