In mammalian cells, mitogen-induced phosphorylation of ribosomal protein S6 by p70 s6k has been implicated in the selective translational upregulation of 5TOP mRNAs. We demonstrate here that the homologous Arabidopsis thaliana protein, AtS6k2, ectopically expressed in human 293 cells or isolated from plant cells, phosphorylates specifically mammalian and plant S6 at 25°C but not at 37°C. When Arabidopsis suspension culture cells are shifted from 25 to 37°C, the kinase becomes rapidly inactivated, consistent with the observation that heat shock abrogates S6 phosphorylation in plants. Treatment with potato acid phosphatase reduced the specific activity of immunoprecipitated AtS6k2 threefold, an effect which was blocked in the presence of 4-nitrophenyl phosphate. In quiescent mammalian cells, AtS6k2 is activated by serum stimulation, a response which is abolished by the fungal metabolite wortmannin but is resistant to rapamycin. Treatment of mammalian cells with rapamycin abolishes in vivo S6 phosphorylation by p70 s6k ; however, ectopic expression of AtS6k2 rescues the rapamycin block. Collectively, the data demonstrate that AtS6k2 is the functional plant homolog of mammalian p70 s6k and identify a new signalling pathway in plants.
Hypocotyls of dark-grown seedlings of Arabidosis thaliana exhibit a strong negative gravitropism, which is reduced by red and also by long-wavelength, far-red light treatments. Light treatments using phytochrome A (phyA)- and phytochrome B (phyB)-deficient mutants showed that this response is controlled by phyB in a red/far-red reversible way, and by phyA in a non-reversible, very-low-fluence response. Crosses of the previously analyzed phyB-1 allele (in the ecotype Landsberg erecta background) to the ecotype Nossen wild-type (WT) background resulted in a WT-like negative gravitropism in darkness, indicating that the previously described gravitropic randomization observed with phyB-1 in the dark is likely due to a second mutation independent of that in the PHYB gene.
These authors contributed equally to this work.
SUMMARYCircadian clocks are gene networks producing 24-h oscillations at the level of clock gene expression that are synchronized to environmental cycles via light signals. The ELONGATED HYPOCOTYL 5 (HY5) transcription factor is a signalling hub acting downstream of several photoreceptors and is a key mediator of photomorphogenesis. Here we describe a mechanism by which light quality could modulate the pace of the circadian clock through governing abundance of HY5. We show that hy5 mutants display remarkably shorter period rhythms in blue but not in red light or darkness, and blue light is more efficient than red to induce accumulation of HY5 at transcriptional and post-transcriptional levels. We demonstrate that the pattern and level of HY5 accumulation modulates its binding to specific promoter elements of the majority of clock genes, but only a few of these show altered transcription in the hy5 mutant. Mathematical modelling suggests that the direct effect of HY5 on the apparently non-responsive clock genes could be masked by feedback from the clock gene network. We conclude that the information on the ratio of blue and red components of the white light spectrum is decoded and relayed to the circadian oscillator, at least partially, by HY5.
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