Biochemical characterization of Arabidopsis wild-type and mutant phytochrome B holoproteins.
Although phytochrome B (phyB) plays a particularly important role throughout the life cycle of a plant, it has not been studied in detail at the molecular level due to its low abundance. Here, we report on the expression, assembly with chromophore, and purification of epitope-tagged Arabidopsis phyB. In addition, we have reconstructed two missense mutations, phyB-4 and phyB-101, isolated in long hypocotyl screens. We show that mutant proteins phyB-4 and phyB-101 exhibit altered spectrophotometric and biochemical properties relative to the wild-type protein. In particular, we demonstrate that phyB-101 Pfr exhibits rapid nonphotochemical (dark) reversion to Pr that results in a lower photoequilibrium level of the active Pfr form. We conclude that this occurs in vivo as well because phyB-101 mutants are shown to lack an end-of-day-far-red hypocotyl elongation response that requires a stable Pfr species. We propose that this Pfr instability may be the primary molecular mechanism underlying the phyB-101 mutant phenotype.
Elich
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TD
Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Chory
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D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics* / Q000528:radiation effects; D029681:Arabidopsis Proteins; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D003624:Darkness; D017343:Genes, Plant; D009154:Mutation; D010641:Phenotype; D010777:Photochemistry; D010786:Photoreceptor Cells; D010834:Phytochrome / Q000737:chemistry* / Q000235:genetics* / Q000528:radiation effects; D051939:Phytochrome B; D010944:Plants / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013053:Spectrophotometry; D014157:Transcription Factors
D029681:Arabidopsis Proteins; D017931:DNA Primers; C497952:PHYB protein, Arabidopsis; D014157:Transcription Factors; D010834:Phytochrome; D051939:Phytochrome B