Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in light-grown Avena sativa L.

Abstract

A method is described for the extraction of phytochrome from chlorophyllous shoots of Avena sativa L. Poly(ethyleneimine) and salt fractionation are used to reduce chlorophyll and to increase the phytochrome concentration sufficiently to permit spectral and immunochemical analyses. The phototransformation difference spectrum of this phytochrome is distinct from that of phytochrome from etiolated shoots in that the maximum in the red region of the difference spectrum is shifted about 15 nm to a shorter wavelength. Immunochemical probing of electroblotted proteins (Western blotting), using a method sensitive to 50 pg, demonstrates the presence of two polypeptides in green tissue that bind antiphytochrome antibodies: a predominant species with a relative molecular mass (Mr) of 118000 and a lesser-abundant 124000-Mr polypeptide. Under nondenaturing conditions all of the 124000-Mr species is immunoprecipitable, but the 118000-Mr species remains in the supernatant. Peptide mapping and immunochemical analysis with monoclonal antibodies show that the 118000-Mr species has structural features that differ from etiolated-oat phytochrome. Mixing experiments show that these structural differences are intrinsic to the molecular species from these two tissues rather than being the result of post-homogenization modifications or interfering substances in the green-tissue extracts. Together the data indicate that the phytochrome that predominates in green-tissue has a polypeptide distinct from the well-characterized molecule from etiolated tissue.

The phytochrome gene family in tomato (Solarium lycopersicum L.)

“…Tokuhisa, Daniels & Quail (1985) and Shimazaki & Pratt (1985) demonstrated subsequently that this initial report did not reflect any one of several possible artifacts. These observations confirmed a prediction published more than three decades ago, when H. W. Siegelman & Warren Butler (1965) small fraction of the phytochrome in dark-grown seedlings must be different or potentially different in some respect from the main bulk of the phytochrome, which is labile.'…”

Novel phytochrome sequences in Arabidopsis thaliana: structure, evolution, and differential expression of a plant regulatory photoreceptor family.

“…Tokuhisa and Quail (1983) presented the first direct evidence that extracts of fully green oat tissue contain two pools of phytochrome: a greatly reduced level of the etiolated-tissue form and a second, immunochemically distinct form. Further experiments confirmed these observations (Shimazaki and Pratt 1985;Tokuhisa et al 1985) and extended them to a dicot species, pea (Abe et al 1985). The green-tissue phytochrome from oats is of lower molecular mass, has different spectral properties, and is more stable in vivo in the presence of light when compared to the etiolated-tissue form (Tokuhisa et al 1985).…”

Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach

“…Gels were stained with Brilliant blue G-colloidal (Sigma). Western blotting procedures were as described previously (Tokuhisa et al, 1985). Immunoprecipitations were done as described by Weretilnyk et al (1989).…”