Native phytochrome: immunoblot analysis of relative molecular mass and in-vitro proteolytic degradation for several plant species

Abstract

The relative molecular mass (M_r) of the native phytochrome monomer from etiolated Cucurbita pepo L., Pisum sativum L., Secale cereale L. and Zea mays L. seedlings has been determined using immunoblotting to visualize the chromoprotein in crude extracts subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single phytochrome band is observed for each plant species when the molecule is extracted under conditions previously demonstrated to inhibit the proteolysis of native Avena sativa L. phytochrome. A comparison among plant species indicates that the M_r of native phytochrome is variable: Zea mays=127000; Secale=Avena=124000; Pisum=121000; Cucurbita=120000. The in-vitro phototransformation difference spectrum for native phytochrome from each species is similar to that observed in vivo in each case and is indistinguishable from that described for native Avena phytochrome. The difference minima between the red- and far-red-absorbing forms of the pigment (Pr-Pfr) are all at 730 nm and the spectral change ratios (ΔA_r/ΔA_fr) are near unity. When incubated in crude extracts, phytochrome from all four species is susceptible to Pr-specific limited proteolysis in a manner qualitatively similar to that observed for Avena phytochrome, albeit with slower rates and with the production of different M_r degradation products. Further examination of the in-vitro proteolysis of Avena phytochrome by endogeneous proteases has identified several additional phytochrome degradation products and permitted construction of a peptide map of the molecule. The results indicate that both the 6000- and 4000-M_r polypeptide segments cleaved by Pr-specific proteolysis are located at the NH₂-terminus of the chromoprotein and are adjacent to a 64000-M_r polypeptide that contains the chromophore.