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

Abstract

The relative molecular mass (Mr) 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 Mr 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 (ΔAr/ΔAfr) 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 Mr 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-Mr polypeptide segments cleaved by Pr-specific proteolysis are located at the NH2-terminus of the chromoprotein and are adjacent to a 64000-Mr polypeptide that contains the chromophore.

Spectral Characterization and Proteolytic Mapping of Native 120-Kilodalton Phytochrome from Cucurbita pepo L.

“…The molecular basis for the biological activity of Pfr remains obscure despite intensive study over the last two decades (see 19,26). This understanding has been hindered in part by the difficulty of purifying the chromoprotein in its native form (28 apparent that phytochrome from all plant species thus far examined is susceptible to a Pr-specific, PMSF3-sensitive protease(s) present in crude plant extracts (8,28,32). This proteolysis, although limited, results in the production of chromoproteins with spectral and biochemical properties that are altered relative to those of the native form (see 21).…”

“…This proteolysis, although limited, results in the production of chromoproteins with spectral and biochemical properties that are altered relative to those of the native form (see 21). Since, prior to 1982, phytochrome was invariably isolated without knowledge of or adequate precautions against this proteolysis, it appears likely that a majority of previously reported biochemical investigations of the molecule have been performed inadvertently with preparations containing at least in part degraded chromoproteins (28,32).…”

“…extraction of the chromoprotein as Pfr in the presence of PMSF, a protocol for the purification of native 124-kD phytochrome from the monocot, Avena sativa, has been developed recently (30) and a detailed characterization of this molecule begun (4,6,12,21,(29)(30)(31)(32). However, as noted by Cordonnier and Pratt (2), an extensive characterization of phytochrome from only a single plant species may yield misleading information, since at least some of the properties observed may be unique only to the molecule from that species.…”

“…However, as noted by Cordonnier and Pratt (2), an extensive characterization of phytochrome from only a single plant species may yield misleading information, since at least some of the properties observed may be unique only to the molecule from that species. This potential problem is underscored by the observations that phytochrome exhibits substantial interspecific polymorphism especially when analyzed using immunochemical methods (2,5,18,23,32). Alternatively, a search for molecular features conserved among phytochromes from evolutionarily divergent plant species may be a potentially more rewarding approach.…”

“…This comparative approach would focus attention on only those molecular properties most likely related to function. To this end, several common features among phytochromes have already been identified including the presence of conserved epitopes (2-4) and domains whose protease sensitivity is modulated by the spectral form of the molecule (32).…”