Comparative Phytochrome Immunochemistry as Assayed by Antisera against Both Monocotyledonous and Dicotyledonous Phytochrome

Cordonnier, Marie-Michèle; Pratt, Lee H.

doi:10.1104/pp.70.3.912

Cited by 28 publications

(20 citation statements)

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“…However, since dark reversion in the presence of dithionite was nearly complete after 2 h, it appears likely that a majority of degraded chromopeptides was capable of dark reversion. Using gel filtration on Bio-Gel A-1.5m, we were able to separate partially the large and small polypeptide groups generated by proteolysis and examine their spectral characteristics individually (Fig.…”

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confidence: 99%

“…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. 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).…”

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confidence: 99%

“…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.…”

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confidence: 99%

“…As Cucurbita phytochrome in agreement with past observations and indicating that no major production of cross-reacting antibodies was elicited by the NH2 terminus of 124-kDAvena phytochrome. (2). By comparing the reactivity of serial dilutions of the antiAvena-serum with phytochrome from the two plant species, we estimate that the native chromoprotein from Cucurbita is onefourth to one-eighth as reactive with this antiserum as the native chromoprotein from Avena.…”

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confidence: 99%

“…Ouchterlony double immunodiffusion was performed as described previously using undiluted serum (2). RESULTS Cucurbita Phytochrome Purification.…”

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confidence: 99%

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

Vierstra¹,

Quail²

1985

Plant Physiol.

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A specral, immunochemical, and proteolytic characterization of native 120-kilodalton (kD) phytochrome from Cucurbita pepo L. is presented and compared with that previously reported for native 124-kD phytochrome from Avena satiia. The molecule was partialy purified ('200-fold) in the phytochrome-far red-absorbing form (Pfr) in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride, using a modification of the procedure initially developed to purify 124-kD Arena phytochrome. The spectral properties of the preparations obtained are indistinguishable from those described for 124-kD Avena phytochrome, including a Pfr X..., at 730 nanometers, a spectral change ratio (AAJ AAe,) of 1.05, and negligible dark reversion of Pfr to the red-absorbing form (Pr) in the presence or absence of sodium dithionite. This lack of dark reversion in vitro contrasts with observations that Cucurbita phytochrome, like phytochrome from most other dicotyledons, exhibits substantial dark reversion in vivo. Ouchterlony double immunodiffusion analysis with polyclonal antibodies indicates that 120-kD Cawurbita phytochrome is immunologicaHly dissimilar to 124-kD Avena phytochrome. However, despite this dissimilarity, immunoblot analyses of proteolytic digests have identified at least three spatially separate epitopes that are common to both phytochromes. Using endogeneous protease(s), a peptide map for Cucurbita phytochrome has been constructed and the role that specific domains play in the overall structure of the photoreceptor has been examined. One domain near the NH2 terminus is critical to the spectrl integrity of the molecule indicating that this domain plays a structural role analogous to that of a domain near the NH2 terminus ofAvena phytochrome. Proteolytic removal of this domain occurs preferentially in Pr and its removal shifts the Pfr X.. to 722 nm, increases the spectral change ratio to 1.3, and substantially enhances the dark reversion rate. The apparent conservation of this domain among evolutionarily divergent plant species and its involvement in a conformational change upon photoconversion makes it potentially relevant to the mechanism(s) of phytochrome action. Preliminary evidence from gel filtration studies suggests that the 55-kD chromophoreless COOHterminal region of the polypeptide contains a domain responsible for dimerization of phytochrome monomers.

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confidence: 99%