Large-scale partial purification of phytochrome from green leaves of Avena sativa L.

Phytochrome from 10 or 11-d-old oat (Avena sativa L. cv. Garry) leaves, which were harvested just prior to sunset from plants grown in a greenhouse in the absence of supplemental illumination, was purified an estimated 250-fold by sequential poly(ethylenimine) and ammonium-sulfate fractionations, followed by linear-gradient hydroxyapatite chromatography. Compared to earlier protocols, the one presented here is substantially more rapid, provides improved yield and purity, can be used with larger quantities of tissue, and eliminates an apparently immunodominant contaminant with a molecular mass of about 115 kDa (kilodalton). Phytochrome obtained by this procedure has an apparent monomer size of 123 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is estimated to be 0.6% pure. This purity permitted spectral analysis at wavelengths below 500 nm, in which region phytochromes from green and etiolated oat shoots do not differ markedly, as they do at longer wavelengths.Abbreviations Da Dalton - HA hydroxyapatite - Pfr, Pr farredand red-absorbing form of phytochrome, respectively - SDS sodium dodecyl sulfate This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). The excellent technical assistance of Mrs. Donna Tucker and Mrs. Danielle Neal is gratefully acknowledged.     

The levels of two distinct species of phytochrome are regulated differently during germination in Avena sativa L.

The abundance and molecular mass of phytochrome in germinating embryos of A. sativa (oat) grown in light or darkness have been monitored using immunoblot and spectrophotometric assays. Immunoblot analysis shows that imbibed but quiescent embryos have two immunochemically distinct species of phytochrome with monomeric molecular masses of 124 and 118 kDa (kdalton). The 118-kDa species has the properties of the 118-kDa phytochrome extracted from fully green oat tissue (J.G. Tokuhisa, S.M. Daniels, P.H. Quail, 1985, Planta 164, 321–332), whereas the 124-kDa polypeptide appears similar to the well-characterized photoreceptor of etiolated tissue. The capacity of antibodies directed against etiolated-oat phytochrome to immunoprecipitate the 124-kDa species but not the 118-kDa species has been exploited to quantitate the levels of each separately over a 72-h time course of germination and seedling development. The abundance of the 124-kDa molecule increases at least 200-fold in etiolated seedlings over 72 h whereas in light-grown seedlings the level of this molecule is relatively constant. In contrast, the amount of the 118-kDa species increases only twofold in both dark- and light-grown seedlings over the same period of time. These data indicate that whereas the abundance of 124-kDa phytochrome is regulated at the protein level by the well-documented, differential stability of the red- and far-red-absorbing forms in vivo, the 118-kDa molecule is present at a low constitutive level, presumably reflecting no such difference in the stability of the two spectral forms.Abbreviations ELISA enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - Pfr, Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Monoclonal antibodies directed to phytochrome from green leaves of Avena sativa L. cross-react weakly or not at all with the phytochrome that is most abundant in etiolated shoots of the same species

Seven monoclonal antibodies (MAbs) have been prepared to phytochrome from green oat (Avena sativa L. cv. Garry) leaves. One of these MAbs (GO-1) cross-reacts with apoprotein of the phytochrome that is most abundant in etiolated oat shoots as assessed by immunoblot assay of fusion proteins expressed in Escherichia coli. The epitope for this MAb is located between amino acids 618 and 686 in the primary sequence of type 3 phytochrome (Hershey et al. 1985, Nucleic Acids Res. 13, 8543–8559), which is one of the predominant phytochromes in etiolated oats. Three other MAbs (GO-4, GO-5, GO-6) immunoprecipitate phytochrome isolated from green oat leaves, as evaluated by photoreversibility assay. GO-1, GO-4, GO-5 and GO-6 are therefore directed to phytochrome. While evidence obtained with the other three MAbs (GO-2, GO-7, GO-8) strongly indicates that they are also directed to phytochrome, this evidence is not as rigorous. Recognition of antigen by any of these seven MAbs is not significantly reduced by periodate oxidation, indicating that their epitopes probably do not include carbohydrate. All but GO-1 bind either very poorly or not at all the phytochrome that is abundant in etiolated oat shoots. These data reinforce earlier observations made with antibodies directed to phytochrome from etiolated oats, indicating (1) that the phytochromes that predominate in etiolated and green oats differ immunochemically and (2) that phytochrome preparations from green oat leaves contain very little of the phytochrome that is abundant in etiolated shoots. An hypothesis that these two immunochemically distinct phytochromes form heterodimers in vitroAbbreviations Da Dalton - DEAE diethylaminoethyl - ELISA enzyme-linked immunosorbent assay - HA hydroxyapatite - Ig immunoglobulin - MAb monoclonal antibody - SDS sodium dodecyl sulfate is supported by comparison of immunoblot data obtained with conventionally purified phytochrome from etiolated oats to that expressed as fusion protein in E. coli. This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). We thank Dr. Lyle Crossland and Ms. Sue Kadwell for their assistance in the construction of the cDNA clones, and Dr. Gyorgy Bisztray for providing us with clone pCBP3712. Dr. Phillip Evans and Dr. Russell Malmberg kindly provided MAbs 4F3, 6F12 and 8C10, as well as a corresponding antigen preparation. The excellent technical assistance of Mrs. Donna Tucker and Mrs. Danielle Neal is gratefully acknowledged.     

Immunoprecipitation of phytochrome from green Avena by rabbit antisera to phytochrome from etiolated Avena

Thirty-nine antiserum preparations from eight rabbits were screened for their ability to precipitate the immunochemically distinct phytochrome that is obtained from green oat (Avena sativa L.) shoots. The antisera were obtained from rabbits immunized with either proteolytically degraded, but still photoreversible, 60-kDa (kilodalton) phytochrome, or approx. 120-kDa phytochrome, both of which were purified from etiolated oat shoots. The ability of these antisera to precipitate phytochrome from green oats was independent of the size of phytochrome used for immunization. While crude antisera immunoprecipitated as much as 80% of the phytochrome isolated from green oat shoots, antibodies immunopurified from these sera with a column of highly purified, approx. 120-kDa phytochrome from etiolated oats precipitated no more than about 5–10%.Abbreviations kDa kilodalton - mU milliunit     

Spectrophotometric phytochrome measurements in light-grown Avena sativa L.

Phytochrome was studied spectrophotometrically in Avena sativa L. seedlings that had been grown for 6 d in continous white fluorescent light from lamps. Greening was prevented through the use of the herbicide San 9789. When placed in the light, phytochrome (P_tot) decreased with first order kinetics (_1/2 2 h) but reached a stable low level (2.5% of the dark level) after 36 h. This concentration of phytochrome remained constant in the light and during the initial hours of a subsequent dark period, but increased significantly after a prolonged dark period. Evidence suggests that the constant pool of phytochrome in the light is achieved through an equilibrium between synthesis of the red absorbing (P_r) and destruction of the far-red absorbing form (P_fr) of phytochrome. It is concluded that the phytochrome system in light-grown oat seedlings is qualitatively the same as that known from etiolated monocotyledonous seedlings, but different than that described for cauliflower florets.Abbreviations P_fr the far-red light absorbing form of phytochroma - P_r the red light absorbing form of phytochrome - P_tot P_r+P_fr - k_s rate constant of P_r synthesis - k_d rate constant of P_fr destruction - MOPS N-morpholino-3-propane-sulfonic acid - IRIS Tris (hydroxymethyl) amino methane - San 9789 4-chloro-5-(methyl amino)-2-(,,-trifluoro-m-tolyl)-3(2H)pyridazinone     

Avena sativa L. contains three phytochromes,only one of which is abundant in etiolated tissue

Phytochrome from leaves of light-grown oat (Avena sativa L. cv. Garry) plants is characterized with newly generated monoclonal antibodies (MAbs) directed to it. The results indicate that there are at least two phytochromes in green oat leaves, each of which differs from the phytochrome that is most abundant in etiolated oat tissue. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with reference to 124-kilodalton (kDa) phytochrome from etiolated oats, the two phytochromes from green oats have monomer sizes of 123 of 125 kDa. Immunoblot analysis of SDS, sample buffer extracts of lyophilized, green oat leaves indicates that neither the 125-kDa nor the 123-kDa polypeptide is a degradation product arising after tissue homogenization. Of the two, the 123-kDa phytochrome appears to be the predominant species in light-grown oat leaves. During SDS-PAGE in the presence of 1 mM Zn²⁺, 123-kDa phytochrome undergoes a mobility shift corresponding to an apparent mass increase of 2 kDa. In contrast, the electrophoretic mobility of 125-kDa phytochrome is unaffected by added Zn²⁺. Some MAbs that recognize 123-kDa phytochrome fail to recognize 125-kDa phytochrome and vice versa, indicating that these two phytochromes are not only immunochemically distinct from 124-kDa phytochrome, but also from each other. It is evident, therefore, that there are at least three phytochromes in an oat plant: 124-kDa phytochrome, which is most abundant in etiolated tissue, plus 123-and 125-kDa phytochromes, which predominate in light-grown tissue.Abbreviations Da Dalton - HA hydroxyapatite - MAb monoclonal antibody - PAb polyclonal antibody preparation - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). We thank Dr. Alan Jones, Department of Biology, University of North Carolina, Chapel Hill, USA, for kindly providing rabbit antiserum 4032, and Mrs. Donna Tucker and Mrs. Danielle Neal for their technical assistance.     

Spatial distribution of three phytochromes in dark- and light-grown Avena sativa L.

We have addressed two issues regarding the spatial distribution of three phytochromes in 3-d-old oat (Avena sativa L.) seedlings. Three monoclonal antibodies, GO-4, GO-7 and Oat-22, were used as probes. Each antibody detects only one of the phytochromes. The first issue is whether any of the phytochromes might be membrane-bound. To address this issue the abundance of each phytochrome in extracts prepared with either a detergent-free or a detergent-containing buffer was compared by immunoblot assay. The detergent-free buffer was formulated to extract only soluble protein, while the detergent-containing buffer was intended to extract both soluble and membrane proteins. None of the data indicate that any of these three phytochromes is membrane-bound in either a dark- or a light-grown seedling. The second issue is whether these three phytochromes are distributed differentially in 3-d-old dark- and light-grown seedlings. When seedlings were dissected into shoots, scutellums, and roots, all three phytochromes were detected in all three fractions from both dark- and light-grown seedlings. Each of the three phytochromes was most abundant in the shoot and least abundant in the root, except that in light-grown seedlings type I, etiolated-tissue phytochrome was more abundant in the root than in either the shoot or the scutellum. When the equivalent fractions dissected from different seedlings were compared, those dissected from dark-grown seedlings contained a higher quantity of each of the three phytochromes than did those dissected from light-grown seedlings, except that green-tissue, type II phytochromes did not differ significantly in the roots. At this level of resolution, no evidence was obtained to indicate a substantive difference among the three phytochromes in their spatial distribution. We thank Drs. Elizabeth Williams and Tammy Sage (Botany Department, University of Georgia, USA) for generously permitting us to use their image-analysis system. This research was supported by USDA NRICGP grant 91-37100-6490.     

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

Discrimination between the red- and far-red-absorbing forms of phytochrome from Avena sativa L. by monoclonal antibodies

A set of rat monoclonal antibodies (ARC MAC 48 to 52 and 54 to 56), raised to phytochrome from dark-grown seedlings of Avena sativa L. was tested for the ability to discriminate between the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of phytochrome by indirect enzyme-linked immunosorbent assay. MAC 50 bound more strongly to Pfr and MAC 49 and 52 showed preferential binding to Pr from extracts of dark-grown Avena seedlings; MAC 50 also bound more strongly to Pfr from brushite-purified phytochrome. The remainder of the monoclonal antibodies and a rabbit polyclonal antiphytochrome preparation did not discriminate between Pr and Pfr. The results provide evidence for conformational changes in defined regions of the phytochrome apoprotein upon photoconversion.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - McAb monoclonal antibody(ies) - PBS phosphate-buffered saline - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - PMSF phenylmethylsulphonylfluoride     

The role of separate molecular domains in the structure of phytochrome from etiolated Avena sativa L.

The spectral properties of peptides generated from etiolated-Avana, 124-kDa (kilodalton) phytochrome by endogenous protease(s) have been studied to assess the role of the amino-terminal and the carboxyl-terminal domains in maintaining the proper interaction between protein and chromophore. The amino-terminal, 74-kDa chromopeptide, a degradation product of the far-red absorbing form of the pigment (Pfr), is shown to be spectrally similar to the 124-kDa, undegraded molecule. The minimum and maximum of the difference spectrum (Pr-Pfr) are 730 and 665 nm, respectively, and the spectral-change ratio is unity. Also, like undegraded, 124-kDa phytochrome, the 74-kDa peptide exhibits minimal dark reversion. These data indicate that the 55-kDa, carboxyl-terminal half of the polypeptide does not interact with the chromophore and may not have a role in the structureal integrity of the amino-terminal domain. The 64-kDa chromopeptide can be generated directly from the 74-kDa species by cleavage of 10 kDa from the amino terminus upon incubation of this species as Pr. Accompanying this conversion are changes in the spectral properties, namely, a shift in the difference spectrum minimum to 722–724 nm and a tenfold increase in the capacity for dark reversion. These data indicate that the 6–10 kDa, amino-terminal segment continues to function in its role of maintaining proper chromophore-protein interactions in the 74-kDa peptide as it does in the undegraded molecule. Conversely, removal of this segment upon proteolysis to the 63-kDa species leads to aberrant spectral properties analogous to those observed when this domain is lost from the full-length, 124-kDa molecule, resulting in the 118/114-kDa degradation products. The data also show that photoconversion of the 74-kDa chromopeptide from Pfr to Pr exposes proteolytically susceptible sites in the same way as in the 124-kDa molecule. Thus, the separated, 74-kDa amino-terminal domain undergoes a photoinducible conformational change comparable to that in the intact molecule.Abbreviations and symbols Da dalton - Pfr far-red-absorbing from of phytochrome - PMSF phenylmethylsulfonyl fluoride - Pr red-absorbing form of phytochrome - R red light - FR lar-red light - A _r/A _fr spectral change ratio - _max ^FR peak maximum (nm) of Pfr absorbance     

Cross-reactivity of monoclonal antibodies against phytochrome from Zea and Avena

The cross-reactivity of diverse monoclonal antibodies against phytochrome from Zea and Avena was tested by enzyme-linked immunosorbentassay (ELISA) and by immunoblotting. About 40 antibodies were selected by means of nondenatured phytochrome; all of them reacted with sodium dodecyl sulfate denatured homologous antigen on immunoblots. The epitopes for 14 antibodies (4 raised against Avena and 10 against Zea phytochrome) were localized in 6 regions of the phytochrome molecule by means of Western blot analysis of proteolytic fragments of known localization. Results of studies on the inhibition of antibody binding by other antibodies were largely compatible with these latter findings. Except in a few cases, inhibition occurred when antibodies were located on the same or a closely adjacent region. As demonstrated by 16 species, cross-reactivity with phytochromes from other Poaceae was high. Greater losses in cross-reactivity were observed only with antibodies recognizing an epitope in the vicinity of the carboxyl terminus of 118-kg · mol^-1 phytochrome. Cross-reactivity with phytochrome from dicotyledons was restricted to a few antibodies. However, phytochrome(s) from plants illuminated for 24 h or more could be detected. One of the antibodies that recognized phytochrome from dicotyledons was also found to recognize phytochrome or a protein of 120–125 kg·mol^-1 from several ferns, a liverwort and mosses. This antibody (Z-3B1), which was localized within a 23.5-kg·mol^-1 section of Avena phytochrome (Grimm et al., 1986, Z. Naturforsch. 41c, 993), seems to be the first antibody raised against phytochrome from a monocotyledon with such a wide range of reactivity. Even though epitopes were recognized on different phytochromes, the strength of antibody binding indicated that these epitopes are not necessarily wholly identical.Abbreviations ELISA enzyme-linked immunosorbent assay - McAb monoclonal antibody - PBS phosphate-buffered saline - Pfr (Pr) far-red-absorbing (red-absorbing) form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A monoclonal antibody specific for the red-absorbing form of phytochrome

Characterisation of a new monoclonal antibody (mAb), designated LAS 41, directed against 124-kilodalton (kDa) etiolated-oat (Avena sativa L.) phytochrome, indicates that it recognises an epitope unique to the red-light-absorbing form, Pr. In a solid-phase enzyme-linked immunosorbent assay (ELISA), LAS 41 exhibits a seven- to eight-fold higher affinity for Pr than for the far-red-light-absorbing form of phytochrome, Pfr. In addition, in immunoprecipitation assays LAS 41 effectively precipitates 100% of phytochrome presented as Pr but only precipitates a maximum of 24.5% of phytochrome presented as Pfr. These values are indicative of binding exclusively to Pr. Peptide-mapping studies show that LAS 41 recognises and epitope located within a region 6–10 kDa from the aminoterminus of the phytochrome molecule. Since binding of LAS 41 to Pr induces alterations in the spectral properties of Pr, this indicates that at least part of the 4 kDa domain to which the antibody binds is essential for protein-chromophore interaction. Subsequent photoconversion of LAS 41-Pr complexes produces native Pfr spectra, with concomitant production of free antibody and antigen, as shown by a modified ELISA. The specificity of LAS 41 for Pr has facilitated the purification of Pfr which is free of contaminating Pr. This has enabled direct determination of the mole fraction of Pfr established by red light to be 0.874.Abbreviations ELISA enzyme-linked immunsorbent assay - kDa kilodalton - mAb monoclonal antibody - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - (A) difference in absorbance (A ₆₆₅ ^Pr –A ₇₃₀ ^Pr )-(A ₆₆₅ ^Pfr –A ₇₃₀ ^Pfr ) - Ar/Afr spectral change ratio (SCR) - max mole fraction of Pfr following saturating red light     

Partial purification of sequestered particles of phytochrome from oat (Avenu sativa L.) seedlings

Sequestered particles of phytochrome (SAPs) were partially purified from red-light-irradiated oat coleoptiles. Phytochrome pelletability was enhanced by using buffers containing 10 mM Mg²⁺ or high concentrations (0.6–0.8 M) of orthophosphate (Pi). Combining the pelletability of phytochrome in the presence of Mg²⁺ with that in the presence of 0.6 Pi resulted in a strong enrichment (about 100-fold) of pelletable phytochrome. Antisera were raised against Mg²⁺-Pi-pellets from darkgrown seedlings. Using these antisera, no evidence was found by Western blotting and immunocytochemistry that SAPs contain major proteins other than phytochrome. The major contamination of these enriched SAP preparations consisted of protein crystals which are probably catalase. The preparations contained methyltransferase and protein-kinase activities which were not associated with SAPs. Phytochrome purified from SAPs served as a substrate for protein-kinase activity but not for the methyltransferase activity. Phytochrome itself did not show any kinase activity.Abbreviations ME 2-mercaptoethanol - PAGE polyacrylamide gel electrophoresis - Pfr far-red-light-absorbing form of phytochrome - PMSF phenylmethylsulfonyl fluoride - SAP sequestered area of phytochrome - SDS sodium dodecyl sulfate This work was supported by Deutsche Forschungsgemeinschaft. The competent technical assistance of Karin Fischer is gratefully acknowledged.     

Intracellular redistribution of phytochrome in etiolated soybean (Glycine max L.) seedlings

The intracellular distribution of phytochrome in hypocotyl hooks of etiolated soybean (Glycine max L.) has been examined by immunofluorescence using a newly produced monoclonal antibody (Soy-1) directed to phytochrome purified from etiolated soybean shoots. Cortical cells in the hook region exhibit the strongest phytochrome-associated fluorescence, which is diffusely distributed throughout the cytosol in unirradiated, etiolated seedlings. A redistribution of immunocytochemically detectable hytochrome to discrete areas (sequestering) following irradiation with red light requires a few minutes at room temperature in soybean, whereas this redistribution is reversed rapidly following irradiation with far-red light. In contrast, sequestering in oat (Avena sativa L.) occurs within a few seconds (D. McCurdy and L. Pratt, 1986, Planta 167, 330–336) while its reversal by far-red light requires hours (J. M. Mackenzie Jr. et al., 1975, Proc. Natl. Acad. Sci. USA 72, 799–803). The time courses, however, of red-light-enhanced phytochrome pelletability and sequestering are similar for soybean as they are for oat. Thus, while these observations made with a dicotyledon are consistent with the previous conclusion derived from work with oat, namely that sequestering and enhanced pelletability are different manifestations of the same intracellular event, they are inconsistent with the hypothesis that either is a primary step in the mode of action of phytochrome.Abbreviations DIC differential interference contrast - FR far-red light - Ig immunoglobulin - Pfr, P far-red- and red-absorbing form of phytochrome, respectively - R red light This work was supported by National Science Foundation grant No. DCB-8703057.     

Temporal and light regulation of the expression of three phytochromes in germinating seeds and young seedlings of Avena sativa L.

An oat (Avena sativa L.) plant contains at least three phytochromes, which have monomeric masses of 125, 124, and 123 kilodaltons (kDa) (Wang et al., 1991, Planta 184, 96–104). The 124-kDa phytochrome is most abundant in dark-grown seedlings, while the other two phytochromes predominate in light-grown seedlings. Using three monoclonal antibodies, each specific to one of the three phytochromes, we have monitored by immunoblot assay the expression of these three phytochromes in the 5 d following onset of imbibition of seeds. On a per-organism basis, each of these three phytochromes increased in abundance for the first 3 d in the light, or for the first 4 d in darkness, after which they each began to decrease in quantity. When 3-d-old dark-grown seedlings were transferred to the light, the abundance of each of these three phytochromes decreased both in absolute amount and relative to the phytochrome levels in control seedlings kept in darkness. In contrast, when 3-d-old light-grown seedlings were transferred to darkness, the abundance of the 124-kDa and 125-kDa phytochromes increased while that of 123-kDa phytochrome remained unchanged. In each case, the level of phytochrome was greater than that of control seedlings maintained in the light. Thus, in addition to temporal regulation, all three phytochromes exhibit photoregulated expression at the protein level, although the magnitude of this photoregulation varies substantially. We thank Drs. Elizabeth Williams and Tammy Sage (Botany Department, University of Georgia, USA) for generously permitting us to use their image-analysis system. This research was supported by USDA NRICGP grant 91-37100-6490.     

Sequence analysis of proteolytic fragments of 124-kilodalton phytochrome from etiolatedAvena sativa L.: Conclusions on the conformation of the native protein

Proteolytic fragments were obtained by limited proteolysis of 124-kDa (kilodalton) phytochrome from etiolatedAvena sativa using trypsin, endoproteinase-Lys-C, endoproteinase-Glu-C and subtilisin. The fragments were separated by sodium dodecyl sulfate gel electrophoresis, blotted onto activated glass-fiber sheets and investigated by amino-acid sequencing in a gas-phase sequencer. Determination of N-terminal sequences in three to six Edman degradation steps allowed the exact localization of the fragments within the published entire amino-acid sequence of 124-kDaAvena phytochrome (H.P. Hershey, R.F. Barker, K.B. Idler, J.L. Lissemore, P.H. Quail (1985), Nucleic Acids Res.13, 8543–8559). From the knowledge of the exact sites for preferred proteolytic cleavage of undenatured phytochrome, conclusions on the conformation of the phytochrome protein were drawn. Sites of preferred cleavage are considered to be freely exposed to the environment whereas potential cleavage sites which are resistant to proteolysis over a long time are considered to be localized in the interior of the native phytochrome. Two different sites which are exposed in the far-red-absorbing form but not in the red-absorbing form of phytochrome are localized at amino-acid residues 354 and 753, respectively. The N-terminal region which is exposed only in the red-absorbing form stretches only as far as amino-acid residue 60.Abbreviations kDa kilodalton - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Dedicated to Professor W. Rau on the occasion of his 60th birthday.     

The kinetics of type 1 phytochrome in green,light-grown wheat (Triticum aestivum L.)

The kinetics of type 1 phytochrome were investigated in green, light-grown wheat. Phytochrome was measured by a quantitative sandwich enzyme-linked immunosorbent assay using monoclonal antibodies. The assay was capable of detecting down to 150 pg of phytochrome. In red light, rapid first-order destruction of the far-red-light-absorbing form of phytochrome (Pfr) with a half-life of 15 min was observed. Following white light terminated by red, phytochrome synthesis was delayed in darkness by about 15 h compared to plants given a terminal far-red treatment. Synthesis of the red-light-absorbing form of phytochrome (Pr) was zero-order in these experiments. Phytochrome synthesis in far-red light was approximately equal to synthesis in darkness in wheat although net destruction occurred in light-grown Avena sativa tissues in continuous far-red light, as has been reported for other monocotyledons. In wheat, destruction of Pfr apparently did not occur below a certain threshold level of Pfr or Pfr/total phytochrome. These results are consistent with an involvement of type 1 phytochrome in the photoperiodic control of flowering in wheat and other long-day plants.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - HIR high-irradiance response - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - Ptot total phytochrome (Pr + Pfr) - R red light The authors wish to thank Prof. Daphne Vince-Prue (University of Reading) for many helpful discussions regarding this work. Hugh Carr-Smith was supported by a Science and Engineering Research Council studentship and Chris Plumpton by an Agricultural and Food Research Council (AFRC) studentship. B. Thomas and G. Butcher were supported by the AFRC.     

Destruction and possible de novo synthesis of phytochrome in subcellular fractions of laminae from Avena sativa L.

Phytochrome was determined in etiolated laminae of Avena sativaL. either without pretreatment or after 5 min of red irradiation followed by different periods of darkness (0–24 h). At given intervals laminae were homogenized and phytochrome was determined spectrophotometrically in the total homogenate and in purified etioplasts and mitochondria. Enhanced specific activity of phytochrome was found in all fractions after the irradiation in comparison to dark controls. Phytochrome destruction was observed in all fractions at the beginning of the subsequent dark period. Whereas the homogenate and the mitochondrial fraction showed a continuous destruction so that phytochrome reached a level far below that in etiolated plants, the phytochrome level in the plastid fraction reacheda minimum at 2 h with a subsequent increase beyond the dark level. This increase was most pronounced between 4 and 8 h after the red irradiation. The results are discussed in terms of the destruction and possible de novo synthesis of phytochrome that may be different in mitochondria and plastids.Abbreviations P_tot total phytochrome - P_r red absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - ER endoplasmic reticulum     

Phytochrome structure: Peptide fragments from the amino-terminal domain involved in protein-chromophore interactions

We have undertaken a study of the structure of the amino-terminal domain of the phytochrome polypeptide purified from Avena sativa L. Amino-acid sequencing was used to indentify arginine 52 as the precise location of a conformation-specific cleavage of phytochrome by subtilisin. The location of the epitopes for a class of monoclonal antibodies designated type 2 has been shown to be located between approx. 10 and 20 kilodaltons (kDa) from the amino terminus. These two new spatial markers, in addition to the chromophore and another epitope recognized by type 1 monoclonal antibodies and located within 6 kDa from the amino terminus, have been used to map the locations of several new protease-accessible sites along the polypeptide. After extensive digestion of phytochrome with subtilisin, a stable spectrally-active group of peptides remains. Within this group is a 16-kDa chromopeptide which, either alone or as part of an assemblage of peptides, elutes from a size-exclusion column under nondenaturing conditions at a volume consistent with a molecular mass of 35–40 kDa. This group of peptides has an absorbance spectrum similar to the red-absorbing form of phytochrome (Pr) and is red/far-red photoreversible between this and a photobleached form. These data indicate that this group of peptides still retains the principal structural requisites for Pr-chromophore-protein interactions and for photoreversibility, but not for Pfr (far-red-absorbing phytochrome)-chromophore-protein interactions. It is uncertain if these structural requisites reside exclusively on the 16-kDa chromopeptide or result from an assemblage of these peptides. However, we have excluded any role for an adjacent 14-kDa fragment (approximately residues 50 to 200) in the observed spectral properties since it can be selectively removed without any effect on the photoreversibility.Abbreviations Da dalton - M_r relative molecular mass - Pr, Pfr red and far-red-absorbing forms of phytochrome, respectively - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis This work was presented, in part, at the XVI Yamada Conference on Phytochrome and Plant Photomorphogenesis, Okazaki, Japan, October 1986     

Proteolysis alters the spectral properties of 124 kdalton phytochrome from Avena

Native phytochrome from Avena sativa L. is homogeneous with a monomeric molecular weight of 124 kdalton; 6–10 kdalton larger than the heterogeneous 120 kdalton preparations previously considered to be undegraded (Vierstra and Quail, 1982, Proc. Natl. Acad. Sci. USA, 79: 5272–5276). The phototransformation difference spectrum (Pr-Pfr) of 124 kdalton phytochrome measured in crude extracts has a minimum in the farred region at 730 nm, the same as that observed in vivo. These spectral properties contrast with those of 120 kdalton phytochrome purified by column immunoaffinity chromatography where the difference minimum is at 724 nm. When 124 kdalton phytochrome is incubated as Pr in crude extracts, the difference minimum shifts progressively to shorter wavelengths (from 730 to 722 nm) concomitant with the proteolytic degradation of the chromoprotein to the mixture of 118 and 114 kdalton species that comprise 120 kdalton phytochrome preparations. These two effects are inhibited in concert by the serine protease inhibitor, phenylmethylsulfonylfluoride, and or maintenance of the phytochrome in the Pfr form. These results provide further evidence that 124 kdalton phytochrome is the native molecule in Avena and indicate that the peptide segments removed by proteolysis of the Pr form are important to the pigment's spectral integrity. The present data thus resolve the previously unsettled question of why the Pfr form of 120 kdalton phytochrome isolated by various procedures from Avena has been found to absorb at shorter wavelengths than that observed in vivo. Previous spectral studies with 120 kdalton phytochrome preparations are open to reexamination.Abbreviations, symbols PMSF phenylmethylsulfonylfluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - Ig immunoglobulin - A_minimum, A_maximum phototransformation difference spectrum (Pr-Pfr) minimum and maximum - A_r/A_fr ratio spectral change ratio