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

Preparation and characterization of antisera against lettuce (Lactuca sativa L., cv. Grand Rapids) and pea (Pisum sativum L., cv. Alaska) phytochrome is described. These antisera, together with previously obtained antisera against zucchini (Cucurbita pepo L., cv. Black Beauty) and oat (Avena sativa L., cv. Garry) phytochrome, were used to compare by Ouchterlony double immunodiffusion phytochrome isolated from etiolated lettuce, pea, bean (Phaseolus vulgaris L., cv. Taylor Horticultural Bush), zucchini, oat and rye (Secale cereale L., cv. Balbo) seedlings. Cross reactivity between monocotyledonous phytochrome and antidicotyledonous-phytochrome serum and between dicotyledonous phytochrome and antimonocotyledonous-phytochrome serum was always weak or not perceptible by this assay. Among the four dicotyledonous phytochromes examined, pea and bean were the most similar immunochemically as anticipated. Pea and lettuce phytochrome somewhat unexpectedly also exhibited similar immunochemical reactivity. Zucchini phytochrome by contrast was immunochemically distinct from pea, bean, and lettuce phytochrome, although it did react with all three antidicotyledonous-phytochrome sera. Initial attempts to identify immunoglobulins that would recognize phytochrome regardless of its source indicated that they may exist. Such immunoglobulins are of interest because they might react with one or more determinants that could be part of an active site of phytochrome. These immunoglobulins, once isolated, could thus serve as a potential probe for the active site of phytochrome.     

Phytochrome immunoaffinity purification

We have developed a phytochrome immunoaffinity purification procedure that yields undegraded oat (Avena sativa L., cv. Garry) phytochrome of greater than 98% purity within 2 hours when starting with a brushite-purified preparation. Immunoaffinity-purified phytochrome, except for its greater purity, is indistinguishable from conventionally purified phytochrome by gel exclusion chromatography, isoelectric focusing, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have also used the immunoaffinity technique to purify phytochrome from crude oat extracts, and from brushite-purified pea (Pisum sativum L., cv. Alaska) and rye (Secale cereale L., cv. Balbo) preparations.     

Phytochrome Characterization by Rabbit Antiserum against High Molecular Weight Phytochrome

Both small and large sizes of phytochrome purified from Garry oat (Avena sativa L. ev. Garry) as well as large phytochrome purified from Newton oat (A. sativa L. cv. Newton), rye (Secale cereale L. cv. Balbo), barley (Hordeum vulgare L. cv. Harrison), and pea (Pisum sativum L. cv. Alaska) seedlings are characterized by a specific antiserum against large Garry oat phytochrome. A spur is observed by double diffusion assay against large and small Garry oat phytochrome indicating only partial identity. In micro-complement fixation assays, large Garry oat phytochrome yields greater activity than small Garry oat phytochrome. In addition, the peak of activity is shifted to a higher antigen concentration with small phytochrome. Phytochrome, red-absorbing form, and phytochrome, far redabsorbing form, are indistinguishable by both double diffusion and micro-complement fixation assay. The different grass phytochromes are antigenically identical by double diffusion assay. Immunoelectrophoretic analyses of oat and rye large phytochrome, after proteolysis, suggest that there are one or a few regions of the molecule especially susceptible to hydrolysis by a wide variety of endopeptidases.     

Comparative immunochemistry of phytochrome

Partially purified high molecular weight preparations of phytochrome, estimated to be close to 440,000 molecular weight based upon chromatography through a calibrated Bio-Gel P-300 column, were obtained from Garry and Newton oats (Avena Sativa L., cv. Garry and cv. Newton), rye (Secale cereale L., cv. Balbo), barley (Horedum vulgare L., cv. Harrison), and pea (Pisum sativum L., cv. Alaska) by a sequence of three chromatographic steps: brushite, diethylaminoethyl cellulose, and Bio-Gel P-300. No significant differences were observed between these preparations during purification or subsequent handling. In addition, a low molecular weight form of phytochrome was purified from Garry oats. Two specific antisera against a low molecular weight form of phytochrome (60,000 molecular weight) obtained from etiolated Garry oat seedlings are characterized and used to compare the phytochrome preparations. Double diffusion assays indicated antigenic identity between all preparations except that pea phytochrome yielded a spur when compared to oat phytochrome. Micro complement fixation assays yielded complete identity between Garry and Newton oat phytochrome, reduced activity with rye and barley phytochrome, and a complete lack of activity with pea phytochrome at the serum dilutions assayed. Immunoelectrophoretic assays indicated that all high molecular weight phytochrome preparations were homogeneous by this criterion and that there were only slight differences between the preparations in electrophoretic mobility. Large and small forms of phytochrome isolated from Garry oats were found to be very similar antigens when tested with the anti-small phytochrome sera, although the small form was observed to electrophorese at a much slower rate than the large.     

Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to pisum and Avena phytochrome

Nine monoclonal antibodies to pea (Pisum sativum L.) and 16 to oat (Avena sativa L.) phytochrome are characterized by enzyme-linked immunosorbent assay against phytochrome from six different sources: pea, zucchini (Cucurbita pepo L.), lettuce (Lactuca sativa L.), oat, rye (Secale cereale L.), and barley (Hordeum vulgare L.). All antibodies were raised against phytochrome with a monomer size near 120,000 daltons. Nevertheless, none of them discriminated qualitatively between 118/114-kilodalton oat phytochrome and a photoreversible, 60-kilodalton proteolytic degradation product derived from it. In addition, none of the 23 antibodies tested discriminated substantially between phytochrome—red-absorbing form and phytochrome—far red-absorbing form. Two antibodies to pea and six to oat phytochrome also bound strongly to phytochrome from the other species, even though these two plants are evolutionarily widely divergent. Of these eight antibodies, two bound significantly to all of the six phytochrome preparations tested, indicating that these two may recognize highly conserved regions of the chromoprotein. Since the molecular function of phytochrome is unknown, these two antibodies may serve as unique probes for regions of this pigment that are important to its mode of action.     

Red light-induced accumulation of ubiquitin-phytochrome conjugates in both monocots and dicots

Phytochrome is rapidly degraded in vivo after photoconversion from the stable red-absorbing (Pr) form to the far red-absorbing (Pfr) form. Previously, we have shown in etiolated oat seedlings that ubiquitin-phytochrome conjugates (Ub-P) appear after Pfr formation suggesting that oat phytochrome is rapidly degraded by a ubiquitin-dependent proteolytic pathway. Here, we extend this observation to etiolated tissue from other monocotyledonous (corn [Zea mays. (L.)] and rye [Secale cereale (L.)] and dicotyledonous species (pea [Pisum sativum (L,)] and zucchini squash [Cucurbita pepo (L.)]). Following Pfr formation by red light, all four species synthesized a heterogeneous series of Ub-P that appeared and disappeared concomitant with the degradation of the chromoprotein. When Pfr was photoconverted back to Pr by a far-red light pulse, degradation of phytochrome ceased and the levels of Ub-P concomitantly dropped. In pea and zucchini squash, loss of Ub-P after photoconversion of Pfr back to Pr was rapid, occurring with a half-life of approximately 5 to 10 minutes. These data indicate that the accumulation of Ub-P after Pfr formation is a general phenomenon in etiolated seedlings of higher plants and further support the hypothesis that plants degrade Pfr via Ub-P intermediates.     

Purification of oat and rye phytochrome

A purification procedure employing normal chromatographic techniques is outlined for isolating phytochrome from etiolated oat (Avena sativa L.) seedlings. Yields in excess of 20% (25 milligrams or more) of phytochrome in crude extract were obtained from 10- to 15-kilograms lots. The purified oat phytochrome had an absorbance ratio (A₂₈₀ nm/A₆₆₅ nm) of 0.78 to 0.85, comparable to reported values, and gave a single major band with an estimated molecular weight of 62,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. A modification of the oat isolation procedure was used to isolate phytochrome from etiolated rye Secale cereale cv. Balbo) seedlings. During isolation rye phytochrome exhibited chromatographic profiles differing from oat phytochrome on diethylaminoethyl cellulose and on molecular sieve gels. It eluted at a higher salt concentration on diethylaminoethyl cellulose and nearer the void volume on molecular sieve gels. Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10- to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants.     

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     

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     

PHYTOCHROME DISTRIBUTION IN ETIOLATED GRASS SEEDLINGS AS ASSAYED BY AN INDIRECT ANTIBODY-LABELLING METHOD

The distribution of phytochrome in several etiolated grass seedlings (Avena saliva L., cvs. Garry and Newton; Secale cereale L., cv. Balbo; Hordeum vulgare L., cv. Harrison; Oryza sativa L; Zea mays L., cv. Golden Cross) was determined, by an indirect antibody-labelling method employing peroxidase as the ultimate label. Although the pattern of phytochrome distribution in etiolated shoots varies widely, it is nevertheless clear that, with the exception of corn, in which phytochrome is relatively uniformly distributed, the distribution of phytochrome is highly specific with respect both to organs and to cell types within an organ for a given species. Oat, rye, barley, and rice shoots all have high concentrations of phytochrome near the tips of their coleoptiles, as well as near the shoot apex itself. Rice, barley, and rye also have high concentrations of phytochrome in their leaf bases, but oat leaves are almost totally devoid of measurable phytochrome. An association of phytochrome with vascular tissue often occurs and is most pronounced in the rice shoot. Dark-grown roots were found to have high levels of phytochrome only in the root caps, with lesser amounts, if any, observed in other parts of the root.     

INTRACELLULAR PHYTOCHROME DISTRIBUTION AS A FUNCTION OF ITS MOLECULAR FORM AND OF ITS DESTRUCTION

The immunocytochemically observed intracellular redistribution of phytochrome as a function of its molecular form is described by utilizing color photomicrography. The reversible change from a diffuse to a discretely localized distribution following photoconversion of the red-absorbing Pr form to the far-red-absorbing Pfr form observed with etiolated oat (Avena sativa L., cv. Garry) coleoptile parenchyma cells is not seen with etiolated wheat (Triticum sativum L., cv. unknown), barley (Hordeum vulgare L., cv. Harrison), or rye (Secale cereale L., cv. Balbo). Whether redistribution in these latter cases does not occur or is below the limit of detection is not known. Upon continuous actinic irradiation, phytochrome, which is discretely localized as Pfr, rapidly disappears by both immunocytochemical and spectral assay. However, after about 90 min irradiation, a new association of phytochrome with nuclei is evident which is more pronounced after 4 or 8 h of irradiation. With longer irradiation times there is a total loss of antigenically detectable phytochrome at the resolution employed in these experiments.     

"Disaggregation" of phytochrome in vitro-a consequence of proteolysis

The relationship between a large molecular weight (9S) and a small molecular weight (4.5S, 60,000 molecular weight) species of phytochrome was examined to determine if the larger species was an aggregate of the smaller. Alterations of pH, salt concentration, or phytochrome concentration did not cause any observable formation of the large form from the small form. However, in partially purified phytochrome extracts from Secale cereale L. and Avena sativa L., the large form was converted to the small form over time at 4 C in the dark. This breakdown was inhibitable by the protease inhibitor phenylmethanesulfonyl fluoride. When highly purified large molecular weight rye phytochrome was incubated with a neutral protease isolated from etiolated oat shoots, the large phytochrome was converted to the small form without qualitative visible absorbancy changes. The effect of the oat protease could be mimicked by a wide variety of commercial endopeptidases, including trypsin. Examination of the trypsin-induced breakdown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that as the size of the photoreversible unit changes from large to small, the size of its constituent polypeptide chains is reduced from 120,000 to 62,000 molecular weight. These experiments provide evidence that the endogenous breakdown observed in extracts is a result of contaminant protease and, consequently, that the small molecular weight species of phytochrome is an artifact due to proteolysis.     

Localization of phytochrome in oats by electron microscopy

Phytochrome was localized by immunoelectron microscopy in cells of the coleoptile tip of etiolated and irradiated oat (Avena sativa L., cv. Konata) seedlings. By using ultrathin frozen sections and immunopurified, monospecific antibodies, both the sensitivity and resolution of the immunocytochemical assay were increased. The results with etiolated plants agree with and extend previously published data. A brief red light illumination caused the redistribution of phytochrome from a diffuse to a more particulate appearance. Areas that accumulated phytochrome were identified as small vacuoles into which phytochrome was sequestered following illumination. In seedlings illuminated for several hours and in normal light-grown plants, the cellular distribution of phytochrome is qualitatively similar to that of nonirradiated, dark-grown material, except that in green plants the nucleus shows a positive immunocytochemical reaction.     

4-amino-5-hexynoic Acid-a potent inhibitor of tetrapyrrole biosynthesis in plants

4-Amino-5-hexynoic acid, a suicide inactivator of the mammalian pyridoxal phosphate-dependent 4-aminobutyric acid:2-oxoglutaric acid aminotransferase, inhibits phytochrome and chlorophyll synthesis in developing oat (Avena sativa L.), corn (Zea mays L.), pea (Pisum sativum L.), and cucumber (Cucumis sativus L.) seedlings. In Avena and Cucumis seedlings, respectively, inhibition of phytochrome and chlorophyll accumulation by 4-amino-5-hexynoic acid can be significantly reversed by application of 5-aminolevulinic acid. These results indicate that 4-amino-5-hexynoic acid inhibits the synthesis of 5-aminolevulinic acid in plants.     

Phytochrome photoreversibility: Empirical test of the hypothesis that it varies as a consequence of pigment compartmentation

Phytochrome of oat (Avena sativa L., cv. Garry) coleoptile cells in the red-light-absorbing form, Pr, is diffusely distributed while after conversion to the far-red-light-absorbing form, Pfr, it is observed only in very small areas within the cell. Comparison of phytochrome photoversibility measurements to the distribution of the pigment within the cell indicates that the spectral assay is not influenced by the observed compartmentalization of the chromoprotein. However, the observed compartmentalization of phytochrome is correlated with a loss in spectrophotometrically detectable Pr.Abbreviations Pr red-absorbing form of phytochrome - Pfr farred-absorbing form of phytochrome - R red light - FR far-red light C.I.W.-D.P.B. Publication No. 622     

Monoclonal antibodies to three separate domains on 124 kilodalton phytochrome from Avena

Forty-six monoclonal antibodies have been prepared against 124 kilodalton phytochrome from Avena sativa cv Garry. Clones grown in mice have yielded ascites fluids with antibodies which bind to three distinct regions of the molecule, as visualized by immunoblot analysis of proteolytically produced peptides of the protein. One antibody group (type 1) recognizes an antigenic domain(s) that lies within 6 kilodaltons of the amino terminus of the molecule, a region critical to correct protein-chromophore interaction. The second group (type 2) binds to an antigenic site(s) present within the chromophore-containing half of the molecule that is adjacent to the domain recognized by the type 1 antibodies. The third group (type 3) recognizes an antigenic site(s) that resides in the nonchromophoric, carboxy terminal end of the molecule between 88 and 97 kilodaltons from the amino terminus. One of the type 1 antibodies cross-reacts with apparently undegraded 120 kilodalton phytochrome from zucchini, and therefore may be useful for identifying conserved domains which are essential to the regulatory role of the photoreceptor.     

Phytochrome destruction: an apparent requirement for protein synthesis in the induction of the destruction mechanism

Examination of the phytochrome destruction reaction as a function of age in etiolated oat (Avena sativa L. cv. Garry) seedlings demonstrates that following illumination of 3-day-old shoots there is a lag, not observed in 4- or 5-day-old oats, prior to the onset of destruction. This light-mediated induction of the phytochrome destruction mechanism in 3-day-old shoots is inhibited by chloramphenicol, actinomycin D, and puromycin suggesting that protein synthesis is required. In 4-day-old shoots, actinomycin D and puromycin do not alter the kinetics of destruction while chloramphenicol partially inhibits the process. Thus, the inhibitors have a specific effect on the induction of the destruction mechanism but not its subsequent operation.     

Phytochrome Levels in Light-Grown Avena Change in Response to End-of-Day Irradiations

The effect of 15-minute end-of-day irradiations on photoreversible phytochrome levels in light-grown oat (Avena sativa L., cv Garry) seedlings was investigated. Oat seedlings were grown in a cycle of 8 hours of natural daylight and 16 hours of complete darkness, from sowing until harvest at day 10. The level of extractable, photoreversible phytochrome per unit fresh weight was 60% higher after end-of-day far-red irradiation than after either end-of-day red irradiation or end-of-day far-red followed by end-of-day red. Seedlings irradiated with end-of-day far-red also exhibited a small but significant increase in shoot height and fresh weight per seedling. Extracts of seedlings given each of these end-of-day treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted, and immunostained with monoclonal antibodies specific to different phytochromes. Regardless of end-of-day light treatment, phytochrome that is abundant in etiolated tissue was below the limit of detection, indicating that one or more of the phytochromes predominating in green tissue changes in abundance.     

Immunological and physical characterization of the products of phytochrome proteolysis

The relationship between high molecular weight (large) and low molecular weight (small) forms of phytochrome has been shown earlier to be one of proteolysis. The products of such proteolysis are characterized here by chromatography through Bio-Gel P-200 using specific antiphytochrome sera as an assay system. Degradation of large oat (Avena sativa L. cv. Garry) phytochrome as phytochrome, red-absorbing form, phytochrome, far red-absorbing form, or under cycling conditions in crude preparations containing one or more proteases, always yields one fragment with the immunochemical, electrophoretic, spectroscopic, and size characteristics of small phytochrome. In addition, other fragments are detected which may account, in part, for the different molecular weight estimates reported by others for purified, photoreversible phytochrome. The small phytochrome produced by proteolysis with trypsin of a purified large phytochrome preparation is similar to that produced by the endogenously derived protease(s). A large (estimated molecular weight = 90,000), apparently nonphotoreversible peptide is also identified which is electrophoretically and immunochemically distinct from small phytochrome. Thus, it seems that small phytochrome may not represent more than approximately one-half of the native molecule.     

Short term phytochrome control of oat coleoptile and pea epicotyl growth

Continuous recordings of the effect of light on oat (Avena sativa L. cv. Victory) coleoptile and pea (Pisum sativum L. cv. Alaska) epicotyl growth were made. Using a single excised coleoptile 10 minutes of red light was found to promote growth after a latent period of 46 minutes. The stimulation was transient and was not far red-reversible. Blue and far red light also promoted growth with similar kinetics. The action of continuous red or far red light was similar to that of 10-minute light. The growth of the intact pea third internode (as well as excised segments) was strongly inhibited by red light, with a latent period of 80 minutes. This effect was far red-reversible, and far red and blue light caused only a slight inhibition of growth.