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.     

Immunochemistry of phytochrome

Purified oat and rye phytochrome were examined by analytical gel chromatography, polyacrylamide gel electrophoresis, N-terminal, and amino acid analysis. Purified oat phytochrome had a partition coefficient on Sephadex G-200 (σ₂₀₀) of 0.350 with an estimated molecular weight of 62,000; sodium dodecyl sulfate polyacrylamide electrophoresis gave an equivalent weight estimate. Purified rye phytochrome had a σ₂₀₀ value of 0.085 with an estimated molecular weight of 375,000; sodium dodecyl sulfate electrophoresis gave a weight estimate of 120,000, indicating a multimer structure for the nondenatured protein. Comparative sodium dodecyl sulfate electrophoresis with purified phycocyanin and allophycocyanin gave a molecular weight estimate of 15,000 for allophycocyanin, and two constituent classes of subunits for phycocyanin with molecular weights of 17,000 and 15,000. Amino acid analysis of oat phytochrome confirmed a previous report; amino acid analysis of rye phytochrome differs markedly from a previous report. Oat phytochome has four detectable N-terminal residues (glutamic acid, serine, lysine, and leucine, or isoleucine); rye phytochrome has two detectable groups (aspartic and glutamic acids). Model experiments subjecting purified rye phytochrome to proteinolysis generate a product with the characteristic spectral and weight properties of oat phytochrome, as it has been described in the literature. It is concluded that the structural characteristics of purified rye phytochrome are likely those of the native protein.     

Spectral properties and chromophore rotation of phytochrome bound to substituted Sepharose

Brushite purified phytochrome from Avena sativa L. cv. Sol II was bound to phenyl Sepharose, octyl Sepharose, CNBr-activated Sepharose and to anti-phytochrome immunoglobulins immobilized on Sepharose. The spectral properties of phytochrome bound to anti-phytochrome immunoglobulins and to phenyl Sepharose were similar to phytochrome in solution. Phytochrome bound to CNBr-activated Sepharose or to octyl Sepharose showed reduced P_fr formation after red irradiation. The reversal to P_r with far-red light was only partial but a further increase at 667 nm took place slowly in the dark. A peak at 657 nm was seen in the difference spectrum between CNBr-activated Sepharose-bound phytochrome kept in darkness and the identical sample immediately after a far-red irradiation.
The change in linear dichroism at 660 nm and 730 nm, induced by plane polarized red or far-red light, was measured. It was computed that the long-wavelength transition moment of phytochrome had an average rotation angle of 31.5° or 180°–31.5°. The substrate used for immobilization had a limited effect on the rotation angle. Phytochrome immobilized on CNBr-activated Sepharose gave an angle of 27.8° and phytochrome immobilized on phenyl Sepharose gave an angle of 32.6°.     

Partial characterization of oat and rye phytochrome

Purified oat and rye phytochrome were examined by analytical gel chromatography, polyacrylamide gel electrophoresis, N-terminal, and amino acid analysis. Purified oat phytochrome had a partition coefficient on Sephadex G-200 (sigma(200)) of 0.350 with an estimated molecular weight of 62,000; sodium dodecyl sulfate polyacrylamide electrophoresis gave an equivalent weight estimate. Purified rye phytochrome had a sigma(200) value of 0.085 with an estimated molecular weight of 375,000; sodium dodecyl sulfate electrophoresis gave a weight estimate of 120,000, indicating a multimer structure for the nondenatured protein. Comparative sodium dodecyl sulfate electrophoresis with purified phycocyanin and allophycocyanin gave a molecular weight estimate of 15,000 for allophycocyanin, and two constituent classes of subunits for phycocyanin with molecular weights of 17,000 and 15,000. Amino acid analysis of oat phytochrome confirmed a previous report; amino acid analysis of rye phytochrome differs markedly from a previous report. Oat phytochome has four detectable N-terminal residues (glutamic acid, serine, lysine, and leucine, or isoleucine); rye phytochrome has two detectable groups (aspartic and glutamic acids). Model experiments subjecting purified rye phytochrome to proteinolysis generate a product with the characteristic spectral and weight properties of oat phytochrome, as it has been described in the literature. It is concluded that the structural characteristics of purified rye phytochrome are likely those of the native protein.     

Expression of functional oat phytochrome A in transgenic rice.

To investigate the biological functions of phytochromes in monocots, we generated, by electric discharge particle bombardment, transgenic rice (Oryza sativa cv Gulfmont) that constitutively expresses the oat phytochrome A apoprotein. The introduced 124-kD polypeptide bound chromophore and assembled into a red- and far-red-light-photoreversible chromoprotein with absorbance spectra indistinguishable from those of phytochrome purified from etiolated oats. Transgenic lines expressed up to 3 and 4 times more spectrophotometrically detectable phytochrome than wild-type plants in etiolated and green seedlings, respectively. Upon photo-conversion to the far-red-absorbing form of phytochrome, oat phytochrome A was degraded in etiolated seedlings with kinetics similar to those of endogenous rice phytochromes (half-life approximately 20 min). Although plants overexpressing phytochrome A were phenotypically indistinguishable from wild-type plants when grown under high-fluence white light, they were more sensitive as etiolated seedlings to light pulses that established very low phytochrome equilibria. This indicates that the introduced oat phytochrome A was biologically active. Thus, rice ectopically expressing PHY genes may offer a useful model to help understand the physiological functions of the various phytochrome isoforms in monocotyledonous plants.     

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     

Identification of photo-inactive phytochrome A in etiolated seedlings and photo-active phytochrome B in green leaves of the aurea mutant of tomato

The contents of spectrophotometrically measurable phytochrome A (PhyA) and phytochrome B (PhyB) and the corresponding immunochemically detectable apoproteins (PHYA and PHYB) were examined in dark- and light-grown tissues of the aurea mutant of tomato and its wild-type (WT). The amount of PHYA in etiolated aurea seedlings was found to be about 20% of that in the WT; this PHYA showed no photoreversible changes in absorbance, no downregulation of the level of PHYA in light-grown seedlings, and no differential proteolysis of Pr and Pfr species in vitro which was seen in the case of the WT. By contrast, the amount of PHYB in aurea seedlings was not significantly different from that in WT seedlings. Phytochrome isolated from green leaves of the aurea mutant and purified by ion-exchange chromatography showed a red/far-red reversible spectral change, and its elution profile during chromatography was essentially similar to that of PHYB. The results indicate that aurea is a mutant that is deficient in photoactive PhyA at the etiolated stage, when it contains a spectrally inactive PHYA. However, the mutant contains spectrally active PhyB in its green tissue as does the WT.     

"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.     

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.     

An enzyme-linked immunosorbent assay for phytochrome

A double-antibody sandwich, enzyme-linked immunosorbent assay has been developed for phytochrome in Avena sativa L. cv. Saladin. An immunoglobulin fraction of rabbit antiserum raised to 118 kdalton phytochrome was used with alkaline phosphatase as the enzyme label. The assay detected as little as 0.2 ng phytochrome in extracts of dark-grown plant material. No evidence for specific or non-specific measurement of proteins other than phytochrome was found. The assay detected phytochrome in extracts of Avena grown in the light. Dilution curves for light-grown phytochrome extracts had a reduced slope and saturated at a lower level of enzyme activity than those for dark extracts. These differences were not caused by an inhibitor in extracts from light-grown plants. Phytochromes from dark- and light-grown plants may be immunologically different.     

Chromophore structure in phytochrome intermediates and bleached forms of phytochrome

Phytochrome (120 kdalton or 60 kdalton) was isolated from etiolated seedlings of Avena sativa L. cv. Pirol (Baywa München). Irradiation with red light of the P_r form at −23°C in aqueous medium or at −40°C in 66% glycerol leads to the intermediate meta-Rb. Acidification of the glycerol solution at −40°C leads to the absorption of the 15(E) phytochrome chromophore (= P_fr chromophore). Subsequent irradiation transforms this into the 15(Z) chromophore (= P_r chromophore). The presence of the 15(E) chromophore was demonstrated by the same methods also in phytochrome bleached either as P_fr in the dark by 4 M urea, methanol, acetone, ethylene glycol, 8-anilinonaphthalene-1-sulfonate, or as P_r by irradiation with red light in the presence of the same agents. Phytochrome bleached by sodium dodecylsulfate or by dehydration was also investigated. It was concluded that bleached phytochrome contains the P_fr chromophore without specific interaction with the protein.     

Circular dichroism of phytochrome intermediates

Phototransformation P_t to P_fr was investigated with 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa L. cv. Pirol) using circular dichroism spectroscopy at -110°C to +30°C. Using absorption spectra of the intermediates formed at the respective temperatures, circular dichroism spectra (300–800 nm) of pure intermediates were calculated.
The sign of the circular dichroic absorption bands changed upon formation of lumi-R, the primary photoproduct of P_r. This would be compatible with a Z→E isomerization taking place at this reaction step. The subsequent intermediates (meta-R_a and meta-R_c) as well as P_fr showed only small circular dichroism. Their absorption spectra were drastically shifted, but had similar spectral shapes. The results are discussed in terms of conformational changes of the phytochrome chromophore presumably taking place at the early steps of phototransformation P_r to P_fr.     

Purification and characterisation of gelonin from seeds of Gelonium multiflorum

Gelonin, a ribosome-inactivating protein has been isolated from the seeds of Gelonium multifluorum of Euphorbiaceae family by two methods and the results are compared. In method-I conventional aqueous extraction, cation-exchange and gel-filtration chromatography has been used. In method-II S-Sepharose fast flow gel has been used to purify the proteins from the seed extract, followed by ammonium sulfate fractionation, cation-exchange and gel-filtration chromatography. Extensive physico-chemical and immunological characterizations show that molecular weight of gelonin as determined by gel-filtration chromatography and SDS-PAGE is approximately 30 kDa. The non-proteinous material which binds to CMC-gel in association with gelonin in method-I is substantially removed when gelonin is purified by method-II. Cation exchange, G-100 chromatography, RP-HPLC and SDS-PAGE show that method-II yields 50% more purified gelonin when compared to the yield by method-I. The immunoreactivity of gelonin obtained by methods I and II vary from 22-26% and 50-66% respectively and the ribosome-inactivating property vary from 46-56% and 70-87% respectively.     

In vitro phytochrome dark reversion process

Thermal reversion of the far-red absorbing form of phytochrome to the red absorbing form in darkness has been investigated in crude and partially purified isolates from a number of etiolated and light grown higher plants. The influence of temperature, aging and urea on the rate of reversion was also determined.

Phytochrome isolated from all higher plants underwent reversion. The reversion proceeded in at least 2 distinct stages; a short rapid initial phase being followed a slow phase which continued for many hours. Reversion rate was highest in phytochrome isolated from green leaves of parsnip (Pastinacea sativa) and lowest in that isolated from etiolated oats (Avena sativa). Although the rate of reversion could be changed by modifying the tertiary structure of the protein component, the large differences in rate appeared to be characteristic of the plant source. Observed in vitro rates of reversion are slower than those occurring in vivo. Removal of other buffer solubilized material during purification had little effect on the rate of reversion of phytochrome isolated from etiolated material.

     

Low temperature spectroscopy of phytochrome: Pr, Pfr and intermediates

Phytochrome (120 kdalton) was isolated from etiolated seedlings of Avena sativa L. cv. Pirol (Baywa, München). Low temperature spectra between −17°C and −160°C are recorded for P_r, P_fr, and irradiated phytochrome samples. The temperature-dependence of the P_r and P_fr absorption spectra is described. Difference spectra of such temperature effects can erroneously be interpreted as difference spectra of intermediates. Probable absorption spectra of intermediates are calculated from the spectra of irradiated P_r or P_fr, respectively. The calculated spectral data are compared with published data on phytochrome intermediates.     

Purification and spectroscopic properties of 124-kDa oat phytochrome

A simplified procedure for the isolation and purification of 124-kDa phytochrome from etiolated Avena seedlings has been developed using the method of ammonium sulfate back-extraction. After hydroxyapatite chromatography of seedling tissue extracts, the pooled phytochrome was subjected to ammonium sulfate back-extraction instead of the usual application to an Affi-Gel Blue column. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280) ranging from 0.85 to 0.95. Subsequent Bio-Gel filtration chromatography yielded highly pure 124-kDa phytochrome with SAR values ranging from 0.99 to 1.13. The absorption maxima of 124-kDa phytochrome were at 280, 379, and 666 nm for the red absorbing form of phytochrome (Pr) and at 280, 400 and 730 nm for the far-red absorbing form (Pfr). The A730/A673 ratio in Pfr was found to be 1.5 to 1.6. The mole fraction of Pfr under red light photoequilibrium was 0.88. No dark reversion was detected within 5 h at 3 degrees C. A photoreversible far-uv-circular dichroism was observable with all phytochrome preparations examined. Fluorescence and phosphorescence lifetimes were measured to further characterize the differences between the phytochromes prepared under different conditions. The Trp fluorescence and phosphorescence lifetimes of Pr and Pfr with the chromophore "X", probably polyphenolic in nature, were significantly shorter than those of phytochrome without the contaminant X. The short lifetime of the fluorescence of the Pr chromophore is attributable to X in the former.     

In vitro cross-linking of extensin precursors by mustard extracellular isoforms of peroxidase that respond either to phytochrome or to wounding

The activities of extracellular peroxidase isoforms A3 and A4 from mustard (Sinapis alba) are known to respond to wounding treatments and to phytochrome status, respectively. To investigate the affinity of A3 for extensin precursors in vitro, these acidic isoforms were extracted by low-speed centrifugation of intact mustard internodes infiltrated with CaCl2 and isolated by chromatofocusing. Extensin precursors from carrot (Daucus carota) roots or mustard stems and leaves were isolated by saline extraction followed by purification on carboxymethyl-cellulose ion exchange and gel-filtration chromatography. Cross-linking of extensin precursors in vitro in the presence of peroxidase isoforms and exogenous H2 O2, was quantified following Sephacryl S-400 gel-filtration, as a shift of extensins to higher molecular mass values. Isoforms A3 A4 had similar affinities for natural extensin precursors. A cationic isoform (previously not characterized) was unable to cross-link extensin precursors. Tissue prints of mustard stems indicate that extensin precursors are present in the cell wall of all the tissues, with maximum staining in vascular bundles and epidermis. Isoforms A3 and A4 were detected in extracts from vascular bundles, cortex and pith. Only A3 was detected in epidermal extracts. The observations are consistent with a role of isoforms A3 and A4 in cross-linking of extensin in muro.Keywords: Peroxidase isoforms, mustard, extensin, carrot, cell wall proteins.      

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.     

Purification of 120-Kilodalton Phytochrome from Oryza Sativa L

The procedures of Grimm and Rüdiger for the purification of 120 kDa phytochrome from oat seedlings were modified to isolate native phytochrome from etiolated rice (Oryza sativa L. subsp, japonica var. nongken 58) seedlings. Approximately l kg of 6d old seedlings (the first 2 days at 33℃, the last 4 days at 27 ℃ in darkness) were frozen in liquid nitrogen and then homogenized in a modified Waring blendor with an extraction buffer, at final pH 8.45 (4 ℃). After polyethylenimine precipitation, phytochrome in extract was converted to Pfr by irradiation of the resulting supernatant for 10 min with red light. The step of ammonium sulfate precipitation was followed by resuspending of resultant pellet in buffer B with the ratio of 10 ml per phytochrome unit. The pellet precipitated with ammonium sulfate at 42% saturation from combined phytochrome cont ning fractions after hydroxyapatite chromatography was washed with 10 mmol/l phosphate buffer in 0.8 ml instead of 0.65 ml per phytochrome unit. Then it was washed successively with 200 mmol/l and 100 mmol/1 phosphate buffer (0.85 ml per phytochrome unit). Native phytochrome (120 kDa) in 12% yield was dissolved in 2 mmol/l EHPES buffer (2.2 ml per phytochrome unit, pH 7.8, containing 5 mmol/l EDTA and 14 mmol/l 2-mercaptoethanol) was proved to be pure in SDS- polyacrylamide electrophoresis and showed typical absorption spectrum as that of native oat phytochrome.