Binding of 124-kilodalton oat phytochrome to liposomes and chloroplasts

Binding of the radio-iodinated 124-kDa oat ( Avena sativa L. cv. Garry) phytochrome to liposomes and chloroplasts was investigated as a model system in order to understand the molecular affinity of phytochrome toward cellular organelles in plants. The binding of intact (124 kDa) phytochrome to liposomes and chloroplasts is hydrophobic in nature, as in the case of the degraded (118/114 kDa) phytochrome, but electrostatic interactions play a greater role in the intact phytochrome. The physiologically active P_fr form of the intact phytochrome showed a binding preference over the inactive P_r form with neutral liposomes and chloroplasts. However, the P_fr form of intact phytochrome exhibits smaller binding preference than the P_fr form of degraded phytochrome over their respective P_r forms (see Kim, I.-S. and Song, P.-S. 1981, Biochemistry 20: 5482–5489, for degraded phytochrome binding). These results indicate that the 6/10 kDa N-terminus segment, which is lost in the degraded phytochrome, plays an important role in determining the protein surface properties of the intact phytochrome. A competitive binding study on phytochrome also suggested that the P_fr form had a greater binding affinity for chloroplasts than the P_r form. However, the physiological activity of the P_fr form may not be explained simply by the observed difference in binding affinity between the two forms of phytochrome.     

Photoreversible association of phytochrome with membranes. I. Distinguishing between two light-induced binding responses

Abstract Two types of association between phytochrome and crude membrane fractions from oat (Avena sativa L.) are distinguished and compared, and that which comprises only a small fraction of the total phytochrome in extracts prepared in the absence of added divalent cations (Watson & Smith. 1982b) has been studied in detail. Extraction in the presence of phenylmethylsulphonyl fluoride shows that proteolysis of P_r (the red-light absorbing form) probably does not account for the lower levels of membrane-associated phytochrome measured after far-red light than after red light. Difference spectra of soluble and membrane-associated phytochrome indicate that the latter is much less susceptible to spectral degradation in vitro than is the soluble pool. The stoichiometry of association with the membranes is such that for each phytochrome molecule associated after far-red light there are three associated after red light and it is argued that this stoichiometry is maintained independent of the extraction pH. The characteristics of this photo-reversible association of phytochrome with membranes are compared to the characteristics of the widely studied light-induced enhancement of phytochrome pelletabilily that is dependent on electrostatic interaction of phytochrome and membranes.     

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.     

Phototransformation of oat phytochrome with millisecond and submillisecond flashes: The level of the photostationary Pfr/Ptot ratio is modified by photoreversible intermediates

Photoconversion of the red-absorbing form of phytochrome (P_r) to the far-red-absorbing form of phytochrome (P_fr) and vice versa has been measured spectrophotometrically at 10°C in immobilized and soluble phytochrome (118 kdalton), prepared from 5-day-old etiolated oat seedlings ( Avena saliva L. cv. Sol II). The photostationary equilibrium φ= P_frP_tot (with P_tot= total amount of phytochrome P_r+ P_fr) for red light depends on whether it is established by repetitive pulses (≥ 5 s) or by repetitive flashes (≥ 4 ms). In the wavelength region around 660 nm, a lower φ is reached with flashes as compared to that with pulses. This difference becomes negligible if the wavelength is shortened to the 600 nm region, and it also disappears if the fluence of each individual flash is reduced. In contrast, in long-wavelength red light and short-wavelength far-red light, a higher φ is reached with flashes than with pulses.
We relate the differences in φ for flash and pulse irradiation to photochromic systems between P_r and photoreversible intermediates in the phototransformation pathway P_r→ P_fr. Thus, light absorption by phytochrome intermediates can be limiting for the quantitative relationship between light signal and P_fr formed.     

After-ripening and phytochrome action in seeds of dormant lines of wild oat (Avena fatua)

The effects of a short exposure to red, far-red or alternate red/far-red light on the germination of seeds after-ripened for different periods of time were studied in dormant lines of wild oat ( Avena fatua L.). Three stages were distinguishable in the after-ripening period in the response of germination to light. Seeds stayed dormant and showed no response to light during stage I. Phytochrome-mediated germination was observed in seeds during stage II. The phytochrome action disappeared during stage III, i.e. seeds fully germinated following treatments of all light qualities. When the seeds were imbibed in polyethylene glycol solutions, dark germination was reduced and phytochrome again had an effect, which suggested the involvement of phytochrome in water uptake of the seed.     

Regulation of phytochrome mRNA abundance in green oat leaves

Abstract. Avena sativa L. (oat) seedings were grown 4 d in continuous white light followed by 3 d in darkness. Probes derived from an oat phytochrome cDNA clone (pAP 3.2) were used in slot blot analyses to measure the abundance of phytochrome mRNA in the distinct etiolated and green portions of the leaves produced by these seedlings. Both the green and etiolated portions accumulated phytochrome mRNA to a level of about 85% of the etiolated seedling level. Subsequent experiments with similar seedlings showed that both the green and etiolated portions were capable of inducing a dramatic decline in phytochrome mRNA abundance in response to a saturating red light pulse. Despite the ability of green portions of oat leaves to accumulate phytochrome mRNA and to down-regulate phytochrome mRNA abundance in response to light, no substantial variation in phytochrome mRNA abundance was observed in green oat seedlings maintained on a 12-h day/12-h night cycle. In the same oat seedlings, the abundance of chlorophyll a/b binding protein mRNA fluctuated dramatically during the day/night cycle.     

Protein rotation and chromophore orientation in reconstituted bacteriorhodopsin vesicles

Bacteriorhodopsin has been reconstituted into lipid vesicles with dipalmitoyl and dimyristoyls phosphatidylcholine. Circular dichroism (CD) measurements show that the proteins are in a monomeric state above the main lipid phase transition temperature (T_c), 41 and 23°C for dipalmitoyl and dimyristoyl phosphatidylcholine, respectively. Below T_c, the CD spectrum is the same as that found for the purple membrane. The latter result implies that the orientation of the chromophore at these temperatures is most likely the same as in the purple membrane ($\text{70° ± 5°}$ from the normal to the membrane plane).Transient dichroism measurements show that below T_c the proteins are immobile, while above this temperature protein rotation around an axis normal to the plane of the membrane is occurring. In addition, from the data the angle of the chromophore for the rotating proteins with respect to the rotational diffusion axis can be calculated. This angle is found to be $\text{30° ± 3°}$ and $\text{29° ± 4°}$ in dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, respectively. This is considerably smaller than the value of $\text{70° ± 5°}$ for the natural biomembrane. A reversible reorientation of the chromophore above and below the respective main T_c transition temperature could explain the change of angle observed provided that all the molecules rotate above T_c.     

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.     

Photoregulation of the Endogenous cGMP Content in Oat Seedlings

The concentration of cGMP in the tissues of oat (Avena sativaL.) seedlings was shown to depend on seedling age and the light regime of their growth. The level of cGMP in the etiolated seedlings was lower than in the green ones and declined with seedling age. Red and blue light recognized by phytochrome and cryptochrome, respectively, affected the cGMP content. The effectors of cGMP metabolism, guanylin, protoporphyrin IX, and zaprinast, elevated the cGMP content in tissue extracts from oat seedlings.     

Adsorption of phytochrome on several substituted sepharose gels

Phytochrome from Avena sativa shows strong adsorption with hydrophobic ligands such as octyl and phenyl Sepharose. The same behaviour was observed for undegraded (MW 400 000) and degraded (MW 60 000) phytochromes in the P_r, or P_fr, form as well. The pigment is photoreversible after adsorption on those gels. Chromatography with amino acid ligands of degraded phytochrome was also tested. The chromoprotein showed the same strong adsorption on tryptophyl Sepharose. A more specific adsorption could be achieved on histidyl Sepharose but with loss of 70% of photoreversibility. This can be interpreted by the accessibility and perturbation of the chromophoric site by the histidyl ligands     

Light-Dependence of Phospholipase D Activity in Oat Seedlings

The effect of light on the activity of phospholipase D (PLD) in oat (Avena sativa L.) seedlings and the dependence of this enzyme activity on the regime of their illumination were studied. The PLD activity in etiolated seedlings was 1.5–2.0-fold higher than in green plants. The illumination of etiolated seedlings with white light resulted in a decrease in PLD activity to its level in the seedlings grown under light. In contrast, the transfer of green seedlings to darkness enhanced the activity of the enzyme up to its level in etiolated seedlings. The illumination of etiolated seedlings with red light inhibited the PLD as well. It was shown that this photoeffect decreased with seedling aging and correlated with a phytochrome content in plants. Far-red light reversed the effect of red light. The involvement of phytochrome in the control of the PLD activity is discussed.     

Photoreversible association of phytochrome with membranes. II. Reciprocity tests and a model for the binding reaction

Abstract A series of fluence-response curves for the binding of phytochrome to membranes in the absence of divalent cations, as described by Watson & Smith (1982), were constructed to demonstrate that the response obeys the law of reciprocity. Analysis of the binding of P_fr (the far-red-absorbing form of phytochrome) showed that two P_fr molecules bind to the membrane for each P_r (the form with an absorption maximum in the red) photoconverted to P_fr in the intrinsic membrane-bound phytochrome pool. Using this stoichiometry we have been able to model the binding curve of Pr and match the binding data. P_r binding can be simulated if P_r binds only as a consequence of the binding of P_fr, i.e. when P_fr is part of a P_r: P_fr dimer. The enrichment of the membranes with P_fr as a result of the binding of P_fr was also accurately simulated. There is no binding cooperativity. Phytochrome binding is a low-fluence response and the possibility that it has physiological significance as a mediator of phytochrome action is discussed.     

Carbon dioxide-independent and -dependent components of light inhibition of the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in oat leaves

Light inhibits while carbon dioxide enhances the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene in oat ( Avena sativa L. cv. Victory) leaf segments. The possibility that the light inhibition is mediated through changes of carbon dioxide has been investigated. The level of CO₂ increases or decreases in the sealed incubation vial in darkness or in light, respectively, which can apparently account for the differences in ACC-dependent ethylene production between the dark and light treatments. However, although the evolution of ethylene from ACC in the dark is reduced upon depletion of CO₂, the difference between light and dark is still very noticeable. Moreover, the production of the ethylene in CO₂-free air in the dark was still higher than in the light, where the concentration of CO₂ was 0.01%. It is proposed that the light effect on the conversion of ACC to ethylene is composed of two distinguishable components: one CO₂-mediated and the other CO₂-independent.     

Intramolecular uncoupling of chromophore photoconversion from structural signaling determinants drive mutant phytochrome B photoreceptor to far-red light perception

The phytochrome (phy) photoreceptor family regulates almost all aspects of plant development in a broad range of light environments including seed germination, onset of the photomorphogenic program in seedling stage, the shade avoidance syndrome in competing plant communities, flowering induction and senescence of adult plants. During evolution two clearly distinct classes of phy-s emerged covering these very different physiological tasks.¹ PhyA is rapidly degraded in its activated state. PhyA functions in controlling seed germination at very low light intensities (very low fluence response, VLFR) and seedling establishment under photosynthetic shade conditions (high irradiance response, HIR) where the far-red portion of the transmitted light to understorey habitats is substantially enhanced. Arabidopsis phyB together with phyC, D and E belongs to the relatively stable sensor class in comparison to the light labile phyA. PhyB functions at all stages of development including seed germination and seedling establishment, mediates classical red/far-red reversible low fluence responses (LFR) as well as red light high irradiance responses, and it is considered to be the dominating phytochrome sensor of its class.     

Solid-phase iodination: in vitro labeling with 125I of proteins bound to nitrocellulose and substituted Sepharose

A solid-phase iodination method is described which employs either nitrocellulose paper, phenyl- and octyl-Sepharose beads, or octyl hydroxylapatite as matrices to adsorb proteins. Nitrocellulose lends itself to cases where denaturation of the iodinated proteins due to the use of chaotropic reagents or strong acids for protein elution can be tolerated. On the other hand, substituted Sepharoses, preferably octyl-Sepharose, should be used when preservation of the biological activity of the iodinated protein molecules is required; immunoglobulins and protein A, for instance, could be recovered as functionally active molecules because they were extracted from the hydrophobic matrices under nondenaturing conditions. Both methods are advantageous if, for example, series of fractions from column chromatographies (including HPLC) are to be iodinated and subsequently analyzed by gel electrophoresis or bioassays. Furthermore, the amount of radioactive waste can be reduced considerably.     

Spectroscopic properties and chromophore conformations of the photomorphogenic receptor: phytochrome.

Fluorescence lifetimes of 'large (mol. wt. 120,000) and 'small' (mol. wt. 60,000) phytochromes isolated from oat and rye seedlings grown in the dark have been measured at 199 K and 298 K. Phytochrome model compounds have also been studied by phase modulation fluorometrically at 77 K for comparison with lifetime data for phytochrome. It was found that the fluorescence lifetime of 'large' phytochrome was significantly shorter than that of 'small' phytochrome and its chromophore models. The phytochrome chromophore of Pr form has been analyzed by fluorescence polarization, CD, and molecular orbital methods. The fluorescence excitation polarization of 'small' phytochrome and the chromophore model in buffer/glycerol mixture (3 : 1, v/v) at 77 K is very hight (0.4) at the main absorption band and is negative (--0.1) and close to 0 in the near ultraviolet band, respectively. Analyses of the spectroscopic data suggest that the chromophore conformation of Pr and Pfr forms of phytochrome are essentially identical. The induced ellipticity of 'large' rye phytochrome in the blue and near ultraviolet regions was found to be significantly higher than that of 'small' phytochrome, indicating that the binding interaction between the phytochrome chromophore and apoprotein is much tighter in the former than in the latter. In addition, the excitation energy transfer does occur from Trp residue(s) to the chromophore in 'large' phytochrome but not in 'small' Pr. This illustrates one feature of the role played by the large molecular weight apoprotein in the binding site interactions and primary photoprocesses of Pr. Finally, a plausible model for the primary photoprocesses and the mechanism of phytochrome interactions triggered by the Pr leads to Pfr phototransformation have been proposed on the basis of the above results.     

Circumnutation of rice coleoptiles: its occurrence, regulation by phytochrome, and relationship with gravitropism

It has been found that coleoptiles of dark-grown rice (Oryza sativa L.) seedlings undergo regular circumnutation in circular orbits with periods of about 180 min. Both clockwise and counter-clockwise movements were observed, but individual coleoptiles continued to rotate only in one direction. Light-grown seedlings did not show circumnutation. In fact, dark-grown seedlings were found to cease circumnutating in response to a pulse of red light (R). This light-induced inhibition of circumnutation was demonstrated to involve both a FR-inducible very-low-fluence response, solely mediated by phytochrome A, and a FR-reversible low-fluence response, mediated by phytochrome B and/or C. The R-induced inhibition of circumnutation showed temporal agreement with the R-induced inhibition of coleoptile growth, suggesting that the former results from the latter. However, about 25% of growth activity remained after R treatment, indicating that circumnutation is more specifically regulated by phytochrome. The R-treated coleoptile showed gravitropism. Investigation of the growth differential for gravitropic curvature revealed that gravitropic responsiveness was rather enhanced by R. The results suggested that gravitropism is not a cause of circumnutation. It remained probable, however, that gravity perception is a part of the mechanism of circumnutation. It is speculated that the circumnutation investigated aids the seedling shoot in growing through the soil.     

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