INTERACTION BETWEEN PHYTOCHROME AND THE BLUE LIGHT PHOTORECEPTOR SYSTEM IN Mougeotia*

Abstract— Face-to-profile chloroplast movement in Mougeotia was induced by sequences of strong blue and red short irradiations. This type of response occured only when blue light was applied prior to or simultaneously with red light, and far-red irradiation was necessary after the sequence to cancel the remaining gradient of the far-red absorbing form of phytochrome P_fr. The dependence of the response magnitude on blue and red light sequences was studied for a wide range of light durations and dark intervals. The relationship between the response and the dark interval points to the lack of direct coupling between phytochrome and blue-absorbing “cryptochrome”. It was postulated that a photoproduct having a life-time of2–3 min is formed by the blue-light-mediated reaction. This photoproduct interacts with phytochrome during its transformation or with its final P_fr form.     

IRRADIATION-ENHANCED PHYTOCHROME PELLETABILITY IN AVENA: IN VIVO DEVELOPMENT OF A POTENTIAL TO PELLET AND THE ROLE OF Mg2+ IN ITS EXPRESSION IN VITRO

The enhanced phytochrome pelletability that results from in vivo irradiation of Avena shoots may be divided into two operationally defined sequential stages: the in vivo development of a “potential to pellet” and the “expression” of this potential in vitro. Kinetic studies confirm previous findings that the generation of this “potential to pellet” is a very rapid (complete in < 10 s, 25°C), genuinely intracellular process, itself photoreversibly induced by P_fr. In addition, it is shown that the sustained development of the “potential to pellet”, that proceeds in the dark at 0°C following a red pulse, requires P_fr continually in the cell over the entire development period. Far red light immediately terminates further development of the red-induced “potential” at any point during the development phase. No immediate reduction is observed, however, in that level of “potential pelletability” already attained at the time of the far red pulse. This indicates that the level of “potential pelletability” established in vivo is insensitive to the form of the pigment at extraction regardless of the level reached. “Expression” of the “potential to pellet” refers to the actual detection in homogenates of an enhanced physical association of phytochrome with pelletable material. Maximum “expression” requires the presence of a divalent cation in the medium during homogenization. Rapid posthomogenization addition of Mg²⁺ to Mg²⁺-free extracts sustains enhanced pelletability but with rapidly declining effectiveness over the fmt 1–2 min after extraction. The rate of decline is faster if the phytochrome is present as P_fr than as P_r in the homogenate. Neither these nor previous data permit a distinction to be made between (a) preservation by the cation of a pre-existing intracellular interaction, and (b) a Mg²⁺-mediated induction of an artifactual, in vitro association predetermined in the cell by a genuine phyto-chrome-controlled process. Various formalistic models are discussed in the context of these and other data.     

Stabilization of phytochrome intermediates by low temperature

Abstract— The photocon versions between the red-absorbing form (P_r) and the far-red absorbing form (P_fr) of phytochrome were examined at low temperatures. Partially purified preparations of the chromoprotein were examined in phosphate buffer and in 25 per cent buffer plus 75 per cent glycerol. Actinic irradiation of P, below – 150°C produces an intermediate with maximum absorbance near 695 nm, R₆₉₅. Actinic irradiation of R₆₉₅ converts it back to P. Above – 150°C R₆₉₅ decays to a low extinction form of phytochrome, R, which in turn decays to P_fr upon further warming. Light absorption by P_fr below – 150°C results in the formation of an intermediate form of phytochrome with maximum absorbance near 660 nm, FR₆₆₀. FR₆₆₀ decays upon warming to a lower extinction form, FR'. which in turn decays to P_r on continued warming. No evidence was obtained to suggest that any of the observed intermediate states are involved in more than one direction of phytochrome photocon version.     

PHYTOCHROME CONTROL OF ITS OWN SYNTHESIS IN Sorghum vulgare AND Avena sativa

The accumulation of phytochrome in the dark was measured for Avena sativa seedlings after a white light pretreatment and for Sorghum vulgare seedlings after continuous red or far-red light treatments, using the herbicide Norflurazon to prevent greening under continuous irradiation. In both cases the accumulation of phytochrome depends on the state of the phytochrome at the light-dark transition: high P_fr levels (red light pulse) led to a slower rate of phytochrome accumulation than lower P_fr levels (long wavelength far-red (RG 9) light pulse). Poly-(A⁺)-RNA was isolated fromA. sativa seedlings grown for 48 h in darkness + 24 h WL + light pulse (5 min) (red, RG 9 light, red followed by RG 9 light or RG 9 followed by red light pulse) + 19 h darkness. The poly-(A⁺)-RNA was translated in a rabbit reticulocyte lysate system and the translation products were immunoprecipitated by specific anti-phytochrome antibodies. It was demonstrated that the activity of mRNA coding for phytochrome was under phytochrome control.     

Phytochrome-dependent photomovement responses mediated by phototropin family proteins in cryptogam plants

In this review, we describe the regulation of photomovement responses by phototropin and phytochrome photoreceptors. The blue light receptor phototropin mediates various photomovement responses such as phototropism, chloroplast movement and stomatal opening. In cryptogamic plants including ferns, mosses and green alga, red as well as blue light mediates phototropism and chloroplast movement. The red/far-red light reversibility suggests the involvement of phytochrome in these responses. Thereby, plant growth is presumably promoted by coordinating these photomovements to capture efficiently light for photosynthesis.     

Comparison of the protein conformations between different forms (Pr and Pfr) of native (124 kDa) and degraded (118/114 kDa) phytochromes from Avena sativa

Abstract— Circular dichroic properties of native, 124 kDa phytochrome from etiolated Avena sativa seedlings have been examined and compared with those of degraded phytochrome (118/114 kDa). The CD spectrum of the P_r form of 124 kDa phytochrome does not differ significantly in the visible region from that of 118/114 kDa P_r. In contrast, the CD spectrum of the P_fr form of 124 kDa phytochrome differs from that of the 118/114 kDa species in the far-red, red and blue regions of the spectrum. This result confirms that the NH₂-terminal polypeptide segment has a critical role in chromophore-protein interaction in the P_fr but not in the P_r form. In the UV region, 124 kDa phytochrome exhibits a photoreversible difference between the CD spectra of P_r and P_fr, whereas no such difference is observed for 118/114 kDa preparations. These data suggest a possible photoreversible change in secondary structure of the 124 kDa phytochrome polypeptide that requires the presence of the 6/10 kDa NH₂-terminal domain to occur.     

PHYTOCHROME PHOTOCONVERSION in vivo. EFFECT OF THE INITIAL Pfr/Ptot RATIO

The equation for phytochrome photoconversion, derived from photoconversion kinetics of purified phytochrome, predicts that the rates of photoconversions starting from low and high Pfr/Ptot₍₀₎, (initial Pfr/Ptot) should be the same for light of the same quality and fluence rate. The situation might be different in vivo. Phytochrome photoconversion rates were measured in excised cotyledons of Cucurbita pepo L. exposed to BL (blue; ?_BL= 0.39; ?, Pfr/Ptot at photoequilibrium) and RI (mixture of red and far red; ?_RI= 0.46) after saturating preirradiations with red and far-red to establish high (0.78) and low (0.02) Pfr/Ptot₍₀₎, respectively. Under BL, the rate of photoconversion is faster when starting from a high than a low Pfr/Ptot₍₀₎; under RI, the rate of photoconversion is faster when starting from a low than a high Pfr/Ptot₍₀₎., No effects of Pfr/Ptot₍₀₎, on photoconversion rates were found in phytochrome solutions exposed to BL and RI. These data provide another indication of the discrepancies between phytochrome photconversion kinetics in vivo and in vitro.     

Phytochrome structure: A new methodological approach

Higher plants use the protein phytochrome as a photosensor. In physiological temperatures phytochrome exists in two forms: Pr and Pfr. The chromophore of phytochrome is an open-chain tetrapyrrole. On the pathway from Pr to Pfr four intermediates (Lumi-R, Meta-Ra, Meta-Rb, and Meta-Rc) can be distinguished, while only two (Lumi-F and Meta-F) can be seen on the way back from Pfr to Pr. We have used the x-ray structure of the C-Phycocyanin protein Fremyella diplosiphon bacteria as a template to build a model (∼200 atoms) that includes only the chromophore and five amino acids of the phytochrome (Arg316–Cys321–His322–Leu323–Gln324) around it. Using the existing experimental evidences, we have proposed a three-dimensional (3D) structure for Pr, Pfr, and intermediates and a mechanism for the photoisomerization as well. Structures were fully optimized using AM1 (Unichem package on a Cray J90-NACAD). Using the INDO/S method of Zerner and co-workers, we calculated the absorption spectra of the model compounds and compared them with the experimental data. The oscillator strength ratio is an indicator of the chomophore conformation in biliproteins. The calculated spectra reproduces well the spectra of the phytochrome (Pr, Pfr, and intermediates) except for the lower energy band. This result is attributed to the small number of amino acids in the models. The calculated ratios (f_VIS/f_UV−f_osc of visible band over f_osc of UV band and f₂/f₁−f_osc of second absorption band over f_osc of first absorption band) for the models match very well the experimental ratios obtained for the phytochrome (Pr, Pfr, and intermediates). This supports the proposed mechanism for the photoisomerization process. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 70: 1145–1157, 1998     

IN VITRO SYNTHESIS OF PHYTOCHROME APOPROTEIN DIRECTED BY mRNA FROM LIGHT AND DARK GROWN AVENA SEEDLINGS*

Abstract— The aim of this work was to compare the translatability of poly-A-RNA from light- and dark-grown Arena seedlings, the product of which should be phytochrome. Polysomal poly-A-RNA was isolated from 4-day-old dark-grown Avena seedlings (Avena sativa L. cv. Garry) and translated in a rabbit reticulocyte lysate system. Immunoprecipitation by anti-phytochrome serum was used for measurements of specific phytochrome translation. Characterization of the translated and immunoprectpitated protein was performed by comparison with [₃₅S]-methionine in vivo-labelled phytochrome. The specificity of the precipitation was shown by parallel use of non-specific serum and by competitive inhibition of precipitation by exogenous unlabelled phytochrome isolated by affinity chromatography.     

THE MODE OF INTERACTION BETWEEN BLUE (UV) LIGHT PHOTORECEPTOR AND PHYTOCHROME IN ANTHOCYANIN FORMATION OF THE SORGHUM SEEDLING

Two non-photosynthetic photoreceptors (phytochrome and a blue light photoreceptor) are involved in light-mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings. The present study was undertaken to investigate the kind of interaction between phytochrome and the blue light photoreceptor. The data show that phytochrome (P_fr) can only act once a blue light effect has occurred. On the other hand, the blue light effect cannot express itself without P_fr. It is concluded that there is an obligatory dependency (or sequential interaction) between the blue light effect and the light effect occurring through phytochrome, although the blue light photoreaction per se is not affected by the presence or absence of phytochrome. The latter statement is based on the results of dichromatic experiments, i.e. simultaneous, high fluence rate irradiation with two kinds of light. Blue light can be replaced by UV light. It is not clarified yet whether the effect of blue and UV light is due to the same photoreceptor.     

SPECTRAL PROPERTIES,PHOTOTRANSFORMATION KINETICS AND PELLETABILITY OF PHYTOCHROME IN Pharbitis nil Chois*

Abstract— An examination has been made of the involvement of phytochrome in the circadian rhythm of flowering in Pharbitis nil Chois. The peak position of P_fr absorption changes with time after a red light pulse. The shortest absorption wavelength of P_fr occurs at the same time as flowering is inhibited by red light in dark grown, red light pretreated plants. Pelletable and supernatant phytochrome show a similar trend with lowest values found at the time of flower inhibition. Neither phototransformation kinetics nor intermediates of phytochrome which accumulate in white light show such a relationship to the circadian rhythm found in flowering of dark grown P. nil.     

ACTION SPECTRA OF PHYTOCHROME IN VIVO*

Abstract— A method is described to determine spectral properties of phytochrome in vivo. For photochrome in 7-day-old dark-grown Cucurbita pepo L. seedlings the mole fraction of the far-red-absorbing form (P_fr) present at photoequilibrium at 664 nm was found to be 0.76 ± 0.02 in vivo. Based on reflectance measurements, the photon fluence rate just below the surface of the cotyledons was calculated. Local rates of photoconversion for known local fluence rates were measured across cotyledons after non-saturating irradiations with wavelengths between 544 and 781 nm and in situ molar photoconversion coefficients were obtained. In contrast to purified oat phytochrome, the in situ molar photoconversion coefficients for P_fr show a strong shoulder between 660 and 700 nm. The maximum of P_fr absorption is at 726 nm. An isosbestic point of phytochrome is found at 686 nm. The mole fraction of P_fr present at photoequilibrium with 686 nm light is 0.58. The ratio of photoconversion quantum yields (that for P_r→ P_fr divided by that for P_fr→ P_r) is 1.38 ± 0.06.     

Results of studies of potential light spectrum effects on human performance

Abstract— Sensitization of the phytochrome-mediated germination at 20°C of lettuce seeds (Lactuca sativa L. cv. Grand Rapids) by pretreatment at 4°C, 28°C, or on 1% ethanol, was studied. The 660 nm fiuence-response characteristics were similarly biphasic for all sensitizing treatments and displayed responses at very low fluences (VLFR) as well as responses characteristic of non-sensitized seeds at 10000-fold higher, low fluences (LFR). Maximum VLFR increased with the duration of sensitizing treatments. However, the fluence ranges required for the two types of responses remained relatively constant. These and additonal responses of sensitized seeds to 730 nm fluences were compared to simulations of a mechanism involving a receptor, X, and based on the dimeric structure of phytochrome in which each monomer is independently phototransformed from the inactive (P_r) to the active (P_fr) form. The fluence requirements for phytochrome photoconversion in seeds were determined to be similar to those of purified Avena phytochrome in vitro, on which photochemical parameters for the simulations were based. The analyses suggest that P_r:P_fr-Xand P_fr:P_fr-X are responsible, respectively, for the VLFR and the LFR, and that sensitization involves membrane influences on the activity of P_r:P_r-X. They also suggest the concentration of X to be about 0.001 that of total phytochrome dimer in this system.     

PHOTOCONTROL OF PHYTOCHROME DESTRUCTION IN GRASS SEEDLINGS. THE INFLUENCE OF WAVELENGTH AND IRRADIANCE

Abstract— –The kinetics of phytochrome destruction in vivo of coleoptiles and mesocotyls of etiolated grass seedlings (Avena sativa L., Zea mays L.) in continuous light were investigated using wavelength and irradiance as experimental variables. In contrast to dicotyledonous seedlings, the destruction reaction of these monocotyledons is saturated at very low levels of the far-red absorbing form of phytochrome, P_fr (e.g. at 1% of total phytochrome, corresponding to the photostationary state established by 727 nm light, in 2.5-day-old dark-grown Avena). On the other hand, the first-order rate constant of monocotyledon destruction may be at least one order of magnitude larger than in dicots, as indicated by the zero-order rate measured in the presence of saturating amounts of P_fr (τ_l/2 1.5 min in Avena). At sub-saturation P_fr levels, the destruction rate was found to be determined by the rate constants of the photoreactions over a wide range of wavelengths and irradiances. These results can be interpreted in terms of a destruction enzyme with high catalytic efficiency but limited availability. Analysis of in vivo binding of phytochrome to a pelletable cell structure during destruction revealed that both the pelletable and the non-pelletable fraction lose photoreversiblility with similar rates and thus provide no useful information with respect to a causal relationship between the two processes. However, due to the short half-life of P_fr at sub-saturation levels (which make the photoreactions and intermediary processes rate-limiting for destruction even at relatively high irradiances) the existence of a similarly rapid dark-reaction between the photoreactions producing P_fr and the destruction reaction could be demonstrated. This dark reaction displays the properties of P_fr binding to a receptor site.     

EVIDENCE FOR THE EXISTENCE OF MEMBRANE-ASSOCIATED PHYTOCHROME IN THE CELL

Abstract Comparative fluorescence and photochemical studies of phytochrome in etiolated seedlings of maize and in soluble and membrane-containing fractions isolated from them were camed out. The membrane fractions prepared in the absence of Mg²⁺ from etiolated coleoptiles contained 13% of total photoreversible phytochrome, which was readily solubilized by mild detergents. Its molecular size was indistinguishable from soluble phytochrome and equal to nondegraded maize phytochrome. Low-temperature fluorescence studies with intact tissue found that the position of the emission maximum at 85 K (λ_max) and the extent of the phototransformation of the red-absorbing form (Pr) into the first stable photoproduct, lumi-R, at 85 K (γ₁), varied in different parts of etiolated seedlings: λ_max and γ₁ reached their maximum values in the tips of coleoptiles and roots, 686 nm and 0.30–0.40, whereas the lowest values, 682 nm and ca 0.05, were observed in the root base. These parameters correlated well with those obtained for the pigment in the soluble and membrane-containing fractions: 684 and 680 nm, and 0.33 and 0.06, respectively. The extent of the Pr phototransformation into the far red-absorbing form (Pfr) (γ₂) did not differ much: values of 0.80–0.85 and 0.70–0.75 correlated with the high and low values of γ₁. These variations of the parameters were interpreted in agreement with our previous observations in terms of two phytochrome A species whose relative concentrations vary depending on the experimental conditions—the longer wavelength bulk light-labile species with high γ₁ (Pr″), and the shorter wavelength minor light-stable species with low γ₁ (Pr″). Close similarity between Pr’and the soluble phytochrome and between Pr″ and the membrane-bound phytochrome points to the possible origin of the native Pr’and PrPrime; species, thus providing evidence for the existence of membrane-bound pigment in the cell.     

THE TEMPERATURE DEPENDENCE OF Pfr ACTION GOVERNS THE UPPER TEMPERATURE LIMIT FOR GERMINATION IN LETTUCE

In most cultivars of lettuce (Lactuca saliva L,), red light acting through the red/far-red reversible phytochrome system promotes full germination within the20–30°C range, but at progressively higher temperatures germination declines sharply. The relationship between this upper ternperature limit for germination and the temperature dependence of phytochrome action was investigated in Grand Rapids lettuce. In fresh seeds the GT₅₀ (temperature giving half maximal germination) was ca 29–30°C. In these seeds, escape from far-red reversibility did not occur at 35°C, a temperature above the GT₅₀, but occurred rapidly at 27°C, a temperature below the upper limit. Increasing periods of dark pretreatment at high temperature (35°C) or increasing concentrations of the germination inhibitor coumarin caused a progressive decline in the GT₅₀, Escape from photoreversibility did not occur at 27°C in seeds in which the GT₅₀ had been reduced to less than 25°C by coumarin or by prolonged high temperature pretreatment. These results indicate that there is a close correlation between the position of the upper temperature limit for germination, and the temperature dependence of phytochrome action. We conclude that factors that alter the upper temperature limit for germination do so by changing the temperature dependence of phytochrome action.     

A MODEL FOR THE MOLECULAR STRUCTURE AND ORIENTATION OF THE CHROMOPHORE IN A DIMERIC PHYTOCHROME MOLECULE*

A model for the molecular structure and orientation of red-light absorbing form of phytochrome (P,) chromophores in a dimeric molecular model of P_r is proposed. A chromophore model with probable molecular structures was generated to reproduce the absorption spectrum produced by its π-electron conjugating system. The model has C₅-Z, syn, C₁₀-E, anti and C₁₅-Z, syn configurations and a protonation at a C-ring nitrogen. Orientation of the chromophore model in the dimeric phytochrome molecular was analyzed by displaying the atoms of the chromophore, the coordinates of which were converted into those with respect to the molecular axes to the dimeric molecule, on a 3-D graphic workstation. The conversions were performed by using the azimuthal angles between the Z axis of the dimeric molecule (axis of 2-fold rotational symmetry) and the dipole moments of the electronic transition at the blue- (384 nm) and red- (667 nm) absorbing bands of the chromophore, which were calculated as 55.5° and 59.3°, respectively, based on linear dichroism of the oriented phytochrome molecules. The result demonstrates that the long axis of the P, chromophore lies almost parallel to the Y axis of the molecular model, and that the tetrapyrrolic chromophore is well contained within the flat chromophoric domain without protruding from it, a configuration that assures that the chromophore is protected against aqueous environments. The model may explain the rotation angle of the transition moment of the red-absorbing band, induced by the phototransformation from P_r to P_rr which we measured as smaller than that measured in nonoriented preparations by a photoselection technique. The model also suggests a molecular basis for the polarotropic response of phytochrome.     

PHOTOREVERSIBLE PROTON DISSOCIATION AND ASSOCIATION IN PEA PHYTOCHROME AND ITS CHROMOPEPTIDES

Abstract— Effect of red-light irradiation on the medium pH at 10d?C was measured and compared among unbuffered solutions of the 121-kDa native pea (Pisum sativum cv. Alaska) phytochrome and its 114- and 62-kDa fragments in a red-light-absorbing form (P_r), all of which converted to far-red-light-absorbing form (Pf_r) on red-light irradiation. Red-light irradiation induced alkalinization in the solutions of the phytochrome and the fragments in the pH range 6.6-7.2 and 6.2-7.8, respectively. The amount of protons taken up by the 121-kDa phytochrome was less than one half of that of the 114-kDa fragment. Red-light irradiation induced acidification in the solutions of the 114- and the 62-kDa fragments above pH 7.8. In the solutions of the 121-kDa phytochrome, however, the irradiation induced no pH change at pH 7.2-8.2, and only a slight acidification at pH 8.2-8.7, which may be ascribed to a small amount of contamination from the 114-kDa fragment. All these red-light-induced pH changes were reversible following exposure to far-red light. The 7-kDa polypeptide(s) of the native 121-kDa phytochrome, which is lacking in the 114-kDa fragment, thus, prohibited proton transfer between phytochrome and the medium. A red-light-induced pH change was also measured in unbuffered solutions of the 39-kDa fragment of the phytochrome and of the 114-kDa fragment in the presence of 0.8 mM soyasaponin I. The 39-kDa fragment showed partially photoreversible conversion between a spectral form having an absorption maximum at 659 nm (P₆₅₉) and a bleached form, P_***. The 114-kDa fragment in the presence of the saponin showed a photoreversible conversion between P65V and Pb,. Exposure of P₆₅₉ from the 39-kDa fragment and from the 114-kDa fragment in the presence of the saponin to red light, caused acidification of the medium in the pH range 6.8-8.8 and 7.2-9.0, respectively, but no change at pH 6.2-6.8 and 6.4-7.2, respectively. The acidification of the latter was reversible following a far-red-light irradiation, but that of the former was only partially photoreversible. Proton uptake of phytochrome was inhibited by tryptic degradation to the 39-kDa fragment and also by the presence of the saponin. Only proton release was observed during the photoconversion from P₆₅₉ and P_***hl. It is suggested that a phytochrome molecule has possible site(s) for both proton release and for uptake and that the proton release reaction may be correlated to the photoconversion process(es) prior to the bleached intermediate (I_***) of phytochrome.     

UVB Irradiation Enhances TiO2 Nanoparticle‐induced Disruption of Calcium Homeostasis in Human Lens Epithelial Cells

Currently, titanium dioxide nanoparticles (TiO₂ NPs) have been widely used in various applications including cosmetics, food additives and biomedicine. However, there are few reports available using TiO₂ NPs to treat ocular diseases. Posterior capsular opacification (PCO) is the most frequent complication after cataract surgery, which is induced by the proliferation and migration of lens epithelial cells. Thus, inhibiting the proliferation of lens epithelial cells will efficiently reduce the occurrence of PCO. In this study, we investigated the effects of TiO₂ NPs on HLE B‐3 cells with or without ultraviolet B (UVB) irradiation in vitro. We found that TiO₂ NPs can inhibit HLE B‐3 cell growth, cause the elevation of intracellular [Ca²⁺], produce excessive reactive oxygen species (ROS), further reduce Ca²⁺‐ATPase activity and decrease the expression of plasma membrane calcium ATPase 1 (PMCA1), finally disrupt the intracellular calcium homeostasis and induce cell damage. Importantly, UVB irradiation can apparently enhance these effects on HLE B‐3 cells in the presence of TiO₂ NPs. Taken together, the generation of excessive ROS and the disruption of intracellular calcium homeostasis may be both involved in TiO₂ nanoparticle‐induced HLE B‐3 cell damage under UVB irradiation.     

PHYTOCHROME IN GREEN-TISSUE: PARTIAL PURIFICATION and CHARACTERIZATION OF THE 118-KILODALTON PHYTOCHROME SPECIES FROM LIGHT-GROWN Avena sativa L.*

The predominant, immunochemically-detectable phytochrome polypeptide rapidly extracted directly into boiling sodium dodecyl sulfate-containing buffer from fresh or freeze-dried green Avena tissue has an apparent molecular mass of 118 kilodaltons (kDa). This result indicates that the 118-kDa phytochrome species obtained from green Avena by extraction and rapid processing under non-denaturing conditions in previous studies was not derived by partial proteolysis of a larger polypeptide present in the cell. Additional data do, however, demonstrate the presence in green tissue homogenates of proteolytic activity that can cause a = 6-kDa reduction in apparent molecular mass and a blue-shift in the P_fr absorbance maximum of phytochrome during handling. This proteolytic activity contrasts with that previously encountered in etiolated tissue in that it is not inhibited by phenylmethylsulfonyl fluoride, but is inhibited by iodoacetamide and leupeptin. This result indicates that the activity is associated with a thiol-like protease. A partial purification procedure that incorporates the use of iodacetamide and a novel chromatographic step is described for green-tissue phytochrome. This procedure provides 50% recovery with a 90-fold enrichment of phytochrome relative to the initial extract in which the chromoprotein is 0.003% of the total soluble protein. The final fraction is apparently free of proteolytic activity. Immunoblot analysis of this fraction demonstrates that the predominant immunoreactive band has a monomeric molecular mass of 118 kDa. Comigration of this band with a band exhibiting zinc-induced fluorescence on blots of the partially purified preparations verifies that the 118-kDa species is the principal tetrapyrrole-bearing polypeptide present. Spectral properties of the final fraction are identical to those published for crude green-tissue extracts, indicating the stability of the molecule's spectral properties throughout the procedure. Size exclusion chromatography under nondenaturing conditions shows that the 118-kDa phytochrome species from green tissue comigrates with the dimeric, etiolated-tissue molecule, and is therefore suggestive of similar quaternary structure. Together these data reinforce previous conclusions that the predominant phytochrome molecule present in the living cells of green tissue is resolvable as a 118-kDa species, distinct from the well-characterized 124-kDa molecule from etiolated tissue (Tokuhisa et al., 1985, Planta 164, 321–332), and indicate that the partial purification protocol described here sustains the green-tissue phytochrome in its native state throughout the procedure.