CHROMOPHORE ROTATION IN 124-kD ALTON A vena sativa PHYTOCHROME AS MEASURED BY LIGHT-INDUCED CHANGES IN LINEAR DICHROISM

Abstract— From light-induced changes in linear dichroism, we have calculated the rotation of the long-wavelength-absorbing transition moment that occurs during phototransformation of 124-kilodalton Avena sativa phytochrome. Phytochrome was purified to homogeneity and immobilized onto Sepharose beads covalently coated with antibodies against A. sativa phytochrome. Changes in linear dichroism were induced by plane-polarized red or far-red light and measured by the absorbance differences at 660 and 730 nm using a dual-wavelength spectrophotometer equipped with polarizing filters in the measuring beams. From such measurements, we calculate a rotation angle of 31^o (or 149^o) during photoconversion of Pr to Pfr and 30^o (or 150^o) during photoconversion of Pfr to Pr. These values are similar to the value of 32^o (or 148^o) reported earlier for the rotation of the transition moment of "large" A. sativa phytochrome (∽ 120 kilodalton) isolated under conditions that did not preclude post homogenization proteolysis of the 124 kilodalton molecule.     

PHOTOTRANSFORMATIONS OF PHYTOCHROME

Abstract— –Phytochrome is the photoreversible chromoprotein that controls many aspects of plant growth and development Phototransformations of the red absorbing form (Pr) and the far red absorbing form (Pfr) involve initial photoreactions followed by dark relaxation reactions. Techniques for the study of intermediates of phototransformation and the present picture of intermediates involved in the phototransformations of Pr and Pfr are outlined. The molecular natures of the phototransformations are reviewed in relationship to knowledge of the chemistry of the chromophore and apoprotein. The significance of phytochrome intermediates in understanding the physiology of phytochrome controlled responses is discussed.     

ON THE SIGNAL-TRANSDUCTION CHAINS OF TWO Pfr- MEDIATED SHORT-TERM PROCESSES: INCREASE OF ANCHORAGE and MOVEMENT OF Mougeotia CHLOROPLASTS

Abstract— We examined two published hypotheses on the signal-transduction chain of the light-oriented chloroplast movements in the fresh-water alga Mougeotia. One hypothesis postulates a Ca²+-influx controlled by a tetrapolar gradient of phytochrome in its far-red light absorbing form (Pfr). The other hypothesis postulates anchorage sites for actin-filaments even at those areas of the plasmalemma where phytochrome is in its inactive form (Pr). Calmodulin and Ca²+-sequestering vesicles are assumed to be essential links of this transduction chain.
To test these hypotheses we have studied the effects of Ca²+-entry blockers, Ca²+ deficiency and calmodulin antagonists on chloroplast movements and on chloroplast anchorage. None of our results support the Ca²+/calmodulin hypotheses mentioned above. The results and their implications (with regard to the role of Ca²', calmodulin and anchorage sites) are discussed.     

LIGHT INDUCED FLUORESCENCE SPECTRAL CHANGES IN NATIVE PHYTOCHROME FROM Secale cereale L. AT LIQUID NITROGEN TEMPERATURE

Abstract— Fluorescence spectra of native rye phytochrome were determined under different light conditions at liquid nitrogen temperature. Fluorescence spectrum of the red-light-absorbing form (Pr) had a major peak at about 685 nm (14 600 cm⁻¹) and a broad sub-peak at about 515 nm (19 400 cm⁻¹). The peak height at 685 nm was reduced by irradiation with monochromatic light of 640 nm, and a new peak became obvious at about 702 nm (14250 cm⁻¹). This spectral change was almost completely reversed by subsequent irradiation with 700-nm light. Fluorescence spectrum of the photoequilibrium mixture of Pr and far-red-light absorbing form under continuous red light showed a sharp peak at about 685 nm having a peak height ca. 12% of Pr, and a broad sub-peak at about 508 nm (19 700 cm⁻¹). Light of 730 nm did not reduce the peak height at about 685 nm but induced a new shoulder at about 699 nm (14300 cm⁻¹). Monochromatic light of 640 and 700 nm given following the light of 730 nm could not reverse the spectral change at 699 nm induced by the irradiation with 730-nm light. Fluorescence spectrum of Pr in partially degraded phytochrome was similar to that in native phytochrome but the peak position in the red region was shifted by about 5 nm (100 cm⁻¹) to the blue.     

PERMEABILITY AND STRUCTURAL CHANGES INDUCED BY PHYTOCHROME IN LIPID VESICLES

Abstract— This paper describes a method for rapidly monitoring early changes in electrolyte permeability induced by phytochrome from salt-loaded liposomes. The method allows for the continuous monitoring of low-level ion efflux from liposomes by measuring the conductivity of a liposome suspension medium which has osmotic and chemical potentials that promote a slow. passive efflux of the compartmented electrolytes. The addition of the far-red absorbing form of phytochrome (Pfr) to this system at 20°C immediately produces efflux rates which are 2–3 times greater than if the red-absorbing form (Pr) is added. This differential effect is not evident at 4°C and varies with the lipid composition of the liposomes. Under conditions in which Pfr induces a 2-fold greater change in the electrolyte permeability of liposomes than Pr. only about 18% more ¹²⁵I-labeled Pfr than ¹²⁵I-labeled Pr binds to the liposomes. At equimolar concentrations. the photochromic small peptide of phytochrome (60 000 dalton monomer) and the more native'large'phytochrome (120000 dalton subunits) induced equivalent changes in the electrolyte permeability of liposomes. No differential leakage of ATP, glucose, or trvpsin from liposomes was observed after Pr and Pfr reacted with vesicles enclosing these substances. The Pfr form of phytochrome promoted greater turbidity in liposome suspensions and a greater degree of aggregation and/or vesiclc fusion than Pr. The kinetics of these changes suggested that they were not the hasis of the differential permeability effects of Pr and Pfr.     

TWO SPECTROSCOPICALLY AND PHOTOCHEMICALLY DISTINGUISHABLE PHYTOCHROMES IN ETIOLATED SEEDLINGS OF MONOCOTS AND DICOTS *

Abstract Fluorescence (F) emission spectra of the red-absorbing phytochrome form (Pr) at 85 K, temperature dependence of the F intensity and the extent of the Pr F changes in the phototransformation of Pr into the first stable photoproduct (lumi-R) at 85 K (γ₁,) and into the far-red-absorbing form (Pfr) at 267 K (γ₂) were investigated in etiolated shoots and roots of monocots (oat, maize, rice) and dicots (pea, cress). These characteristics monotonously changed as a function of the phytochrome content, [Ptot]: with its decrease to 3-5% of the maximal values, the F spectrum shifts from 686 nm to 682 nm, its half-band width rises from 22 nm to ca 25 nm, temperature dependence of Pr F changes its character, γ¹, drops down from ca > 0.45 to ca 0.05-0.10 and γ₂ from 0.80–0.82 to ≤0.70. These data were interpreted in terms of two different phytochromes whose relative concentration varies with [Ptot]: (1) a longer wavelength type with the F maximum at 686 nm, low activation energy of the photoreaction (Ea ≤ 3–4 kj/ mol) and high extent of the phototransformation at 85 K (0.49 ± 0.03) and at 267 K (ca 0.85) (Pra); (2) a shorter wavelength type practically inactive at 85 K with F maximum at 682 nm, higher Ea (ca 35 kj/mol) and lower extent of the Pr & Pfr phototransformation (≤0.70) (Pri). [Pra] widely varies in different parts of the seedlings (up to 100 times) and Pra dominates when [Ptot] is high. The [Pri] is much more constant (variations, <10 times), and it becomes the major one when [Ptot] drops down. The two species are likely to belong to the labile (type 1) and stable pools of pigment and not to be connected with the localization of the pigment in the cell since red-far-red preillu-mination, which is believed to bring about sequestering of the pigment, does not change their relative concentration and properties.     

PHOTOPHYSIOLOGY OF TURION GERMINATION IN Spirodela polyrhiza (L.) SCHLEIDEN–V. DEMONSTRATION OF A CALCIUM-REQUIRING PHASE DURING PHYTOCHROME-MEDIATED GERMINATION

Abstract— Etiolated turions of Spirodela polyrhiza are positively photoblastic and show a phytochrome-mediated low fiuence germination response. The far-red light (FR) reversibility decreased with the delay of FR irradiation (lag phase 1.06 ± 0.03 days after red light irradiation; half-maximal response 1.9 days). The action of the far-red-absorbing form of phytochrome (Pfr) was only realized by a germination response if exogenously applied Ca²+ was present. Calcium step-down (from 1 mM to 0.9 μ M Ca²+) and Ca²+ step-up (from 0.9 μ M to 1 m M Ca²+) experiments were carried out to determine the Ca²+-sensitive phase. There was no time gap between the two phases determined by the step-down and step-up experiments but a clear coincidence of both curves. Pulse treatments (24 h) with Ca²+ (1 m M ) showed the upper part of this common curve to represent the most Ca²+-sensitive phase. The Ca²+-sensitive phase was within the Pfr-requiring phase. After reversion of Pfr by FR pulses there was only a negligible response to the high Ca²+-concentration, independent of the delay between the red light (R) and FR pulses. These results are compatible with the assumption of Ca²+ acting as a second messenger of Pfr. However, the Ca²+-insensitivity in the first 12 h after the R pulse points against this hypothesis.     

PHOTOCONVERSION OF PHYTOCHROME IN VIVO STUDIED BY DOUBLE FLASH IRRADIATION IN Mougeotia AND Avena

Abstract— The kinetics of phytochrome phototransformation from the red-absorbing form (P_r) to the far-red-absorbing form (P_fr) in vivo at 22°C were studied using a double flash apparatus with 1-ms flashes. Photoconversion by simultaneous flashes of red light saturates at a low P_fr level, indicating the possible attainment of a photoequilibrium between the excitation of P_r and the photoreversion of intermediates in the course of the I-ms flashes. At saturation energy, simultaneous flashes resulted in about 50% as much P_fr as was produced by saturating irradiation with 5 s red light. Intermediates of the phototransformation pathway were analysed by separating two red or a red and a far-red flash by variable dark intervals. In both plants phototransformation intermediates with half-lives < 1 ms occur, but they are too short-lived to characterize by our method. The subsequent intermediates have half-lives of about 7 ms and 150 ms in A vena , 2 ms and 10 ms in Mougeotia. The conversion from P_r to P_fr seems to be completed 1 s after the red flash in Avena. In the alga Mougeotia , P_fr formation seems to be finished within only 50 ms after the inducing red flash. The kinetics obtained from physiological and spectrophotometric experiments with Avena mesocotyls are almost identical. These observations indicate that the physiological response corresponds directly to the amount of P_fr produced and not to phototransformation intermediates or "cycling" between P_r and P_fr.     

PHYTOCHROME-MEDIATED THRESHOLD CONTROL OF HYPOCOTYL GROWTH IN PARTLY DE-ETIOLATED MUSTARD (Sinapis alba L.) SEEDLINGS

Abstract— Hypocotyl elongation in etiolated mustard ( Sinapis alba L.) seedlings is known to be controlled by phytochrome (Pfr) through a threshold mechanism. The Pfr threshold value required to suppress hypocotyl growth was low (3 times 10⁻²% Pfr, based on total phytochrome in the hypocotyl at 36 h after sowing = 100%). In the present study the question was addressed whether the threshold control by Pfr of hypocotyl elongation also operates in light-pretreated, partly de-etiolated seedlings after transfer to darkness. The experimental results show that this is the case. Calculation of the threshold level in far-red light pretreated seedlings led to a very low value (3 times 10⁻⁷%) compared to etiolated seedlings (3 times 10⁻²%). In red light pretreated seedlings the threshold level was calculated to be 9 times 10⁻⁷%. Since the light pretreatment affected the rate of degradation of phytochrome strongly (half-life of Ptot in continuous red light was found to be 35 min in far-red pretreated instead of 47 min in etiolated material), the difference in threshold level between far-red and red pretreated material cannot be interpreted unambiguously. However, the conclusion can be drawn that light nretreatment strongly increases the degradation rate of Pfr and decreases the threshold level.     

ON THE PRIMARY PHOTOPROCESS OF 124-kdalton PHYTOCHROME

The photoreaction between P_τ and the first detectable intermediate, lumi-R, of 124-kdalton oat phytochrome has been investigated at low temperatures. The temperature dependence of the quantum yields of the photoreactions, P_τ to lumi-R and lumi-R to P_τ, has been determined. From measurements over a temperature range from 119 to 155 K, an activation barrier of 3.6 ± 0.5 kJ mol ₁ is found for the photoreaction of P_τ with 661-nm actinic light. A higher value (5.7 ± 0.7 kJ mol ^-1) is found for the photoreaction of lumi-R to P^τ. with 698-nm actinic light. Increased quantum yields are found in deuterated buffer solutions at low temperatures. The activation energies for deuterated phytochrome (3.2 ± 0.7 kJ mol^–1 for P_τ with 661-nm irradiation and 6.2 ± 1.2 kJ mol^-1 for lumi-R at 698-nm irradiation) are identical within the limits of error with those of protonated phytochrome. The lack of a deuterium effect for the activation energies favors the Z,E-isomerization rather than proton transfer or tautomerization for the chromophore photochemistry during P_τ⇄lumi-R conversion.     

ACTION SPECTRUM FOR PHOTOREVERSION OF THE ACTIVE Pfr-FRACTION OF Mougeotia in vivo

Abstract— The dichroic oriented fraction of the far-red light absorbing form of phytochrome (Pfr) in the green alga Mougeotia was characterized by action spectroscopy. Microbeam irradiations had to be used for the induction of chloroplast movement in Pfr-containing cells, because of the special dichroic absorption characteristics of the red light absorbing form of phytochrome (Pr) and Pfr in the alga. Fluence-response curves were elaborated especially in the far-red spectral region by reverting Pfr to Pr at the flanks of the cells and thus generating Pfr-gradients. Linearly polarized light vibrating perpendicularly to the cell axis was used, thus corresponding to the S,-transition moments of Pfr at the flanks of the cells. The action spectrum is characterized by a peak at approximately 715 nm and a very pronounced decrease towards 728 and 734 nm. The data indicate that the spectral absorption of the active Pfr-fraction in green Mougeotia is shifted towards shorter wavelengths as compared to extracted phytochrome from etiolated or even green higher plants. This "blue shift" seems to be typical for Pfr from green lower plants.     

ULTRAVIOLET RESONANCE RAMAN SPECTRA OF PHYTOCHROME: A COMPARISON OF THE ENVIRONMENTS OF TRYPTOPHAN SIDE CHAINS BETWEEN RED LIGHT-ABSORBING AND FAR-RED LIGHT-ABSORBING FORMS

Ultraviolet resonance Raman spectra of phytochrome in the red light-absorbing form (Pr) and the far-red light-absorbing form (Pfr) are reported. The spectra excited at 240-nm provide structural information about the protein part of phytochrome. The protein contains only a very small amount of β-sheet structure and most of the tyrosine side chains are located in hydrophobic environments. Indole rings of tryptophan (Trp) interact with neighboring groups in the Pr form and these interactions become weaker with the conversion from Pr to Pfr. Some Trp side chains of Pfr are surrounded by aliphatic groups but such is not the case in Pr. These changes in the environment occur at the same time as changes in orientation of Trp side chains. Our observations suggest that interactions between Trp residues and the tetrapyrrolic chromophore occur in the Pr form and that the strength of these interactions diminishes in the Pfr form.     

INTERPRETATION OF THE 'DICHROIC ORIENTATION' OF PHYTOCHROME

The dichroic orientation of phytochrome observed both in the phytochrome-mediated phototropism in Adiantum protonemata and in the phytochrome-mediated chloroplast movement in Mougeotia were analyzed in terms of the orientation of the transition moment associated with the long-wavelength absorption band, assuming that phytochrome, associated with the plasma membrane, rotates around the normal to the membrane. The orientation of the long-wavelength transition moment of the phytochrome chromophore was calculated using the zero-differential overlap approximation of the molecular orbital theory for ir-electrons. The results indicate that the orientation of the long-wavelength transition moment mainly changes later than 2 ms after red light excitation of P_r, and that the different dichroic orientations of P_r and P_fr can be attributed to the change in the angle of the long-wavelength transition moment of phytochrome with the plasma membrane from 18^o to 72^o during phototransformation.     

FLUORESCENCE QUANTUM YIELDS OF 124-kDa PHYTOCHROME FROM OAT UPON EXCITATION WITHIN DIFFERENT ABSORPTION BANDS

Abstract— A fluorescence quantum yield (emission at650–850 nm) of π= (2.3 ± 0.3)10⁻³ was measured for the red-absorbing form (Pr) of 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa ) upon excitation in the Soret band at Λ^exc= 380 nm. The small difference between this value and the previously determined quantum yield with Λ^exc= 640 nm, π= (3.5 ± 0.4)10⁻³is attributed to a blue-absorbing emitter responsible for the "anomalous" or "blue" emission of the chromoprotein in the region from ca. 400 to 550 nm. The absorption of Pr at 380 nm is consequently somewhat lower than that measured directly from the spectrum. Processes from upper excited states of the Pr phytochromobilin-derived chromophore other than rapid relaxation to the emitting state are not important. A quantum yield of Φ ' 1.2 times 10⁻³ is estimated for the blue fluorescence. The proportion of the blue emitters relative to Pr appears to be relatively high.     

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.     

APPARENT INCREASE IN PHOTODETECTABLE PHYTOCHROME IN THE PRESENCE OF CALCIUM CARBONATE*

Abstract— –Using squash seedling extracts ( Cucurbita pepo L.), we describe an apparent increase in photodetectable phytochrome content that is depended upon the use of either CaCO₃ or starch as a scattering agent and is also a function of preirradiation with red light of either intact tissue or crude extracts prior to sample preparation for spectrophotometric assay. This apparent increase in photodetectable phytochrome content requires about 1 h for full expression after addition of the scattering agent. If irradiations are given in vitro , the increase is partially reversed by far red light when using squash extracts, and is fully reversed with Aoena and Zea extracts. For squash extracts the magnitude of this increase. which is typically between 20 and 40%. is quantitatively correlated with both the amount of Pfr produced by a brief red irradiation and with the proportion of the total phytochrome pool that is pelletable upon centrifugation at 20,000 g . The correlation with pelletable phytochrome does not hold for Auena and Zea , at least when irradiations are gwen in uitro . The increase in photodetectable phytochrome may result from changed phytochrome extinction, changed phototransformation quantum yields, and/or specific trapping of phytochrome by the scattering agent as it settles. An important consequence of these data is that they indicate a need for caution when using a scattering agent during spectrophotometric assay of phytochrome.     

Heterologous expression and characterization of recombinant phytochrome from the green alga Mougeotia scalaris

The full-length apoprotein (124 kDa) and the chromophore-binding N-terminal half (66 kDa) of the phytochrome of the unicellular green alga Mougeotia scalaris have been heterologously expressed in the methylotrophic yeast Pichia pastoris. Assembly with the tetrapyrrole phycocyanobilin (PCB) yielded absorption maxima (for the full-length protein) at 646 and 720 nm for red- and far-red absorbing forms of phytochrome (Pr and Pfr), respectively, whereas the maxima of the N-terminal 66 kDa domain are slightly blueshifted (639 and 714 nm, Pr and Pfr, respectively). Comparison with an action spectrum reported earlier gives evidence that in Mougeotia, as formerly reported for the green alga Mesotaenium caldariorum, PCB constitutes the genuine chromophore. The full-length protein, when converted into its Pfr form and kept in the dark, reverted rapidly into the Pr form (lifetimes of 1 and 24 min, ambient temperature), whereas the truncated chromopeptide (66 kDa construct) was more stable and converted into Pr with time constants of 18 and 250 min. Also, time-resolved analysis of the light-induced Pfr formation revealed clear differences between both recombinant chromoproteins in the various steps involved. The full-length phytochrome showed slower kinetics in the long milliseconds-to-seconds time domain (with dominant Pfr formation processes of ca 130 and 800 ms), whereas for the truncated phytochrome the major component of Pfr formation had a lifetime of 32 ms.     

PHOTOREVERSIBLE Ca2+-DEPENDENT AGGREGATION OF PURIFIED PHYTOCHROME FROM ETIOLATED PEA AND RYE SEEDLINGS

The aggregation of phytochrome purified from etiolated pea ( Pisum satirum cv. Alaska) and rye ( Secale cereale cv. Cougar) tissues was investigated by centrifugation and turbidimetry. Purified pea phytochrome (A₆₆₉/A₂₈₀= 0.88), if irradiated with red light, became precipitable in the presence of CaCl₂. The precipitation upon red-light irradiation was optimal at a Ca^2- or Mg²⁺ concentration of 10–20 m M , was greater at increased phytochrome concentration or lower pH values, and was inhibited by 0.1 M KG. The precipitated phytochrome slowly became soluble after far-red light exposure.
Turbidity of pea phytochrome solutions after red-light irradiation also increased rapidly in the presence of either Ca²⁺ or Mg²⁺. Far-red light exposure after the red light cancelled the turbidity increase. Rye phytochrome showed less turbidity increase than pea phytochrome and occurred only in the presence of Ca²⁺. Partially degraded pea phytochrome produced by endogenous proteases in the extract did not show the turbidity increase. Undegraded pea phytochrome also associated with microsomal fractions under conditions similar to those described above, but the partially degraded phytochrome did not.     

EVIDENCE FOR A SEQUENTIAL PATHWAY FROM Pr TO Pfr OF THE PHOTOTRANSFORMATION OF 124-kDa OAT PHYTOCHROME

Abstract. Phototransformation kinetics of 124-kDa oat phytochrome at 298 K after a red (660-nm) laser flash excitation were recorded at different wavelengths. The kinetics of the dark relaxation processes for lumi-R to P_fr can be satisfactorily described by only 3 rate constants: k = 28000 s^-1 370 s^-1 and 20 s^-1. The first rate constant is due to the decay of lumi-R to meta -Ra. The latter two rate constants correspond to processes establishing the far-red (>700 nm) absorption band. No meta -Rb could be detected. From the wavelength dependency of the amplitudes of these two rates, parallel pathways in the formation of P_fr could be excluded. A unique sequential pathway for the dark relaxation leading to P_fr seems to be an intrinsic property of 124-kDa phytochrome, however. Assuming a sequential pathway, molar extinction coefficients for intermediates have been calculated. These values agree with molar extinction coefficients obtained from low-temperature spectra. The process with a rate constant of 370 s^-1 corresponds to absorbance changes for the formation of meta -Rc from meta -Ra and the rate constant of 20 s^-1 describes the absorbance changes due to the transformation of meta -Rc to P_fr.     

THE USE OF A COMPUTERIZED SPECTRORADIOMETER TO PREDICT PHYTOCHROME PHOTOEQUILIBRIA UNDER POLYCHROMATIC IRRADIATION

Abstract A method is described for predicting the effect of polychromatic irradiation upon the photo-stationary equilibrium of the plant photoreceptor phytochrome. This method follows from the rate equations for phototransformation and utilizes the in vivo action spectra for phytochrome phototransformation (Pratt and Briggs, 1966). A scanning spectroradiometer interfaced with a microcomputer is used to determine a spectral photon distribution from 360 to 800 nm. The products of the photon fluence rate and the relative quantum efficiencies at 2-nm intervals are summed over the entire visible range to yield a predicted percentage of the pigment in the Pfr form. This value was determined under eight different polychromatic light sources and was generally within 7% Pfr of the value measured in etiolated corn coleoptiles under the same light sources.