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.     

PHYTOCHROME STATES IN ETIOLATED PEA SEEDLINGS: FLUORESCENCE AND PRIMARY PHOTOREACTIONS AT LOW TEMPERATURES

Emission spectra of the red phytochrome form (Pr) and fluence time-response curves of the Pr fluorescence intensity changes were measured in etiolated pea seedlings at low temperatures (80–150 K) in connection with its phototransformations into the initial photoproduct (Lr) and back upon actinic red (667 nm) and far-red (696 nm) illumination. The variable fluorescence reaches 45% at 85 K and decreases with the rise of temperature. Three kinetic components of the changes were found in the direct (Pr→Lr) and back (Lr→Pr) photoreactions belonging to three states of phytochrome: “slow”, “fast” and “very fast” (respective indices: s, f and vf). The amplitudes of the components and rate constants to reach photoequilibrium were determined in the direct and back photoreactions at different temperatures, and from this, their quantum yields, extent of the Pr?Lr phototransformation and activation energy of the reactions were evaluated for the three Pr and Lr states. The yields differ from each other by approximately a factor of 10 and those for the direct and back photoreactions are close to each other. The proportion of the amplitudes of the variable fluorescence of the three phytochrome states changes with temperature and upon the Pr→Lr photo-transformation and the Pr states differ in the position of their emission spectra by 3–5 nm. A close similarity between the Pr and Lr properties was observed, which implies a symmetrical scheme of their photoreactions. It is suggested that the three phytochrome species may originate in different conformational states of the chromophore and they independently transform in parallel photoreactions into the respective photoproducts: Prvf?Lrvf, Prf?Lrf and Prs?Lrs.     

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.     

Fluorescence Analysis of Oat phyA Deletion Mutants Expressed in Tobacco Suggests that the N-Terminal Domain Determines the Photochemical and Spectroscopic Distinctions between phyA' and phyA"†

Abstract— In vivo low-temperature (85 K) fluorescence spectroscopy has defined two phytochrome A (phyA) subpopulations, designated phyA' and phyA", in etiolated seedlings (V. A. Sineshchekov, J. Photochem. Photobiol. 28, 53–55, 1995). Phytochrome A' is the more abundant but light-labile species characterized by longer wavelength emission/absorption maxima (687/673 nm) and by a higher extent of the photoconversion of its red-absorbing form (Pr) into photoproduct (lumi-R) at 85 K (γ₁≈ 0.5). Phytochrome A" is the minor but relatively light-stable species, characterized by shorter wavelength maxima (682/668 nm) and by a lower γ₁ (<0.05). To help define domains within phyA responsible for these differences, the low-temperature spectral properties of transgenic tobacco expressing full-length (FL) oat phyA and C-and N-terminally truncated versions (CD [Δ786–1129] and NA [Δ7–69], respectively) were compared. Oat phytochrome expression was more pronounced than that of tobacco in the basal section of etiolated seedlings following 2 h irradiation with white light. Seedlings expressing FL and CD phyA had spectral properties for phyA' and phyA" that were indistinguishable from that of wild-type tobacco. Conversely, expression of NA phyA generated an abundant phy species that behaved like phyA". From this we conclude that the N-terminal domain of phyA is involved in determining the photochemical and spectroscopic distinctions between the native phyA' and phyA" species.     

Conformation of 18-Crown-5 and Its Influence on Complexation with Alkali and Ammonium Cations: Why 18-Crown-5 Binds More Than 1000 Times Weaker Than 18C6

Stability constants of potassium, sodium, and benzylammonium salts with 18C5 are determined in water, methanol, and acetonitrile by potentiometric titrations. The corresponding free energies DeltaG agree within the error with those obtained from calorimetric titrations. In comparison to 18C6 the DeltaG values are lower by 14 to 16 kJ/mol, with methanol or acetonitrile as solvent and K(+) or benzylammonium salts. Differences in the calorimetrically determined binding enthalpies DeltaH between 18C6 and 18C5 are usually even larger. In water, however, the DeltaG differences between the 18C5 and 18C6 complexes become almost negligible. The D(3)d-like conformation of such crown ethers can be evaluated for the first time by NOE methods using the less symmmetrical 18C5. The NMR data indicate also the absence of significant conformational changes upon complexation, in line with molecular mechanics calculations (CHARMm). These show that the low binding constants of K(+) with 18C5 are due to the expulsion of the cation due to one C-H bond pointing toward the cavity, leading to larger K(+).O distances. The CHARMm calculated gas phase energy difference between the K(+) crown complexes of 26 kJ/mol agrees approximately with experimental differences.