Fluorescence and photochemical characterization of phytochromes A and B in transgenic potato expressing Arabidopsis phytochrome B

Phytochrome in etiolated sprouts of wild type (WT) potato and its transgenic strains (DARA5 and DARA12) expressing Arabidopsis thaliana phytochrome B (phyB) was investigated using low-temperature (85 K) fluorescence spectroscopy and photochemistry. Phytochrome content, [Ptot], position of the Pr emission and excitation spectra, lambda(max), and extent of the Pr-->lumi-R, gamma1, and Pr-->Pfr, gamma2, phototransformations (at 85 and 273 K, respectively) were shown to vary in the transgenic lines and WT depending on tissue used (upper vs. lower parts of etiolated sprouts) and light-induced phytochrome depletion. Differences in the parameters between the transgenic lines and WT were detected which were interpreted in terms of the two phenomenological Pr types: a labile Pr' with gamma1 approximately 0.5 consisting of a major phytochrome A (phyA) fraction (phyA') and a relatively conserved Pr" with gamma1 = 0 comprising a minor phyA fraction (phyA") and phyB. Both DARA lines had higher [Pr"] as compared with WT in the lower parts of etiolated stems, especially after light-induced phytochrome depletion (residual phytochrome in DARA5 and DARA12 made up to one-third of its initial level vs. <5% in WT). These differences were associated with the expression of Arabidopsis phyB in the DARA lines and its higher light stability than that of phyA. Arabidopsis phyB expressed in potato was characterised by lambda(max) = 683/669 nm in the emission/excitation (absorption) spectra and gamma1 = 0. PhyB also revealed a relatively low gamma2 (approx. 0.5) and its early red drop as compared with the gamma2 wavelength dependence for phyA. This is believed to contribute to the lower signalling ability of phyB and to confine the region (red) of its physiological activity.     

Fluorescence and Photochemistry of Recombinant Phytochrome from the Cyanobacterium Synechocystis

Fluorescence and photochemical properties of phytochrome from the cyanobacterium Synechocystis were investigated in the temperature interval from 293 to 85 K. The apoprotein was obtained by overexpression in Escherichia coli and assembled to a holophytochrome with phycocyanobilin (PCB) and phytochromobilin (PφB), Syn(PCB)phy and Syn(PφB)phy, respectively. Its red-absorbing form, Pr, is characterized at 85 K by the emission and excitation maxima at 682 and 666 nm in Syn(PCB)phy and at 690 and 674 nm in Syn(PφB)phy. At room temperature, the spectra are blue shifted by 5–10 nm. The fluorescence intensity dropped down by ?15–20-fold upon warming from 85 to 293 K and activation energy of the fluorescence decay was estimated to be ca 5.4 and 4.9 kJ mol^?1 in Syn(PCB)phy and Syn(PφB)phy, respectively. Phototransformation of Pr upon red illumination was observed at temperatures above 160–170 K in Syn(PCB)phy and above 140–150 K in Syn(PφB)phy with a 2–3 nm shift of the emission spectrum to the blue and increase of the intensity of its shorter wavelength part. This was interpreted as a possible formation of the photoproduct of the meta-Ra type of the plant phytochrome. At ambient temperatures, the extent of the Pr phototransformation to the far-red-absorbing form, Pfr, was ca 0.7–0.75 and 0.85–0.9 for Syn(PCB)phy and Syn(PφB)phy, respectively. Fluorescence of Pfr and of the photoproduct similar to lumi-R was not observed. With respect to the photochemical parameters, Syn(PCB)phy and Syn(PφB)phy are similar to each other and also to a small fraction of phyA (phyA″) and to phyB. The latter were shown to have low photochemical activity at low temperatures in contrast to the major phyA pool (phyA″), which is distinguished by the high extent (ca 50%) of Pr photoransformation at 85 K. These photochemical features are interpreted in terms of different activation barriers for the photoreaction in the Pr excited state.     

Recombinant phytochrome A in yeast differs by its spectroscopic and photochemical properties from the major phyA' and is close to the minor phyA": evidence for posttranslational modification of the pigment in plants.

Previously, two pools of phytochrome A (phyA' and phyA") have been detected by in situ low-temperature fluorescence spectroscopy and photochemistry; it was suggested that they might differ in the nature of their posttranslational modification. In order to verify this possibility Arabidopsis and rice (Oryza) phyA were expressed in yeast and the pigments were assembled in vivo with phycocyanobilin (PCB) and phytochromobilin (P phi B). The resulting recombinant phytochromes in the red-light-absorbing form (Pr) were characterized in the yeast cell by (1) the fluorescence emission spectra; (2) the temperature dependence of Pr fluorescence intensity and activation energy of fluorescence decay; and (3) the extent of photoconversion of Pr into photoproduct lumi-R (gamma 1) or far-red-light absorbing form (Pfr) (gamma 2). Both Arabidopsis phyA/PCB and Oryza phyA/P phi B had low gamma 1 of ca 0.05, allowing their attribution to the Pr" phenomenological type of phytochrome comprising phyA", phyB and cryptogam phytochromes. The spectroscopic properties of Oryza phyA/P phi B were also very close to phyA". However, both investigated holoproteins differed from phyA", both with respect to the character of temperature dependence of the fluorescence yield and activation energy. Thus, recombinant Oryza phyA/P phi B is similar but not identical to phyA". The data demonstrate that the low-abundance-fraction plant phyA (phyA") comes from the same gene as the major (phyA') fraction. Because both endogenous phyA fractions differ from the phytochrome expressed in yeast, they appear to be posttranslationally modified and/or bound to partner proteins or cellular substructures. However, the character of the presumed chemical modification is different in phyA' and phyA" and its extent is more profound in the case of the former.     

Fluorescence investigation of the recombinant cyanobacterial phytochrome (Cph1) and its C-terminally truncated monomeric species (Cph1Delta2): implication for holoprotein assembly,chromophore-apoprotein interaction and photochemistry

Recombinant dimeric full-length Cph1 holophytochrome and its C-terminally-truncated monomeric species [Cph1Delta2, comprising the chromophore-bearing N-terminal sensory module (residues 1 to 514)] from the cyanobacterium Synechocystis expressed in E. coli and reconstituted in vitro with phycocyanobilin (PCB) were investigated with the use of fluorescence spectroscopy and photochemistry in the temperature range from 85 to 293 K. Holoprotein assembly in Cph1 apparently proceeds via intermediate states with the emission maximum at 680-690 nm (I685) and 700 nm (I700) and a half-life time, at room temperature, of < or =5 s. Conversion of the putative I685 into mature Cph1 involves relaxation of the chromophore into a more flexible conformation. Cph1 and Cph1Delta2 were closely similar in their spectroscopic and photochemical characteristics (position of the emission band and its width, character of the temperature dependence of the fluorescence and activation energy of the fluorescence decay, kinetics and extent of the Pr conversion at low and ambient temperatures), suggesting that there is no immediate effect of the C-terminus on the photochemical properties of the chromophore in Cph1 and that chromophore-chromophore interactions in the dimer are not significant. The latter is also supported by the lack of energy transfer from the phycoerythrobilin (PEB) to PCB in the mixed PEB/PCB adduct of Cph1. At the same time, certain variations in the fluorescence and photochemical parameters of Cph1 with temperature of the sample and intensity of the excitation light and dependence of the emission spectra on excitation wavelength were observed. These variations are interpreted as a manifestation of the Cph1 heterogeneity which may be due to the existence of different conformers of the chromophore and photoproduct formation under excitation light.     

Two native pools of phytochrome A in monocots: Evidence from fluorescence investigations of phytochrome mutants of rice

Fluorescence investigations of phytochrome (phy) in rice (Oryza sativa L. cv. Nipponbare) mutants deficient in phyA, phyB and phyA plus phyB were performed. Total content of the pigment (P(tot)) and its spectroscopic and photochemical characteristics were determined in different parts of the dark-grown and far-red light (FR)-grown coleoptiles. Spectroscopically, phyA in the phyB mutant was identical to phyA in the wild-type (WT) and the extent of the conversion from Pr to lumi-R at 85 K was the same for phyA in both lines and varied similarly, depending on the part of the coleoptile used. The latter finding proved that phyA in rice is heterogeneous and comprises two phyA populations, phyA' and phyA". Functional properties of phyA were also determined. In the dark the phyB mutant had a higher content of phyA, inactive protochlorophyllide (Pchlide633) and active protochlorophyllide (Pchlide655) than WT and its coleoptile was longer, indicating that phyB may affect the development of WT seedlings in the dark. Constant FR drastically reduced the content of phyA, Pchlide633 and Pchlide655 and brought about coleoptile shortening and appearance of the first leaf, whereas pulsed FR of equal fluence was less effective. This suggested that the reactions were primarily of the high irradiance responses type, which are likely to be mediated by phyA'. The effects on protochlorophyllide biosynthesis and growth responses type were more pronounced in the phyB mutant than in the WT seedlings, which can be connected with the higher phyA' content in the phyB mutant and/or phyB interference with its action in WT seedlings. In the phyA mutant induction of Pchlide633 and Pchlide655 biosynthesis was observed under constant FR, indicating that phyC may be responsible for this effect.     

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.     

Phytochrome-mediated inhibition of coleoptile growth in rice: age-dependency and action spectra

Phytochrome has been shown to be the major photoreceptor involved in the photo-inhibition of coleoptile growth in Japonica-type rice (Oryza sativa L.). We have characterized this typical photomorphogenetic response of rice using mutants deficient in phytochrome A (phyA) and phytochrome B (phyB) and with respect to age-dependency and action spectra. Seedlings were irradiated with a pulse of light 40 h or 80 h after germination (i.e. at an early or late developmental stage) and the final coleoptile length of these seedlings was determined. A saturating pulse of red light (R) had a stronger effect when it was given in the late stage than in the early stage. It was found that the photoinhibition is mediated by both the phyA and the phyB in the late stage but predominantly by phyB in the early stage. Consistent with many other reported responses, the photo-inhibition in the phyA mutant, which was observed in the early and late developmental stages and is thought to be mediated mainly by phyB, occurred in the low-fluence range (10(1)-10(3) micromol m(-2)) of R and was far-red-light (FR)-reversible; the photo-inhibition in the phyB mutant, which was observed in the late developmental stage and is thought to be mediated mainly by phyA, occurred in the very-low-fluence range (10(-2)-10(0) micromol m(-2)) and was FR-irreversible. The action spectra (350-800 nm at 50 nm intervals) obtained at the two developmental stages using phyA and phyB mutants indicated that both the phyB-mediated low-fluence response and the phyA-mediated very-low-fluence response have a major peak at 650 nm and a minor peak at 400 nm.     

The photoreactions of recombinant phytochrome CphA from the cyanobacterium Calothrix PCC7601: a low-temperature UV-Vis and FTIR study

The photoreactions of recombinant phytochrome CphA from cyanobacterium Calothrix sp. PCC7601 reconstituted with phycocyanobilin were investigated using UV–Vis and Fourier transform infrared (FTIR) difference spectroscopy, stabilizing intermediates at low temperature. The yield of the forward reaction strongly depends on temperature, unlike the backward reaction. Because of the very fast thermal relaxation processes in the Pr to Pfr pathway, no pure difference spectra of the Pr photoconversion products could be directly measured. Thus, the contribution of the Pfr:Pr pathway was taken into account by applying an appropriate correction procedure both in the UV–Vis and FTIR experiments. Three intermediates have been trapped at −25, −45 and −120°C, which show the characteristic vibrational band pattern of the plant phytochrome phyA intermediates meta-Rc, meta-Ra and lumi-R, respectively. In the backward reaction, two intermediates corresponding to meta-F and lumi-F were trapped at −70 and −140°C, respectively. FTIR spectra of all intermediates, as well as of the Pfr state, show remarkable similarities with the corresponding spectra of Cph1 phytochrome from cyanobacterium Synechocystis and the 59 kDa N-terminal fragment of Cph1, and, albeit not so pronounced, also with plant phyA. The spectral similarities and differences between the various phytochromes are discussed in terms of structural changes of the chromophore and the chromophore–protein interactions.     

Extreme dehydration of plant tissues irreversibly converts the major and variable phyA' into the minor and conserved phyA'

Effect of dehydration of plant tissues on the two native phenomenological phytochrome A (phyA) pools - major, variable and soluble phyA' and minor, relatively conserved and presumably membrane(protein)-associated phyA' - was investigated on etiolated seedlings of barley and maize. With the use of in situ low-temperature fluorescence spectroscopy and photochemistry, it was found that even a considerable loss of water (up to 75-85% of the initial fresh weight) by coleoptiles does not bring about noticeable alterations of the spectroscopic and photochemical parameters of phytochrome pointing to a relative stability of the phyA'/phyA' system in this regard. However, extreme dehydration (loss of weight 90%) of plant tissues including freeze-drying caused dramatic changes of the phytochrome properties - blue shift of the emission maximum and its widening and reduction in the extent of the Pr photoconversion into lumi-R at 85 K and into Pfr at 273 K. Rehydration of the dried tissues did not reverse the spectroscopic changes and did not recover the Pr-->lumi-R phototransformation at 85 K but restored the ability of Pr to photoconvert into Pfr at ambient temperatures. At the same time, the total phytochrome content was not affected by these treatments. These effects were interpreted as an irreversible transformation of phyA' into phyA' upon extreme loss of water by plant tissues suggesting that water may play a role in stabilizing the conformation of the major and soluble phyA' species. The data also imply that phyA in dry and imbibing seeds is likely represented primarily by its phyA' isoform.     

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.     

Formation of the early photoproduct lumi-R of cyanobacterial phytochrome cph1 observed by ultrafast mid-infrared spectroscopy

The photoreactions of the Pr ground state of cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 have been investigated by picosecond time-resolved mid-infrared spectroscopy at ambient temperature. With femtosecond excitation of the Pr state at 640 nm, the photoisomerized Lumi-R product state is generated with kinetics and associated difference spectra indicative of vibrational cooling with tau(1) = 3 ps time constant and excited state decay with tau(1) = 3 ps, tau(2) = 14 ps, and tau(3) = 134 ps time constants. The Lumi-R state is characterized by downshifted absorption of three C=C modes assigned to C(15)=C(16), C(4)=C(6), and a delocalized C=C mode, in addition to the downshifted C(19)=O mode. The Lumi-R minus Pr difference spectrum is indicative of global restructuring of the chromophore on the ultrafast timescale, which is discussed in light of C(15) Z/E photoisomerization in addition to changes near C(5), which could be low bond order torsional angle changes.     

Two modes of the light-induced phytochrome A decline--with and without changes in the proportion of its isoforms (phyA' and phyA''): evidence from fluorescence investigations of mutant phyA-3D pea

Different modes of the phytochrome function are connected with its polymorphism, the major isoforms being phytochromes A and B (phyA and phyB). In its turn, phyA comprises two native species, phyA' and phyA', whose precise nature and functions remain obscure. With the use of in situ fluorescence spectroscopy, we investigated their properties in a mutant of pea, phyA-3D, characterized by exaggerated photoresponses and impaired photodestruction of phyA. The mutation is a substitution of alanine by valine at the position 194 in phyA. The phyA-3DphyB and phyB mutants were also investigated. In dark-grown plants, all the lines had the content and properties of the two phyA species very similar to the wild type. However, a considerably more intense reduction in [phyA] without changes in the phyA'/phyA' equilibrium was found in far-red grown mutant plants suggesting a hypersensitivity of phyA-3D with regard to its autoregulation. On the contrary, under red illumination, a higher stability of phyA-3D was observed confirming our earlier findings. This allows a conclusion that the A194V substitution in phyA-3D not only impairs its destruction but also enhances its signaling ability, suggesting a role of this locus in modulation of its activity.     

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.     

Phytochromes, cryptochromes, phototropin: photoreceptor interactions in plants

In higher plants, natural radiation simultaneously activates more than one photoreceptor. Five phytochromes (phyA through phyD), two cryptochromes (cry1, cry2) and phototropin have been identified in the model species Arabidopsis thaliana. There is light-dependent epistasis among certain photoreceptor genes because the action of one pigment can be affected by the activity of others. Under red light, phyA and phyB are antagonistic, but under far-red light, followed by brief red light, phyA and phyB are synergistic in the control of seedling morphology and the expression of some genes during de-etiolation. Under short photoperiods of red and blue light, cry1 and phyB are synergistic, but under continuous exposure to the same light field the actions of phyB and cry1 become independent and additive. Phototropic bending of the shoot toward unilateral blue light is mediated by phototropin, but cry1, cry2, phyA and phyB positively regulate the response. Finally, cry2 and phyB are antagonistic in the induction of flowering. At least some of these interactions are likely to result from cross talk of the photoreceptor signaling pathways and uncover new avenues to approach signal transduction. Experiments under natural radiation are beginning to show that the interactions create a phototransduction network with emergent properties. This provides a more robust system for light perception in plants.     

Single- and two-photon excited fluorescence in organic nonlinear optical single crystal 3-(1,1-dicyanoethenyl)-1-phenyl-4,5-dihydro-1H-pyrazole

Fluorescence of nonlinear optical organic single crystal of 3-(1,1-dicyanoethenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (DCNP) excited by a nonabsorbed light pulses from Q-switched Nd:YAG laser λ = 1064 nm as well as absorbed λ = 532 nm light is reported. Two mechanisms of two-photon excited fluorescence are considered: (i) direct two-photon excited fluorescence and (ii) single-photon excitation due to reabsorption of light generated in process of second harmonic generation (SHG) by the crystal due to its nonlinear optical properties. Strong anisotropy of fluorescence that has been observed is linked with uniaxial molecular alignment. Fluorescence decay profile shows two- exponential decay with lifetimes of emitting species of 3.7 and 5.6 ns at 293 K. The excitation and fluorescence spectra of the DCNP single crystal have been measured at 294 K and in function of temperature down to 77.4 K. The strong bathochromic shift of fluorescence spectrum in crystal with respect to fluorescence of DCNP molecule in solution is observed and interpreted with possible formation of molecular aggregates.     

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.     

Tyrosine 263 in Cyanobacterial Phytochrome Cph1 Optimizes Photochemistry at the prelumi‐R→lumi‐R Step

We report a low‐temperature fluorescence spectroscopy study of the PAS‐GAF‐PHY sensory module of Cph1 phytochrome, its Y263F mutant (both with known 3D structures) as well as Y263H and Y263S to connect their photochemical parameters with intramolecular interactions. None of the holoproteins showed photochemical activity at low temperature, and the activation barriers for the Pr→lumi‐R photoreaction (2.5–3.1 kJ mol^?1) and fluorescence quantum yields (0.29–0.42) were similar. The effect of the mutations on Pr→Pfr photoconversion efficiency (Φ_Pr→Pfr) was observed primarily at the prelumi‐R S₀ bifurcation point corresponding to the conical intersection of the energy surfaces at which the molecule relaxes to form lumi‐R or Pr, lowering Φ_Pr→Pfr from 0.13 in the wild type to 0.05–0.07 in the mutants. We suggest that the E_a activation barrier in the Pr* S₁ excited state might correspond to the D‐ring (C19) carbonyl – H290 hydrogen bond or possibly to the hindrance caused by the C13¹/C17¹ methyl groups of the C and D rings. The critical role of the tyrosine hydroxyl group can be at the prelumi‐R bifurcation point to optimize the yield of the photoprocess and energy storage in the form of lumi‐R for subsequent rearrangement processes culminating in Pfr formation.     

Emission and excitation spectra of Ce3+ and Pr3+ ions in hexafluoroelpasolite lattices

The emission and excitation spectra of Ce(3+) and Pr(3+) doped into the cubic host Cs(2)NaYF(6) have been recorded at room temperature and ～10 K using synchrotron radiation. The two 5d(1) T(2g) states of Ce(3+) have been located from the excitation spectra, whereas the E(g) state is placed above the host band gap. Decay measurements of the 5d(1) → 4f(1) Ce(3+) emission, and spectra collected using selective excitation, indicate the occupation of more than one type of site by Ce(3+) in this host lattice. By contrast, the location of features in the 4f(1)5d(1) → 4f(2) emission of Pr(3+) is independent of the excitation wavelength. Assignments are presented for some of the 4f(1)5d(1) levels and for the Pr(3+)-F(-) charge transfer band. The 5d emission lifetimes for Ce(3+) and Pr(3+) in the Cs(2)NaYF(6) host are 42 and 29 ± 1 ns, respectively, and are not temperature-dependent.     

Two-photon excitation of a phytofluor protein

Phytofluors are highly fluorescent proteins in which the chromophore in a phytochrome is replaced with phycoerythrobilin (PEB), the pigment precursor of the cyanobacterial light harvesting protein phycoerythrin. We examined the fluorescence spectra of the N-terminal region of the cyanobacterial phytochrome 1 from cyanobacterium Synechocystis sp. Pcc 6803 bound to PEB. This protein, Cph1(N514)-PEB, displayed a good two-photon cross-section of 20–30 GM for excitation at 792 nm. This phytofluor also exhibits a high fundamental anisotropy at most practical two-photon excitation (2PE) wavelengths from 700 to 900 nm. Identical lifetimes and correlation times with one and 2PE indicates that the phytofluor is not adversely affected by the intensities needed for 2PE. The one-photon absorption extends well beyond the absorption spectrum and even beyond the emission spectrum to 700 nm. The phytofluor thus appears to be a suitable probe for 2PE and/or cellular imaging.     

Laser-induced UV photodissociation of 2-bromo-2-nitropropane: dynamics of OH and Br formation

Photoexcitation of 2-bromo-2-nitropropane (BNP) at 248 and 193 nm generates OH, Br, and NO(2) among other products. The OH fragment is detected by laser-induced fluorescence spectroscopy, and its translational and internal state distributions (vibration, rotation, spin-orbit, and Λ-doubling components) are probed. At both 248 and 193 nm, the OH fragment is produced translationally hot with the energy of 10.8 and 17.2 kcal∕mol, respectively. It is produced vibrationally cold (v" = 0) at 248 nm, and excited (v" = 1) at 193 nm with a vibrational temperature of 1870 ± 150 K. It is also generated with rotational excitation, rotational populations of OH(v" = 0) being characterized by a temperature of 550 ± 50 and 925 ± 100 K at 248 and 193 nm excitation of BNP, respectively. The spin-orbit components of OH(X(2)Π) are not in equilibrium on excitation at 193 nm, but the Λ-doublets are almost in equilibrium, implying no preference for its π lobe with respect to the plane of rotation. The NO(2) product is produced electronically excited, as detected by measuring UV-visible fluorescence, at 193 nm and mostly in the ground electronic state at 248 nm. The Br product is detected employing resonance-enhanced multiphoton ionization with time-of-flight mass spectrometer for better understanding of the dynamics of dissociation. The forward convolution analysis of the experimental data has provided translational energy distributions and anisotropy parameters for both Br((2)P(3∕2)) and Br?((2)P(1∕2)). The average translational energies for the Br and Br? channels are 5.0 ± 1.0 and 6.0 ± 1.5 kcal∕mol. No recoil anisotropies were observed for these products. Most plausible mechanisms of OH and Br formation are discussed based on both the experimental and the theoretical results. Results suggest that the electronically excited BNP molecules at 248 and 234 nm relax to the ground state, and subsequently dissociate to produce OH and Br through different channels. The mechanism of OH formation from BNP on excitation at 193 nm is also discussed.