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1.
Fluorescence lifetimes of 'large (mol. wt. 120,000) and 'small' (mol. wt. 60,000) phytochromes isolated from oat and rye seedlings grown in the dark have been measured at 199 K and 298 K. Phytochrome model compounds have also been studied by phase modulation fluorometrically at 77 K for comparison with lifetime data for phytochrome. It was found that the fluorescence lifetime of 'large' phytochrome was significantly shorter than that of 'small' phytochrome and its chromophore models. The phytochrome chromophore of Pr form has been analyzed by fluorescence polarization, CD, and molecular orbital methods. The fluorescence excitation polarization of 'small' phytochrome and the chromophore model in buffer/glycerol mixture (3 : 1, v/v) at 77 K is very hight (0.4) at the main absorption band and is negative (--0.1) and close to 0 in the near ultraviolet band, respectively. Analyses of the spectroscopic data suggest that the chromophore conformation of Pr and Pfr forms of phytochrome are essentially identical. The induced ellipticity of 'large' rye phytochrome in the blue and near ultraviolet regions was found to be significantly higher than that of 'small' phytochrome, indicating that the binding interaction between the phytochrome chromophore and apoprotein is much tighter in the former than in the latter. In addition, the excitation energy transfer does occur from Trp residue(s) to the chromophore in 'large' phytochrome but not in 'small' Pr. This illustrates one feature of the role played by the large molecular weight apoprotein in the binding site interactions and primary photoprocesses of Pr. Finally, a plausible model for the primary photoprocesses and the mechanism of phytochrome interactions triggered by the Pr leads to Pfr phototransformation have been proposed on the basis of the above results. 相似文献
2.
The photoprocesses of native (phyA of oat), and of C-terminally truncated recombinant phytochromes, assembled instead of the native phytochromobilin with phycocyanobilin (PCB-65 kDa-phy) and iso-phycocyanobilin (iso-PCB-65 kDa-phy) chromophores, have been studied by femtosecond transient absorption spectroscopy in both their red absorbing phytochrome (Pr) and far-red absorbing phytochrome (Pfr) forms. Native Pr phytochrome shows an excitation wavelength dependence of the kinetics with three main picosecond components. The formation kinetics of the first ground-state intermediate I700, absorbing at ∼690 nm, is mainly described by 28 ps or 40 ps components in native and PCB phytochrome, respectively, whereas additional ∼15 and 50 ps components describe conformational dynamics and equilibria among different local minima on the excited-state hypersurface. No significant amount of I700 formation can be observed on our timescale for iso-PCB phytochrome. We suggest that iso-PCB-65 kDa-phy either interacts with the protein differently leading to a more twisted and/or less protonated configuration, or undergoes Pr to Pfr isomerization primarily via a different configurational pathway, largely circumventing I700 as an intermediate. The isomerization process is accompanied by strong coherent oscillations due to wavepacket motion on the excited-state surface for both phytochrome forms. The femto- to (sub-)nanosecond kinetics of the Pfr forms is again quite similar for the native and the PCB phytochromes. After an ultrafast excited-state relaxation within ∼150 fs, the chromophores return to the first ground-state intermediate in 400-800 fs followed by two additional ground-state intermediates which are formed with 2-3 ps and ∼400 ps lifetimes. We call the first ground-state intermediate in native phytochrome Ifr·750, due to its pronounced absorption at that wavelength. The other intermediates are termed Ifr·675 and pseudo-Pr. The absorption spectrum of the latter already closely resembles the absorption of the Pr chromophore. PCB-65 kDa-phy shows a very similar kinetics, although many of the detailed spectral features in the transients seen in native phy are blurred, presumably due to wider inhomogeneous distribution of the chromophore conformation. Iso-PCB-65 kDa-phy shows similar features to the PCB-65 kDa-phy, with some additional blue-shift of the transient spectra of ∼10 nm. The sub-200 fs component is, however, absent, and the picosecond lifetimes are somewhat longer than in 124 kDa phytochrome or in PCB-65 kDa-phy. We interpret the data within the framework of two- and three-dimensional potential energy surface diagrams for the photoisomerization processes and the ground-state intermediates involved in the two photoconversions. 相似文献
3.
4.
Phytochrome behaves as a dimer in vivo 总被引:2,自引:2,他引:0
J. BROCKMANN S. RIEBLE N. KAZARINOVA-FUKSHANSKY M. SEYFRIED E. SCHÄFER 《Plant, cell & environment》1987,10(2):105-111
Abstract It is well established that phytochrome exists as a dimer in vitro. A comparison of the relative photoequilibrium concentrations of PrPr, PrPfr and PfrPfr, with the relative sizes of the Pfr-pools which undergo dark reversion in the intact plant, leads to the hypothesis that phytochrome also exists as a dimer in vivo, This hypothesis is in accordance with kinetic properties of the phytochrome system under continuous irradiation. Additional support for this view is provided by the observation that Pfr-destruction after a red light flash, which should favour the formation of PrPfr dimers, is paralleled by a decay of Pr, even if the presence of Pr cycled through Pfr can be excluded. Preliminary observations could indicate an interaction of the subunits of a phytochrome dimer during the process of phototransformation. 相似文献
5.
Spectrophotometric characterization of phytochromes in dimorphic cocklebur seeds in relation to capacity for germination 总被引:1,自引:0,他引:1
Phytochrome was measured spectrophotometrically in different tissues of the upper (positively photoblastic) and lower (negatively photoblastic) seeds of the cocklebur (Xanthium pennsylvanicum Wallr.). Axial parts of the seeds, in particular parts of the radicle, contained high levels of phytochrome, while cotyledonary parts contained only low levels. These results were consistent with the distribution of the light-sensitive areas of the seeds that were associated with germination. Phytochrome levels in both types of dimorphic seeds increased gradually with increasing duration of dark imbibition for 4–8 h, then the rates of increase in levels of phytochrome accelerated. In both types of seed, some phytochrome was measurable even before imbibition. In the lower seeds, up to 20% of the phytochrome was occasionally observed as Pfr in samples imbibed in darkness for a short time (up to 12 h). A slight blue shift of the peak of PT in the difference spectrum of phytochrome was observed in the case of lower seeds imbibed for 0–2 h. These results suggest that, to some extent, the lower axes contain dehydrated Pfr or intermediate(s) in the photoconversion of phytochrome. The dark reactions of Pfr were also examined in excised axes of both types of dimorphic seed after they had been pre-imbibed for 16 h in darkness. Dark destruction of Pfr was observed in both types of seed. In addition, net increases in levels of Pr were observed in the dark controls and in the samples irradiated with red light after the level of Pfr diminished. No ‘inverse’ dark reversion from Pr to Pfr was detected. Thus, after 16 h of imbibition, there were no differences in terms of properties of phytochrome between the two types of seed, and the different responses to light of upper and lower seeds might depend mainly on a difference in the physiological state of the two types of seed rather than the properties of phytochrome. 相似文献
6.
Abstract A series of fluence-response curves for the binding of phytochrome to membranes in the absence of divalent cations, as described by Watson & Smith (1982), were constructed to demonstrate that the response obeys the law of reciprocity. Analysis of the binding of Pfr (the far-red-absorbing form of phytochrome) showed that two Pfr molecules bind to the membrane for each Pr (the form with an absorption maximum in the red) photoconverted to Pfr in the intrinsic membrane-bound phytochrome pool. Using this stoichiometry we have been able to model the binding curve of Pr and match the binding data. Pr binding can be simulated if Pr binds only as a consequence of the binding of Pfr, i.e. when Pfr is part of a Pr: Pfr dimer. The enrichment of the membranes with Pfr as a result of the binding of Pfr was also accurately simulated. There is no binding cooperativity. Phytochrome binding is a low-fluence response and the possibility that it has physiological significance as a mediator of phytochrome action is discussed. 相似文献
7.
In the green algaMougeotia, the dichroic orientation of the red-absorbing form of phytochrome (Pr) is parallel of the cell surface, whereas the far-red-absorbing form (Pfr) is oriented normal to it. The time course of the change from parallel to normal was investigated by double-flash irradiation with polarized red and far-red light. The results obtained by two different methods indicate that most of the phytochrome intermediates existing in the first 5 ms after the inducing red flash are still oriented parallel to the cell surface, similar to Pr. At increasing intervals between the red and the far-red flashes, more and more phytochrome molecules turn their transition moments to the Pfr orientation. This reaction is finished after approximately 30 ms. We conclude that the change in dichroic orientation of the phytochrome molecules inMougeotia occurs during the last relaxation steps of the intermediates on the way from Pr to Pfr. It cannot be decided yet, whether the first surface-normal phytochrome species is an intermediate or Pfr itself.Abbreviations Pr
red-absorbing form of phytochrome
- Pfr
far-red-absorbing form of phytochrome
A preliminary report of this work was presented at the European Symposium on Photomorphogenesis, University of Reading, UK (Kraml et al. 1982) 相似文献
8.
Differential reactivity of the red-and far-red-absorbing forms of phytochrome to [14C] N-ethyl maleimide 总被引:1,自引:1,他引:0
Summary The red-absorbing form (P
r
) and the far-red absorbing form (P
fr
) of undergraded, high-molecular-weight phytochrome from rye (Secale cereale L.) seedlings were examined for their reactivity toward N-ethyl-[14C]maleimide ([14C]-NEM). After pre-treatment of P
r
with cold NEM and extensive dialysis, photoconversion to P
fr
and treatment with [14C]NEM resulted in an approximately 70% increase in incorporation of radioactivity over the dark control. These results are discussed in relation to the view that phytochrome undergoes a protein conformational change upon phototransformation. 相似文献
9.
Merten Jabben 《Planta》1980,149(1):91-96
The phytochrome system is analyzed in light-grown maize (Zea mays L.) plants, which were prevented from greening by application of the herbicide SAN 9789. The dark kinetics of phytochrome are not different in the first, second or third leaf. It is concluded that in light-grown maize plants phytochrome levels are regulated by Pr formation and Pfr and Pr destruction, rather than by PfrPr dark reversion. Pr undergoes destruction after it has been cycled through Pfr. The consequences of this Pr destruction on the phytochrome system are discussed.Abbreviations SAN 9789
4-chloro-5-(methylamino)-2-(,,-trifluoro-m-tolyl)-3(2H) pyridazinone
- Pfr
far-red absorbing form of phytochrome
- Pr
red absorbing form of phytochrome
- Ptot
Pfr+Pr 相似文献
10.
Katrin Anders Alexander Gutt Wolfgang G?rtner Lars-Oliver Essen 《The Journal of biological chemistry》2014,289(37):25590-25600
Phytochromes are photoreceptors using a bilin tetrapyrrole as chromophore, which switch in canonical phytochromes between red (Pr) and far red (Pfr) light-absorbing states. Cph2 from Synechocystis sp., a noncanonical phytochrome, harbors besides a cyanobacteriochrome domain a second photosensory module, a Pr/Pfr-interconverting GAF-GAF bidomain (SynCph2(1-2)). As in the canonical phytochromes, a unique motif of the second GAF domain, the tongue region, seals the bilin-binding site in the GAF1 domain from solvent access. Time-resolved spectroscopy of the SynCph2(1-2) module shows four intermediates during Pr → Pfr phototransformation and three intermediates during Pfr → Pr back-conversion. A mutation in the tongue''s conserved PRXSF motif, S385A, affects the formation of late intermediate R3 and of a Pfr-like state but not the back-conversion to Pr via a lumi-F-like state. In contrast, a mutation in the likewise conserved WXE motif, W389A, changes the photocycle at intermediate R2 and causes an alternative red light-adapted state. Here, back-conversion to Pr proceeds via intermediates differing from SynCph2(1-2). Replacement of this tryptophan that is ∼15 Å distant from the chromophore by another aromatic amino acid, W389F, restores native Pr → Pfr phototransformation. These results indicate large scale conformational changes within the tongue region of GAF2 during the final processes of phototransformation. We propose that in early intermediates only the chromophore and its nearest surroundings are altered, whereas late changes during R2 formation depend on the distant WXE motifs of the tongue region. Ser-385 within the PRXSF motif affects only late intermediate R3, when refolding of the tongue and docking to the GAF1 domain are almost completed. 相似文献
11.
Photoperiodism and rhythmic response to light 总被引:2,自引:1,他引:1
Abstract. Seedlings of Pharhitis nil show a circadian rhythm in the capacity to flower in response to the timing of a second red light pulse given at various times after a first saturating exposure to red when this is given together with a benzyladeninc spray. There are also changes in the photon irradiance required for half maximum response to the second red pulse. The photochemical properties of phytochrome in the photoperiodically sensitive cotyledons were also shown to change rhythmically. Oscillations in both pr→ Pfr and Pfr→ Pr photoconversion characteristics persisted over at least two circadian cycles with a periodicity of about 12 h. There were, however, no significant oscillations in either Pfr peak absorbance or in Δ(ΔA). The changes in sensitivity for the photoconversion of Pr→ Pfr did not parallel the much larger changes in sensitivity of the flowering response to red light. The amplitude of the Pr→ Pfr rhythm was at least as great as that for Pr→ Pfr, but the flowering response to far-red light was not rhythmic, nor was there any large change in sensitivity. The changes in photoconversion properties may reflect a basic biochemical oscillation which affects both photoreceptor properties and sensitivity to photoreceptor input. There was also a marked rhythm in the Pfr/P ratio that would be established by a saturating pulse of red light and this too may have affected the flowering response to such a pulse. Far-red light inhibited flowering when given at any time during the inductive night. After 14 h in darkness, Pfr could still be measured in the cotyledons and it was concluded that far-red light inhibited flowering by removing Pfr As red light also inhibited flowering at this time, there may be two pools of phytochrome with different kinetic properties. 相似文献
12.
In a membrane fraction isolated from hypocotyls of Phaseolus aureus Roxb. the activity of a number of enzymes was regulated by red and far-red irradiation in vitro, provided that the tissue received a brief red light treatment before extraction. Other enzymes showed no photoregulation. There were two types of photocontrol, neither of which could be detected in the solute fraction, nor in extracts from completely etiolated material. One (Type I) was a red/far-red reversible regulation of the rate of enzyme activity, depending on the light given (in vivo or in vitro) before the assay was begun. The second (Type II) was a promotion of enzyme activity by red or far-red light given during the assay. The action spectra for type II responses do not coincide with either the phytochrome absorption or difference spectra. However, the effectiveness of red and far-red was correlated with the Pfr/P ratio present at the beginning of the assay, such that far-red was more efficient at high Pfr/P and red at low Pfr/P ratios. All enzymes that were regulated involved ATP. In samples that showed enzyme regulation, small changes in fluorescence yield of tryptophan and the covalent probe Fluram (Roche) accompanied the photoconversion of phytochrome, but no fluorescence changes could be measured after briefly incubating the membrane fraction with ATP. The results indicate that light may affect the interaction of ATP with the membrane fraction.Abbreviations F
far-red light
- Pr and Pfr
phytochrome in the red and far-red absorbing forms
- Ptot
total phytochrome
- R
red light
- RNP
ribonucleoprotein 相似文献
13.
Ohára Augusto Giuseppe Cilento Jing Jung Pill-Soon Song 《Biochemical and biophysical research communications》1978,83(3):963-969
Enzymatically generated triplet acetone transfers its energy to the ground state phytochrome and promotes to some extent, in the dark, the conversion of Pr into Pfr and of Pfr into Pr. This is the first report of inverse dark reversion “in vitro”. 相似文献
14.
Flavonoid synthesis in cell suspension cultures of parsley (Petroselinum hortense Hoffm.) occurs only after irradiation with ultraviolet light (UV), mainly from the UV-B (280–320 nm) spectral range. However, it is also controlled by phytochrome. A Pfr/Ptot ratio of approximately 20% is sufficient for a maximum phytochrome response as induced by pulse irradiation. Continuous red and far red light, as well as blue light, given after UV, are more effective than pulse irradiations. The response to blue light is considerably greater than that to red and far red light. Continuous red and blue light treatments can be substituted for by multiple pulses and can thus probably be ascribed to a multible induction effect. Continuous irradiations with red, far red and blue light also increase the UV-induced flavonoid synthesis if given before UV. The data indicate that besides phytochrome a separate blue light photoreceptor is involved in the regulation of the UV-induced flavonoid synthesis. This blue light receptor seems to require the presence of Pfr in order to be fully effective.Abbreviations HIR
high irradiance response
- Pfr
far red absorhing form of phytochrome
- Ptet
total phytochrome
- UV
ultraviolet light 相似文献
15.
《BBA》2023,1864(4):148996
Using ultrafast spectroscopy and site-specific mutagenesis, we demonstrate the central role of a conserved tyrosine within the chromophore binding pocket in the forward (Pr → Pfr) photoconversion of phytochromes. Taking GAF1 of the knotless phytochrome All2699g1 from Nostoc as representative member of phytochromes, it was found that the mutations have no influence on the early (<30 ps) dynamics associated with conformational changes of the chromophore in the excited state. Conversely, they drastically impact the extended protein-controlled excited state decay (>100 ps). Thus, the steric demand, position and H-bonding capabilities of the identified tyrosine control the chromophore photoisomerization while leaving the excited state chromophore dynamics unaffected. In effect, this residue operates as an isomerization-steric-gate that tunes the excited state lifetime and the photoreaction efficiency by modulating the available space of the chromophore and by stabilizing the primary intermediate Lumi-R. Understanding the role of such a conserved structural element sheds light on a key aspect of phytochrome functionality and provides a basis for rational design of optimized photoreceptors for biotechnological applications. 相似文献
16.
Phytochromes are photoreceptors in plants which can exist in two different conformations: the red light‐absorbing form (Pr) and the far‐red light‐absorbing form (Pfr), depending on the light quality. The Pfr form is the physiologically active conformation. To attenuate the Pfr signal for phytochrome A (phyA), at least two different mechanisms exist: destruction of the molecule and dark reversion. Destruction is an active process leading to the degradation of Pfr. Dark reversion is the light‐independent conversion of physiologically active Pfr into inactive Pr. Here, we show that dark reversion is not only an intrinsic property of the phytochrome molecule but is modulated by cellular components. Furthermore, we demonstrate that dark reversion of phyA may be observed in Arabidopsis ecotype RLD but not in other Arabidopsis ecotypes. For the first time, we have identified mutants with altered dark reversion and destruction in a set of previously isolated loss of function PHYA alleles (Xu et al. Plant Cell 1995, 7, 1433–1443). Therefore, the dynamics of the phytochrome molecule itself need to be considered during the characterization of signal transduction mutants. 相似文献
17.
Carotenoid accumulation in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by light. Besides the stimulatory function of phytochrome in carotenogenesis the experiments reveal the significance of chlorophyll accumulation for the accumulation of larger amounts of acrotenoids. A specific blue light effect was not found. The data suggest that light exerts its control over carotenoid biogenesis through two separate mechanisms: A phytochrome regulation of enzyme levels before a postulated pool of free carotenoids, and a regulation by chlorophyll draining the pool by complex-formation.Abbreviations Chl
chlorophyll(s)
- PChl
protochlorophyll(ide)
- HIR
high irradiance reaction (of phytochrome)
- Pfr
far-red absorbing, physiologically active form of phytochrome
- Pr
red absorbing, physiologically inactive form of phytochrome
- Pfof
total phytochrome, i.e. [Pr]+[Pfr]
-
[Pfr]/[Pfof], wavelength dependent photoequilibrium of the phytochrome system
- red
red light
- fr
far-red light 相似文献
18.
The ability to respond to phytochrome (Pfr, the far-red light absorbing from of phytochrome) with anthocyanin synthesis appears first in some marginal regions of the abaxial epidermis of the mustard cotyledons and from there spreads gradually over the entire tissue (transient phase). The pertinent pattern is independent of environmental influences such as light quality and nutritional culture conditions. The competence for Pfr in the epidermal cells, with regard to the initial action of Pfr (concerning anthocyanin synthesis), appears considerably earlier than the ability for actual anthocyanin synthesis. An electron microscopical study of the ultrastructural changes occurring in vacuoles and plastids of the epidermal cells during the transient phase showed that a correlation only exists between the differentiation of central cell vacuoles, originating from the aleurone vacuoles, and the appearance of the ability to accumulate anthocyanin. It is suggested that the formation of a central cell vacuole is a prerequisite for anthocyanin accumulation in the epidermal cells of the mustard seedling cotyledons.Abbreviations Pr, Pfr
red and far-red absorbing forms of phytochrome
- HS
Hoagland's nutrient solution 相似文献
19.
Peter H. Quail 《Planta》1974,118(4):357-360
Summary The binding of phytochrome to a particulate fraction in extracts from hypocotyl hooks of etiolated Cucurbita pepo L. seedlings has been examined as a function of the light dose and P
fr
level established in vitro. As the steady-state level of P
fr
transiently established in the 500×g supernatant is increased, so the level of P
r
subsequently pelletable at 20 000×g increases up to a saturation level. Increasing both the time and irradiance parameters of the light dose while holding the steady-state P
fr
level constant, results similarly in increasing P
r
pelletability. This agrees with results obtained previously with in-vivo irradiations of maize coleoptiles. Thus, like the in-vivo response, phytochrome binding in vitro appears to be a function of the total number of molecules converted to the P
fr
form during the irradiation period.Abbreviations
P
fr
far-red-absorbing form of phytochrome
-
P
r
red-absorbing form of phytochrome 相似文献
20.
Two phytochromes, CphA and CphB, from the cyanobacterium Calothrix PCC7601, with similar size (768 and 766 amino acids) and domain structure, were investigated for the essential length of
their protein moiety required to maintain the spectral integrity. Both proteins fold into PAS-, GAF-, PHY-, and Histidine-kinase
(HK) domains. CphA binds a phycocyanobilin (PCB) chromophore at a “canonical” cysteine within the GAF domain, identically
as in plant phytochromes. CphB binds biliverdin IXα at cysteine24, positioned in the N-terminal PAS domain. The C-terminally
located HK and PHY domains, present in both proteins, were removed subsequently by introducing stop-codons at the corresponding
DNA positions. The spectral properties of the resulting proteins were investigated. The full-length proteins absorb at (CphA)
663 and 707 nm (red-, far red-absorbing P
r and P
fr forms of phytochromes) and at (CphB) 704 and 750 nm. Removal of the HK domains had no effect on the absorbance maxima of
the resulting PAS–GAF–PHY constructs (CphA: 663/707 nm, CphB: 704/750 nm, P
r/P
fr, respectively). Further deletion of the “PHY” domains caused a blue-shift of the P
r and P
fr absorption of CphA (λ
max: 658/698 nm) and increased the amount of unproperly folded apoprotein, seen by a reduced capability to bind the chromophore
in photoconvertible manner. In CphB, however, it practically impaired the formation of P
fr, i.e., showing a very low oscillator strength absorption band, whereas the P
r form remains unchanged (702 nm). This finding clearly indicates a different interaction between domains in the “typical”,
PCB binding and in the biliverdin-binding phytochromes, and demonstrates a loss of oscillator strength for the latter, most
probably due to a strong conformational distortion of the chromophore in the CphB P
fr form.
Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September
2006. 相似文献