Photoconversion Of Phytochrome Research Articles

The Photoconversion of Phytochrome Includes an Unproductive Shunt Reaction Pathway.

Phytochromes are modular bimodal photoswitches that control gene expression for morphogenetic processes in plants. These functions are triggered by photoinduced conversions between the inactive and active states of the photosensory module, denoted as Pr and Pfr, respectively. In the present time-resolved resonance Raman spectroscopic study of bacterial representatives of this photoreceptor family, we demonstrate that these phototransformations do not represent linear processes but include a branching reaction back to the initial state, prior to (de)activation of the output module. Thus, only a fraction of the photoreceptors undergoing the phototransformations can initiate the downstream signaling process, consistent with phytochrome's function as a sensor for more durable changes of light conditions.

Phytochrome regulation of plant immunity in vegetation canopies.

Plant immunity against pathogens and herbivores is a central determinant of plant fitness in nature and crop yield in agroecosystems. Plant immune responses are orchestrated by two key hormones: jasmonic acid (JA) and salicylic acid (SA). Recent work has demonstrated that for plants of shade-intolerant species, which include the majority of those grown as grain crops, light is a major modulator of defense responses. Light signals that indicate proximity of competitors, such as a low red to far-red (R:FR) ratio, down-regulate the expression of JA- and SA-induced immune responses against pests and pathogens. This down-regulation of defense under low R:FR ratios, which is caused by the photoconversion of the photoreceptor phytochrome B (phyB) to an inactive state, is likely to help the plant to efficiently redirect resources to rapid growth when the competition threat posed by neighboring plants is high. This review is focused on the molecular mechanisms that link phyB with defense signaling. In particular, we discuss novel signaling players that are likely to play a role in the repression of defense responses under low R:FR ratios. A better understanding of the molecular connections between photoreceptors and the hormonal regulation of plant immunity will provide a functional framework to understand the mechanisms used by plants to deal with fundamental resource allocation trade-offs under dynamic conditions of biotic stress.

Phytochrome-controlled level of growth substances in etiolated oat seedlings

Irradiation with red light of coleoptiles and leaves of etiolated oat seedlings, causing photoconversion of phytochrome mainly into P<sub>fr</sub>, leads to the release of free auxins and free gibberellins from conjugated forms. The effect of red light is reversible by far-red light irradiation. A correlation between the photostationary state of phytochrome and endogenous abscisic acid content was not found.

Phytochrome and endogenous gibberellin-like substances in etiolated and irradiated oat seedlings

The level of gibberellin-like substances was investigated in oat coleoptiles with different stationary states of phytochrome and in leaf segments which had been etiolated and irradiated with red light. Etiolated coleoptiles and leaves containing maximum amounts of the PR form of phytochrome were characterized by an increased level of bound gibberellins. Irradiation with red light resulting in the photoconversion of phytochrome into the Pt R form caused the appearance of a high content of free gibberellins. It seems that the releasing the hormones from bound forms correlated with the formation of phytochrome PFR may be an important aspect of the mechanism of phytochrome action in the processes of seedling deetiolation. The interrelation between phytochrome and plant hormones in the control of photomorphogenesis of young monocotyledonous seedlings is also discussed.

Lack of influence of low temperature, light and growth substances on phytochrome resynthesis in coleoptiles of irradiated oat seedlings

The photoconversion of phytochrome P<sub>R</sub> into the P<sub>FR</sub> form causes at the same time the destruction of the initial large fraction of phytochrome found in the coleoptiles of etiolated oat seedlings. Factors such as low temperature, light of different wavelengths or growth substances are not capable of preventing the progressive destruction and restore the synthesis of phytochrome. Thus an abnormally high level of phytochrome is found only in etiolated seedlings. Such seedlings, on the other hand, are characterized by a very high rate of elongation growth. The role of phytochrome in the control of deetiolation of seedlings is discussed.

Altered Dark- and Photoconversion of Phytochrome B Mediate Extreme Light Sensitivity and Loss of Photoreversibility of the phyB-401 Mutant

The phyB-401 mutant is 103 fold more sensitive to red light than its wild-type analogue and shows loss of photoreversibility of hypocotyl growth inhibition. The phyB-401 photoreceptor displays normal spectral properties and shows almost no dark reversion when expressed in yeast cells. To gain insight into the molecular mechanism underlying this complex phenotype, we generated transgenic lines expressing the mutant and wild-type phyB in phyB-9 background. Analysis of these transgenic lines demonstrated that the mutant photoreceptor displays a reduced rate of dark-reversion but normal Pfr to Pr photoconversion in vivo and shows an altered pattern of association/dissociation with nuclear bodies compared to wild-type phyB. In addition we show (i) an enhanced responsiveness to far-red light for hypocotyl growth inhibition and CAB2 expression and (ii) that far-red light mediated photoreversibility of red light induced responses, including inhibition of hypocotyl growth, formation of nuclear bodies and induction of CAB2 expression is reduced in these transgenic lines. We hypothesize that the incomplete photoreversibility of signalling is due to the fact that far-red light induced photoconversion of the chromophore is at least partially uncoupled from the Pfr to Pr conformation change of the protein. It follows that the phyB-401 photoreceptor retains a Pfr-like structure (Pr *) for a few hours after the far-red light treatment. The greatly reduced rate of dark reversion and the formation of a biologically active Pr * conformer satisfactorily explain the complex phenotype of the phyB-401 mutant and suggest that amino acid residues surrounding the position 564 G play an important role in fine-tuning phyB signalling.

Femtosecond Kinetics of Photoconversion of the Higher Plant Photoreceptor Phytochrome Carrying Native and Modified Chromophores

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 (P r) and far-red absorbing phytochrome (P fr) forms. Native P r phytochrome shows an excitation wavelength dependence of the kinetics with three main picosecond components. The formation kinetics of the first ground-state intermediate I 700, 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 I 700 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 P r to P fr isomerization primarily via a different configurational pathway, largely circumventing I 700 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 P fr 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 I fr·750, due to its pronounced absorption at that wavelength. The other intermediates are termed I fr·675 and pseudo-P r. The absorption spectrum of the latter already closely resembles the absorption of the P r 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.

Agrobacterium Phytochrome as an Enzyme for the Production of ZZE Bilins

Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2.

Protonation state and structural changes of the tetrapyrrole chromophore during the Pr --> Pfr phototransformation of phytochrome: a resonance Raman spectroscopic study.

The photoconversion of phytochrome (phytochrome A from Avena satina) from the inactive (Pr) to the physiologically active form (Pfr) was studied by near-infrared Fourier transform resonance Raman spectroscopy at cryogenic temperatures, which allow us to trap the intermediate states. Nondeuterated and deuterated buffer solutions were used to determine the effect of H/D exchange on the resonance Raman spectra. For the first time, reliable spectra of the "bleached" intermediates meta-R(A) and meta-R(C) were obtained. The vibrational bands in the region 1300-1700 cm(-)(1), which is particularly indicative of structural changes in tetrapyrroles, were assigned on the basis of recent calculations of the Raman spectra of the chromophore in C-phycocyanin and model compounds [Kneip, C., Hildebrandt, P., Németh, K., Mark, F., Schaffner, K. (1999) Chem. Phys. Lett. 311, 479-485]. The experimental resonance Raman spectra Pr are compatible with the Raman spectra calculated for the protonated ZZZasa configuration, which hence is suggested to be the chromophore structure in this parent state of phytochrome. Furthermore, marker bands could be identified that are of high diagnostic value for monitoring structural changes in individual parts of the chromophore. Specifically, it could be shown that not only in the parent states Pr and Pfr but also in all intermediates the chromophore is protonated at the pyrroleninic nitrogen. The spectral changes observed for lumi-R confirm the view that the photoreaction of Pr is a Z --> E isomerization of the CD methine bridge. The subsequent thermal decay reaction to meta-R(A) includes relaxations of the CD methine bridge double bond, whereas the formation of meta-R(C) is accompanied by structural adaptations of the pyrrole rings B and C in the protein pocket. The far-reaching similarities between the chromophores of meta-R(A) and Pfr suggest that in the step meta-R(A) --> Pfr the ultimate structural changes of the protein matrix occur.

Soil water thresholds for photoinduction of redroot pigweed germination

Perception of light by phytochrome is a mechanism that triggers weed seed germination in response to soil disturbance. Photoconversion of phytochrome from the red light absorbing form to the active far-red absorbing form depends on hydration of phytochrome. This research was conducted to determine the soil water threshold for the photoinduction of germination by the brief exposure of light that occurs during soil disturbance, and to determine how this threshold is affected by the fluence of the light stimulus and fluence sensitivity of the seed population. Redroot pigweed seedling emergence and germination response to red light (R) was studied for a range of water potentials. Water potential gradients were established by incubating seeds in soils wetted to various water contents, or in polyethylene glycol 8000 (PEG) solutions. After imposing the light treatments, seeds were returned to a fully hydrated state. Seedling emergence in response to R increased as the volumetric water content (θv) of soils increased. At volumetric water contents of 4.0%, R-induced seedling emergence was inhibited 50% compared to photoinduced seedling emergence at the highest soil water contents tested. Attenuation of photoinduction was more pronounced at low vs. high R fluences in freshly imbibed seeds, but was unaffected in seeds that exhibited enhanced fluence sensitivity. In ecosystems where seasonal soil moisture extremes are prevalent, the photoinduction of seed germination may be limited in dry microsites such as surface crusts or under extreme drought conditions.

Photoreversible change in the conformation of phytochrome as probed with a covalently bound fluorescent sulfhydryl reagent, N-(9-acridinyl)maleimide

A fluorescent SH reagent, N-(9-acridiny1)maleimide (NAM), was used as a fluorescent probe to detect changes in the conformation of phytochrome from pea ( Pisum sativum) seedlings during photoconversion between the red-light absorbing form (P r) and the far-red-light absorbing form (P fr). NAM- Cys conjugates emitted weaker fluorescence in non-polar solvent than in polar solvent. The fluorescence intensity (FI) of NAM-phytochrome conjugates depended on the absorbing form of phytochrome: the FI of NAM- rwas greater than that of NAM-P fr, indicating that the Cys residues modified by NAM were in more hydrophobic environment in P fr than in P r. The FI of the conjugate prepared from a red-light-irradiated sample of phytochrome was greater than that for the conjugate prepared from P r, indicating that more Cys residues were modifiable in P fr than P r. The fluorescence polarization of the conjugate (0.0251) indicates that the modified Cys residues may be located at the surface of the phytochrome molecule. The FI of phytochrome conjugates with 8-anilinonaphthalene-1-sulfonate (ANS) did not change with the photoconversion of phytochrome. The FI of a mixture of ANS and phytochrome increased upon the first photoconversio n of P r to P fr. However, it did not change upon subsequent photoconversion between P r and P fr. These results suggest that the initial increase in FI may have resulted from the binding of additional ANS to P fr and that the microenvironment of bound ANS may not be influenced by the photoconversion of phytochrome.

Phosphopeptide mapping of Avena phytochrome phosphorylated by protein kinases in vitro

We previously demonstrated that protein kinases are useful probes of conformational changes that occur upon photoconversion of phytochrome [Wong, Y.-S., Cheng, H.-C., Walsh, D. A., & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097]. Here we present phosphopeptide analyses of oat phytochrome phosphorylated by three mammalian protein kinases and by a polycation-stimulated, phytochrome-associated protein kinase. Phosphorylation of the Pr form by the cAMP-dependent protein kinase occurs predominantly on Ser17 while Ser598 is the preferred phosphorylation site on Pfr. The cGMP-dependent and Ca2(+)-activated, phospholipid-dependent protein kinases, which phosphorylate only the Pr form of phytochrome, recognize the same region on the phytochrome polypeptide as the cAMP-dependent protein kinase. Polycation-stimulated phytochrome phosphorylation reveals that, in contrast to the mammalian enzymes, the plant kinase recognizes the serine-rich, blocked N-terminus of phytochrome. The potential regulatory role of phytochrome phosphorylation, particularly in the structurally conserved serine/threonine-rich N-terminal region of the phytochrome polypeptide, is suggested by these results.

Water content and the conversion of phytochrome regulation of lettuce dormancy.

In an effort to determine which biological reactions can occur in relation to the water content of seeds, the regulation of lettuce seed dormancy by red and far red light was determined at various hydration levels. Far red light had an inhibiting effect on germination for seeds at all moisture contents from 4 to 32% water. Germination was progressively stimulated by red light as seed hydration increased from 8 to 15%, and reached a maximum at moisture contents above 18%. Red light was ineffective at moisture contents below 8%. Seeds that had been stimulated by red light and subsequently dried lost the enhanced germinability if stored at moisture contents above 8%. The contrast between the presumed photoconversion of phytochrome far red-absorbing (Pfr) to (Pr) occurring at any moisture content and the reverse reaction occurring only if the seed moisture content is greater than 8% may be explained on the basis of the existence of unstable intermediates in the Pr to Pfr conversion. Our results suggest that the initial photoreaction involved in phytochrome conversion is relatively independent of water content, while the subsequent partial reactions become increasingly facilitated as water content increases from 8 to 18%.

Single- and Double-flash Photoconversion of Phytochrome in vivo and in vitro

Native (124 kDa) phytochrome was isolated from etiolated oat seedlings ( Avena sativa L., cv. «Pirol»). Photoconversion of the red absorbing form (P r ) to the far-red absorbing form (P fr ) by single- and double-flash or pulse irradiation was compared with induction of germination of positive photoblastic lettuce seeds ( Lactuca sativa L., cv. «Perax») by the same irradiation program. Fluence-response curves for single-flash irradiation (12 ms) in vitro were recorded for 618, 666 and 697 nm, saturating at 200 to 500 Jm −2 with the saturation levels of P fr /P tot around 48 %, 52 %, and 43 %, respectively. The corresponding values for pulse irradiation in the range of minutes were 90 %, 87 %, and 67 %, respectively. The low saturation levels, for single-flash irradiation, are explained by a photoequilibrium P r ⇌ meta-Ra, established during the ms-flash. Double-flash irradiation with 10 s dark interval increased the saturation level, for all tested wavelengths, over that measured for single-flash irradiation. This is explained by assuming that for double-flash irradiation the photoequilibrium P r ⇌ meta-Ra can be established twice. For induction of seed germination similar characteristics were reported using single- and double-flash or pulse irradiation. Kinetics for double-flash irradiation show a biphasic shape. Extending the dark interval between the two flashes from about 10 ms to 1 s, first the effect increases, but from 1 to 10 s it decreases. The same pattern is obtained if the initial red flash is followed by a 710 nm flash. If the second flash is at 744 nm, the kinetics show a permanent drop from about 10 ms to 10 s. The effects are explained by photoreversibility of the intermediates meta-Ra and meta-Rc as well as of P r and P fr . Kinetic data, obtained in vitro at 8 or 10 °C, show similar pattern to the photoinduced germination in lettuce at 24.5 °C. An expected temperature effect may be partly compensated for by deviating viscosity of the phytochrome environment in vivo (in seeds) versus in vitro (in solution).

Effect of malformin on phytochrome reactions in <italic>Vigna</italic> and <italic>Avena</italic>

The rate of destruction of the far red absorbing form of phytochrome (Pfr) in green or etiolated cuttings of Vigna radiata was slower in the presence of malformin than in its absence. Malformin had no effect on the accumulation of total phytochrome in the dark, or on the reaccumulation of phytochrome after destruction in red light. The amount of photoconversion of the red absorbing form of phytochrome (Pr) to Pfr or Pfr to Pr by given doses of red or far red radiation was slightly but consistently less in malformin-treated cuttings of V. radiata than in controls. Malformin had no effect on the rate of destruction or photoconversion of phytochrome in etiolated shoots of Avena sativa. The decrease in destruction rate of Pfr by malformin in V. radiata may contribute to the inhibition of dark abscission by malformin after light treatment.

Phytochrome and internode elongation in Chenopodium polyspermum L. sites of photoreception

The elongation of the fourth internode of 'fully green' Chenopodium polyspermum L. is modulated by far-red light (FR) given in addition to the main light period. Two different types of organs are responsible for the photoreception of FR producing the end-of-day effect; the stem and the leaves situated just above and below the reacting internode. Photoreversibility can be obtained within certain limits in the two organs. Evidence is presented which shows that in the 'fully green' plant there is an interorgan reaction whose primary reaction is the photoconversion of phytochrome.

Red/far-red modulation in vitro of enzyme activity in a membrane fraction from Phaseolus aureus

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.

Particle-bound phytochrome from maize and pumpkin.

PHYTOCHROME-induced changes in membrane properties have been inferred from physiological studies for some time1–7. Hendricks and Borthwick6 have proposed that the primary action of phytochrome is indeed the induction of such membrane changes and Smith7 has proposed a mechanism for phytochrome-membrane interaction. The interest in a search for evidence of a direct association between phytochrome and cellular membranes has therefore been considerable. The polarotropic response of Dryopteris8, chloroplast movement in Mougeotia9 and the photoconversion of phytochrome in corn coleoptiles in polarized light10 have all been taken to indicate that at least some phytochrome is located and orientated in the periphery of the cytoplasm or the plasmalemma itself. An immunological approach has been attempted11 and Ptr-induced changes in black lipid membrane permeability have recently been reported12 (Pr and Ptr are the two photo-reversible forms of phytochrome). Rubinstein et al.13 reported that about 4% of the total extractable phytochrome from Avena could be pelleted at 40,000g from a 1,500g supernatant, pH 7.4. Here we report the presence of particle-bound phytochrome in extracts from maize coleoptiles and pumpkin hooks and suggest a mechanism for the interaction of the pigment with its binding site(s).