Red-absorbing Form Of Phytochrome Research Articles

Assembly of Synthetic Locked Chromophores with Agrobacterium Phytochromes Agp1 and Agp2

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15=C16 double bond is fixed in either the E or Z configuration and the C14-C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4=C5 double bond is fixed in the Z configuration, and the C5-C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4=C5 Z C5-C6 syn C15=C16 Z C14-C15 anti stereochemistry and that in the Pfr chromophore the C15=C16 double bond has isomerized to the E configuration, whereas the C14-C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5-C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs.

Sterically Locked Synthetic Bilin Derivatives and Phytochrome Agp1 from Agrobacterium tumefaciens Form Photoinsensitive Pr- and Pfr-like Adducts

Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15=C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15=C16 double bond is in either the Z or E configuration and the C14-C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.

Protonated 2,3-Dihydrobilindiones—Models for the Chromophores of Phycocyanin and the Red-Absorbing Form of Phytochrome

Protonated 2,3-Dihydrobilindiones—Models for the Chromophores of Phycocyanin and the Red-Absorbing Form of Phytochrome

PHYTOCHROME ACTION AT HIGH PHOTON FLUENCE RATES: RAPID EXTENSION RATE RESPONSES OF LIGHT‐GROWN MUSTARD TO VARIATIONS INFLUENCE RATE and RED: FAR‐RED RATIO*

Abstract— Extension growth rate of light‐grown mustard (Sinapis alba L.) seedlings was monitored continuously using a sensitive linear displacement transducer system. When high fluence rates (ca 2 mmol m−2 s_1) of mixed red and far‐red light were presented to the growing internodes from fibre optic probes, fluctuations in extension rate occurred during the first 30 min. High red: far‐red ratios (R: FR) caused growth deceleration, whilst low R: FR caused transitory growth acceleration. These changes in extension rate were not exactly as predicted from the proportions of Pr (the red‐absorbing form of phytochrome) and Pfr (the far‐red absorbing form of phytochrome) calculated to be established by the light sources. Nevertheless, the data demonstrate that phytochrome is able to control extension growth at fiuence rates approaching those of summer sunlight, thereby providing the capacity to sense the presence of neighbouring vegetation before shading seriously compromises photosynthesis.Varying fiuence rate over two orders of magnitude whilst maintaining R: FR constant evoked transient fluctuations in extension rate. At high R: FR, a 100‐fold step down in fiuence rate led, after a lag of ca 10 min, to a transient (i.e. 20 min) deceleration of extension that was followed by a marked transient (i.e. 20 min) acceleration. After a 100‐fold step up in fiuence rate, a transient (i.e. 20 min) acceleration only was observed, beginning after a lag of ca 10 min. When R: FR was low, neither a step‐down nor a step‐up in fluence rate resulted in appreciable fluctuations in extension rate.The data are discussed in relation to the possible role played by the accumulation of photoconversion intermediates using a simple computer model for simulating active phytochrome concentrations at high fluence rates. The possibility that the mechanism for the photoperception of light quality by phytochrome may be capable of rapid adaptation to fluence rate fluctuations is proposed.

Control Processes in the Induction and Relief of Thermoinhibition of Lettuce Seed Germination

Germination of lettuce seeds (Lactuca sativa L. cv Grand Rapids) in the dark was nearly 100% at 20 degrees C but was inhibited at 27 degrees C and higher temperatures (thermoinhibition). A single 5-minute exposure to red light completely overcame the inhibition at temperatures up to 28 degrees C, above which the effectiveness of single light exposures gradually declined to reach a negligible level at 32 degrees C. However, the promotive effect of light could be extended to 34 degrees C by repeated irradiations. At any one temperature, increased frequency of irradiations increased germination percentage, and with each degree increase in temperature, increasingly frequent irradiations were necessary to elicit maximal germination. Loss of the effectiveness of single irradiations with increase in temperature may result either from acceleration of the thermal reversion of the far red-absorbing form of phytochrome or decrease in seed sensitivity toward a given percentage of the far red-absorbing form of phytochrome. Using continuous red light to induce germination, the role of endogenous C(2)H(4) in germination at 32 degrees C was studied. Ethylene evolution from irradiated seeds began to increase 2 hours prior to radicle protrusion, whereas the dark-incubated (nongerminating) seeds produced a low, constant amount of C(2)H(4) throughout the 24 hour incubation period. Inhibition of C(2)H(4) synthesis with 2-aminoethoxyvinyl glycine and/or inhibition of C(2)H(4) action with 2,5-norbornadiene blocked the promotive effect of light. Exogenous C(2)H(4) overcame these blockages. The results showed that participation by endogenous C(2)H(4) was essential for the light-induced relief of thermoinhibition of lettuce seed germination. However, light did not act exclusively via C(2)H(4) since exogenous C(2)H(4) alone in darkness did not promote germination.

Polyclonal antibodies raised to phycocyanins contain components specific for the red-absorbing form of phytochrome

Polyclonal antibodies raised in rabbits to a mixture of sodium-dodecyl-sulphate-denatured C- and allo-phycocyanin, isolated from Anabaena cylindrica, cross-react with 124-kilodalton (kDa) phytochrome from etiolated oats, in enzyme-linked immunosorbent assays and on Western blots. The component(s) of the anti-phycocyanin serum that cross-reacts with phytochrome appears to be specific for the red-absorbing form of phytochrome (Pr). These antibodies can be detached from Pr by irradiation with red light, and thus show photoreversible binding. This property has been used to immunopurify the anti-phytochrome component from the antiserum using red light as the eluting agent. Competition assays and epitope-mapping studies indicate that the anti-phytochrome component may bind to a site located between 6 and 10 kDa from the amino-terminus of etiolated oat phytochrome.

Sequence analysis of proteolytic fragments of 124-kilodalton phytochrome from etiolatedAvena sativa L.: Conclusions on the conformation of the native protein

Proteolytic fragments were obtained by limited proteolysis of 124-kDa (kilodalton) phytochrome from etiolatedAvena sativa using trypsin, endoproteinase-Lys-C, endoproteinase-Glu-C and subtilisin. The fragments were separated by sodium dodecyl sulfate gel electrophoresis, blotted onto activated glass-fiber sheets and investigated by amino-acid sequencing in a gas-phase sequencer. Determination of N-terminal sequences in three to six Edman degradation steps allowed the exact localization of the fragments within the published entire amino-acid sequence of 124-kDaAvena phytochrome (H.P. Hershey, R.F. Barker, K.B. Idler, J.L. Lissemore, P.H. Quail (1985), Nucleic Acids Res.13, 8543-8559). From the knowledge of the exact sites for preferred proteolytic cleavage of undenatured phytochrome, conclusions on the conformation of the phytochrome protein were drawn. Sites of preferred cleavage are considered to be freely exposed to the environment whereas potential cleavage sites which are resistant to proteolysis over a long time are considered to be localized in the interior of the native phytochrome. Two different sites which are exposed in the far-red-absorbing form but not in the red-absorbing form of phytochrome are localized at amino-acid residues 354 and 753, respectively. The N-terminal region which is exposed only in the red-absorbing form stretches only as far as amino-acid residue 60.

A Kinetic Analysis of Phytochrome Controlled Mesocotyl Growth in Zea mays Seedlings

Mesocotyl elongation in 4 day old etiolated seedlings immediately following 3 hours of white light (3 h W) is reversibly controlled by phytochrome. Time-lapse video measurements were made of the 5 millimeter zone just below the coleoptile which is the main growth region of the mesocotyl. The growth kinetics were determined for five contiguous 1 millimeter zones subtending the coleoptile node for nonirradiated seedlings, for seedlings given 3 h W, and 3 h W followed by terminal far-red (FR) or red subsequent to the far-red (FR/R) irradiation. Each zone in nonirradiated seedlings exhibits exponential elongation kinetics during the early stages of elongation. This finding suggests that during elongation, a growth limiting factor is also exponentially increasing. Following 3 h W differences in the kinetic responses were found for each zone. In all zones, the inhibitory effect following the 3 h W is totally FR reversible. The effect of FR is reversed by R. The upper zone exhibits the fastest response and is the most plastic in its growth response. The three upper zones all exhibit spontaneous and sharp recoveries with time. It is suggested that the control by phytochrome is not inductive but rather continuous, the controlling factor being either the level of the far red-absorbing form of phytochrome (Pfr) or the ratio Pfr to total phytochrome.

Studies of the Mechanism of Enhancement of Phytochrome-dependent Lettuce Seed Germination by Prechilling

Temperature and kinetic studies were performed to examine the mechanism by which prechilling stimulates phytochrome-dependent seed germination in lettuce, Lactuca sativa, L. cv. Grand Rapids. Imbibed seeds were given a short far red irradiation and one day of dark incubation at 20 C to establish very low levels of the far red-absorbing form of phytochrome-(Pfr). Germination was greatly stimulated by subsequent prechilling treatments when they were followed by a second short far red irradiation. Prechilling therefore increased germination sensitivity to the low, normally inhibitory Pfr levels established by far red irradiation. This sensitivity increased with lowered prechilling temperature to a maximum near 4 C. It was linearly dependent upon duration of prechilling at 4 C up to a near maximal response at 10 hours, and it decayed in a converse manner when seeds were returned to 20 C after 10 hours at 4 C. Prechilling also increased germination responses to subsequent periods of high levels of Pfr which were initiated by red and terminated by far red irradiations. High Pfr periods adequate to promote the germination of unchilled seeds produced sharp inflections at 18 C in the dependence of germination on prechilling temperature. Rates of phytochrome potentiation of germination were not affected by prechilling. The response to prechilling fit a mechanism involving homeoviscous adaptation of membrane lipids to temperature.

Photocontrol of Anthocyanin Synthesis

Under continuous far red light, anthocyanin synthesis in young, dark-grown cabbage seedlings (Brassica oleracea cv. Red Acre) is irradiance-dependent and fails to follow the reciprocity (irradiance x time = constant) relationships. Under intermittent far red treatments extended over a prolonged period of time, anthocyanin synthesis becomes dose dependent, and reciprocity relationships are valid. Intermittent far red treatments with short dark intervals between successive irradiations are as effective as continuous treatments, if the total radiation doses applied with the two types of treatments are equal and are applied over equally long periods of time. The high effectiveness of inter-mittent treatments, the dose dependence, and the validity of the reciprocity relationships suggest that cycling between red-absorbing form of phytochrome and far red-absorbing form of phytochrome and the formation of electronically excited far red-absorbing form of phytochrome, or the involvement of a second photoreactive system, besides phytochrome, may play only a minor role in high irradiance reaction anthocyanin synthesis brought about by prolonged exposures to far red irradiation.

Photomanipulation of Phytochrome in Lettuce Seeds

Seeds of lettuce (Lactuca sativa L. cv. Grand Rapids) were imbibed and given either short irradiation with red or far red light prior to drying or dried under continuous red or far red light. Seeds treated with either short or continuous red germinate in darkness, whereas seeds treated with either short or continuous far red require a short exposure to red light, after a period of imbibition, to stimulate germination. Irradiation of dry red seeds with far red light immediately before sowing results in a marked inhibition of germination. This result was predicted since far red-absorbing form phytochrome can be photoconverted to the intermediate P650 (absorbance maximum 650 nm) in freeze-dried tissue. A similar far red treatment to continuous red seeds is less effective and it is concluded that in these seeds a proportion of total phytochrome is blocked as intermediates between red-absorbing and far red-absorbing form phytochrome, which only form the far red-absorbing form of phytochrome on imbibition. The inhibition of dry short red seeds by far red light can be reversed by an irradiation with short red light given immediately before sowing, confirming that P650 can be photoconverted back to the far red-absorbing form of phytochrome. The results are discussed in relation to seed maturation (dehydration) on the parent plant.

Acceleration of Dark Reversion of Phytochrome in Vitro by Calcium and Magnesium

Rates of dark reversion of the far red-absorbing form of phytochrome, Pfr, to the red-absorbing form, Pr, have been determined in the presence of several salts. Low concentrations of calcium chloride and magnesium chloride (up to 3 mm) accelerated the rate of dark reversion at all stages of purification of phytochrome from etiolated rye (Secale cereale L. cv. Balbo) seedlings. The complex kinetics of the dark reversion could be resolved into two first-order components. The effect of the added divalent cations was on the relative proportion of the fast and slow reacting components, rather than on the rate constants of the two populations. It was possible to reverse the effects of the cations by adding the chelating agents ethylene-bis-(oxyethylene-nitrilo) tetraacetic acid or ethylenediaminetetraacetate. The effect of the divalent cations is not a nonspecific ionic strength effect. The relative proportion of the two populations was also affected by the degree of purity of the phytochrome samples.

Inhibition of Prechill-induced Dark Germination in Sorghum halepense (L.) Pers. Seeds by Phytochrome Transformations

A 10 C dark prechilling of johnsongrass [Sorghum halepense (L.) Pers.] seeds, when terminated by a 2-hr, 40 C temperature shift, potentiates about 40% germination at 20 C in darkness. Irradiation of the seeds before, during, and at the end of prechilling with far red light reduces the subsequent germination, although red irradiation after the far red can overcome some of the inhibition. However, either brief red or far red irradiation given immediately after the temperature shift inhibits subsequent germination by one-third to one-half. The results suggest that the far red-absorbing form of phytochrome is a factor in the prechill-induced dark germination and that phytochrome participates in the inhibition of germination by irradiations immediately after the temperature shift.

Loss of Phytochrome Photoreversibility in Vitro

Crude pea (Pisum sativum L. var. Alaska) phytochrome extracts contain a substance, "Killer," which interacts with the far red-absorbing form of phytochrome causing a net loss of spectrophotometrically detectable phytochrome in vitro. Killer is absent from crude extracts of Avena phytochrome, is separable from pea phytochrome by gel filtration, and is alcohol-extractable from etiolated pea seedlings. Killer activity in alcohol extracts behaved, during partial purification, in a manner identical to that derived from pea phytochrome preparations. The mass extraction and partial purification of Killer are described.

Dark Reversion of Phytochrome in Lettuce Seeds Stored in a Water-saturated Atmosphere

Dark reversion of the far red-absorbing form of phytochrome, which does not occur in dry lettuce (Lactuca sativa var. Grand Rapids) seeds, appears to take place in seeds stored in a water-saturated atmosphere. The water content (approximately 70% after 10 days) of such seeds is insufficient to support germination; however the treatment enhances germination in seeds stored for 1 to 5 days, but this enhancement subsequently disappears, and the effect of extended storage (up to 28 days) is inhibiting. The half-time for dark far red-absorbing phytochrome reversion is 7 to 8 days, and at this time it can be completely reversed by exposing the seeds to a flash of red light. Storage of more than 7 to 8 days decreases red light enhancement of germination.

Phytochrome Control of Germination of Rumex crispus L. Seeds Induced by Temperature Shifts

High germination of curly dock (Rumex crispus L.) seeds is evident after suitable imbibition and temperature shift treatment, but germination at constant temperatures fails without an input of far red-absorbing form of phytochrome. Preliminary imbibitions at high temperatures (30 C) sharply reduce germination induced by temperature shifts. High germination may be restored by low energies of red radiation, or by brief far red adequate for the photosteady state. Prolonged far red during imbibition also nullifies temperature shift-induced germination. After prolonged far red, high germination may be restored by red radiation of an energy dependent upon the duration of the far red treatment. The evidence supports the conclusion that dark germination induced by temperature shifts arises from the interaction of pre-existent far red-absorbing form of phytochrome in the mature seeds with the temperature shift.

Phytochrome Control of the Level of Extractable Ribonuclease Activity in Etiolated Hypocotyls

AN increase in RNA degrading activity of hypocotyl homo-genates can be detected 6–10 h after transferring 5 day old, dark-grown seedlings of Lupinus albus L. to continuous white light1. This increase is concomitant with the onset of inhibition of hypocotyl extension1, 2. The ribonuclease component whose level of activity is affected by light treatment (tentatively identified as a ribonucleate nucleotido-2′-transferase (cyclizing) (EC 2.7.7.17)) is associated with the ribosomes (and polysomes)1 in hypocotyl homogenates. This is in contrast to the bulk of RNA degrading activity in homogenates which is associated with the soluble fraction1. Isolation of the ribosomal fraction therefore makes it possible to follow changes in the level of activity of this ribonuclease which would be too small to detect in unfractionated cell homogenates. The results reported here indicate that the photoconversion of the red-absorbing form of phytochrome (Pr) to the physiologically-active far-red-absorbing form (Pfr) leads to a specific increase in ribosome-bound ribonuclease activity.