Action spectra for the inhibition of growth in radish hypocotyls

In etiolated seedlings of Raphanus sativus L. the inhibition of hypocotyl elongation by continuous light showed a major bimodal peak of action in the red and far-red, and two minor peaks in the blue regions of the spectrum. It is argued that, under conditions of prolonged irradiation, phytochrome is the pigment controlling the inhibition of hypocotyl elongation by red and far-red light, but that its mode of action in far-red is different from that in red. A distinct pigment is postulated for blue light.Abbreviations B blue - FR far red - G green - R red - HIR high irradiance reaction - P_r and P_fr red and far red absorbing forms of phytochrome - R red     

Kinetics of the dichroic reorientation of phytochrome during photoconversion inMougeotia

In the green algaMougeotia, the dichroic orientation of the red-absorbing form of phytochrome (P_r) is parallel of the cell surface, whereas the far-red-absorbing form (P_fr) 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 P_r. At increasing intervals between the red and the far-red flashes, more and more phytochrome molecules turn their transition moments to the P_fr 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 P_r to P_fr. It cannot be decided yet, whether the first surface-normal phytochrome species is an intermediate or P_fr itself.Abbreviations P_r red-absorbing form of phytochrome - P_fr 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)     

Light-induced changes in the photoresponses of plant stems the loss of a high irradiance response to far-red light

De-etiolation results in phytochrome destruction, greening, and the loss of the far-red high irradiance responses (HIR). Evidence is presented against the hypothesis that the loss of the far-red HIR is a direct consequence of phytochrome destruction. Loss of the far-red HIR for the inhibition of elongation in hypocotyls of Raphanus sativus involves two different, but linked, actions of phytochrome. An induction reaction requires the far-red absorbing form of phytochrome for about 20 min after which accumulation of its product depends only on time. A second reaction requires continuous light or frequent short irradiations and involves cycling of the phytochrome system. This acts on the product of the induction reaction. It is proposed that in green plants an important mode of operation of phytochrome in the light depends on pigment cycling, and that during de-etiolation this system is established under phytochrome control.Abbreviations HIR high irradiance response - R red - FR farred light - P_tot phytochrome, P_r its red absorbing form, P_fr its far-red absorbing form A.M. Jose was the holder of Ministry of Agriculture, Fisheries and Food award AE 6819     

Phototransformation of phytochrome in the dark

Enzymatically generated triplet acetone transfers its energy to the ground state phytochrome and promotes to some extent, in the dark, the conversion of P_r into P_fr and of P_fr into P_r. This is the first report of inverse dark reversion “in vitro”.     

The phytochrome system in light-grown Zea mays L.

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 P_r formation and P_fr and P_r destruction, rather than by P_frP_r dark reversion. P_r undergoes destruction after it has been cycled through P_fr. The consequences of this P_r destruction on the phytochrome system are discussed.Abbreviations SAN 9789 4-chloro-5-(methylamino)-2-(,,-trifluoro-m-tolyl)-3(2H) pyridazinone - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot P_fr+P_r     

Photoreversible absorption change and domain structure of phytochrome

Phytochrome is a proteinaceous pigment that acts as a photoreceptor for photomorphogenetic responses in plants. It exists as two stable absorbing forms, P_r and P_fr, which are interconvertible reversibly by irradiation with red or far-red light. The present review discusses (i) the primary and higher-order structures of phytochrome that permit the reversible photoreaction; (ii) the molecular properties which change accompanying the phototransformation; and (iii) the four-leaved shape model which has recently been proposed as a model of quaternary structure of phytochrome.     

Photoreversible association of phytochrome with membranes. II. Reciprocity tests and a model for the binding reaction

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 P_fr (the far-red-absorbing form of phytochrome) showed that two P_fr molecules bind to the membrane for each P_r (the form with an absorption maximum in the red) photoconverted to P_fr 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. P_r binding can be simulated if P_r binds only as a consequence of the binding of P_fr, i.e. when P_fr is part of a P_r: P_fr dimer. The enrichment of the membranes with P_fr as a result of the binding of P_fr 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.     

Light requirement,phytochrome and photoperiodic induction of flowering of Pharbits nil Chois

The low chlorophyll content of cotyledons of Pharbitis nil grown for 24 h in far-red light (FR) or at 18° C in white light from fluorescent lamps (WL) allows spectrophotometric measurement of phytochrome in these tissues. The (A) measurements utilize measuring beams at 730/802 nm and an actinic irradiation in excess of 90 s. The constancy of the relationship between phytochrome content and sample thickness confirms that, under these conditions of measurement, a true maximum phytochrome signal was obtained. These techniques have been used to follow changes in the form and amount of phytochrome during an inductive dark period for flowering. Following exposure to 24h WL at 18° C with a terminal 10 min red (R), P_fr was lost rapidly in darkness and approached zero in less than 1 h; during this period there was no change in the total phytochrome signal. Following exposure to 24 h FR with a terminal 10 min R, P_fr approached zero in 3 h, and the total phytochrome signal decreased by about half. The relevance of these changes to photoperiodic time measurement is discussed.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps     

Properties of phytochrome in gymnosperms

Summary Addition of water to dry seeds of Pinus spp. increased the detectable phytochrome immediately and the level reached after 2 h in darkness was retained for at least 20 h at 20° C. The in-vivo difference spectra of phytochrome in Pinus seeds showed absorption maxima at approximately 656 nm and at 710 nm to 715 nm. An isosbestic point was observed at about 680 nm. Shifts towards longer wavebands were obtained especially with tissue containing substantial amounts of chlorophyll and are, therefore, not due to diverse types of phytochrome. Embryo tissue of Ginkgo biloba showed also a maximum in R at 655 nm but the peak in FR occurred at a longer wavelength, 725 nm. This was confirmed by determining action spectra for the phototransformations P_rP_fr.The dark reactions of phytochrome in Pinus differed from those in Ginkgo. Following a short exposure to R light, the total quantity of photoreversible pigment in Pinus seeds remained constant for several hours in darkness at room temperature. Dark reversion of P_fr occurred extremely rapidly and tP_fr 50 was only 0.3 h. In Ginkgo embryos total phytochrome in darkness following a brief exposure to R light was not completely stable. Reversion of P_fr was much slower and tP_fr 50 was slightly less than 2 h.It is concluded that, at least as regards the spectral qualities, the phytochrome in Gymnospermae differs from that of Angiospermae and is apparently also not identical in Coniferae and Ginkgoinae. Abbreviations. R = red; FR = far-red; R/FR ratio = (A) red max./(A) far-red max. of difference spectrum. The peak positions and the isosbestic point are estimated from the difference spectra and are approximate only. P_r = red-absorbing form of phytochrome, P_fr = far-red absorbing formThis work was carried out with financial support from the Netherlands Organisation for Pure Scientific Research (Z.W.O.).312th Communication.     

Control by light of hypocotyl growth in de-etiolated mustard seedlings

After sowing, mustard (Sinapis alba L.) seedlings were grown for 48 h in white light (25°C). These fully de-etiolated, green seedlings were used as experimental material between 48 and 72 (84) h after sowing. The question researched was to what extent control by light of hypocotyl elongation is due to phytochrome in these seedlings. It was found that the light effect on hypocotyl growth is very probably exerted through phytochrome only. In particular, we found no indication for the involvement of a specific blue light photoreceptor pigment.Abbreviations HIR high irradiance reaction - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - Pot total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_tot] - red red light - fr far-red light - wl white light - bl blue light - di dichromatic irradiation - l hypocotyl length     

Kinetics of phytochrome decay in Amaranthus seedlings

Summary In Amaranthus seedlings the disappearance of the unstable P _fr form of phytochrome does not involve dark reversion to P _r . The rate constant for the decay of total phytochrome under continuous illumination is directly related to the proportion in the P _fr form. This relationship allows calculations to be made of the proportion of P _fr under continuous far-red illumination where the amount is too low to be measured directly.     

Gel filtration of particle-bound phytochrome

Pelletable phytochrome from hypocotyl hooks of Cucurbita pepo L. seedlings has been separated into two fractions by gel filtration on Sepharose CL-2B. One fraction with a K _av of 0.7 was detected only after red irradiation (in vivo or in vitro). This separated from a ribonucleoprotein fraction during gel filtration. The weak interaction with ribonucleo-protein which required magnesium (optimal at 10 mM) was overcome by high salt concentrations and prevented by ribonuclease treatment. The second phytochrome fraction was strongly associated with a high molecular weight material with a K _av of less than 0.1. Low levels of this complex were detected in extracts from dark grown tissue but were increased by red irradiation of excised hooks or crude extracts. The binding of phytochrome to the high molecular weight material did not require magnesium, was unaffected by ribonuclease treatment, and was much more resistant to high salt concentrations than was the phytochrome—ribonucleoprotein association. These results suggest that the association of phytochrome with this membrane—containing fraction is not electrostatic.The separation by agarose-gel filtration offers a useful technique for the preparation of membraneassociated phytochrome for physiological studies.Abbreviations EDTA ethylenediaminetetraacetic acid - P _r and P_fr phytochrome in the red and far-red absorbing forms - RNase ribonuclease - RNP ribonucleoprotein     

The role of phytochrome in photoperiodic time measurement and its relation to rhythmic timekeeping in the control of flowering in Chenopodium rubrum

Summary To follow changes in the status of phytochrome in green tissue and to relate these changes to the photoperiodic control of flowering, we have used a null response technique involving 1.5-min irradiations with mixtures of different ratios of R and FR radiation.Following a main photoperiod of light from fluorescent lamps that was terminated with 5 min of R light, the proportion of P_fr in Chenopodium rubrum cotyledons was high and did not change until the 3rd hour in darkness; at this time, P_fr disappeared rapidly. When the dark period began with a 5-min irradiation with BCJ or FR light to set the proportion of P_fr low P_fr gradually reappeared during the first 3 h of darkness and then disappeared again.The timing of disappearance of P_fr is consistent with the involvement of phytochrome in photoperiodic time measurement. Reappearance of P_fr after an initial FR irradiation explains why FR irradiations sometimes fail to influence photoperiodic time measurement or only slightly hasten time measurement. A R light interruption to convert P_r to P_fr delayed, the timer by 3 h but only for interruptions after and not before the time of P_fr disappearance. Such 5-min R-light interruptions did not influence the operation of the rhythmic timekeeping mechanism. Continuous or intermittent-5 min every 1.5 h-irradiations of up to 6 h in duration were required to rephase the rhythm controlling flowering. A skeleton photoperiod of 6 h that was began and terminated by 5 or 15 min of light failed to rephase the rhythm.The shape of the curves for the rhythmic response of C. rubrum to the length of the dark period are sometimes suggestive of clocks operating on the principle of a tension-relaxation mechanism. Such a model allows for separate timing action of a rhythm and of P_fr disappearance over the early hours of darkness. Separate timing action does not, however, preclude an interaction between the rhythm and phytochrome in controlling flowering.Abbreviations FR far-red - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - R red - BCJ photographic ruby-red irradiation A grant in aid of research from the National Research Council of Canada to B. G. Cumming is gratefully acknowledged.     

In-vitro binding of phytochrome to a particulate fraction: A function of light dose and steady-state P fr level

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     

Analysis of light-controlled accumulation of carotenoids in mustard (Sinapis alba L.) seedlings

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) - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - P_fof total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_fof], wavelength dependent photoequilibrium of the phytochrome system - red red light - fr far-red light     

Immunochemical and spectroscopic evidence for protein conformational changes in phytochrome transformations

Phytochrome was examined by immunochemical and spectroscopic techniques to detect differences between the protein moieties of red- and far red-absorbing phytochrome (P_r and P_fr). No differences in the reaction of P_r and P_fr with phytochrome antibody were discernible on Ouchterlony double diffusion plates. However, the microcomplement fixation assay showed a greater degree of antibody reaction with P_fr than with P_r, indicating some difference in the surface characteristics of the two forms. Circular dichroism spectroscopy between 300 and 200 nanometers revealed differences between P_r and P_fr which may reflect differences in the protein conformation. The circular dichroism spectrum of P_r showed a negative band at 285 nanometers which was not present in the spectrum of P_fr, and the large negative circular dichroism band at 222 nanometers with P_fr, associated with the α-helical content, was shifted 2 nanometers to shorter wave length with P_r although there was no change of magnitude of this band. The absorbancy of P_r and P_fr is very nearly the same in the 280 nanometer spectral region, but sensitive difference spectra between P_r and P_fr did reveal spectra which were similar to solvent perturbation spectra obtained by others with different proteins. In total, the experiments indicate that there are conformational differences between the protein moieties of P_r and P_fr but that these differences are rather slight from a standpoint of gross structure.     

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Stability of phytochrome concentration in dicotyledonous tissues under continuous far-red light

Summary The phytochrome concentration in dark-grown seedlings of Pisum sativum, Phaseolus aureus and Sinapis alba remained constant under continuous far-red illumination for periods of up to 6 hours. Similar treatment of Zea mays seedlings reduced the phytochrome concentration by more than 60 percent. The results in the dicotyledonous seedlings may be due to the reversion of P_fr to P_r at a rate sufficient to prevent P_fr destruction; no evidence for reversion has been detected in Zea. Typical photomorphogenic responses were observed in the dicotyledonous seedlings in the absence of P_fr destruction.Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission.     

Phytochrome behaves as a dimer in vivo

Abstract It is well established that phytochrome exists as a dimer in vitro. A comparison of the relative photoequilibrium concentrations of P_rP_r, P_rP_fr and P_frP_fr, with the relative sizes of the P_fr-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 P_fr-destruction after a red light flash, which should favour the formation of P_rP_fr dimers, is paralleled by a decay of P_r, even if the presence of P_r cycled through P_fr can be excluded. Preliminary observations could indicate an interaction of the subunits of a phytochrome dimer during the process of phototransformation.     

Spectrophotometric phytochrome measurements in light-grown Avena sativa L.

Phytochrome was studied spectrophotometrically in Avena sativa L. seedlings that had been grown for 6 d in continous white fluorescent light from lamps. Greening was prevented through the use of the herbicide San 9789. When placed in the light, phytochrome (P_tot) decreased with first order kinetics (_1/2 2 h) but reached a stable low level (2.5% of the dark level) after 36 h. This concentration of phytochrome remained constant in the light and during the initial hours of a subsequent dark period, but increased significantly after a prolonged dark period. Evidence suggests that the constant pool of phytochrome in the light is achieved through an equilibrium between synthesis of the red absorbing (P_r) and destruction of the far-red absorbing form (P_fr) of phytochrome. It is concluded that the phytochrome system in light-grown oat seedlings is qualitatively the same as that known from etiolated monocotyledonous seedlings, but different than that described for cauliflower florets.Abbreviations P_fr the far-red light absorbing form of phytochroma - P_r the red light absorbing form of phytochrome - P_tot P_r+P_fr - k_s rate constant of P_r synthesis - k_d rate constant of P_fr destruction - MOPS N-morpholino-3-propane-sulfonic acid - IRIS Tris (hydroxymethyl) amino methane - San 9789 4-chloro-5-(methyl amino)-2-(,,-trifluoro-m-tolyl)-3(2H)pyridazinone