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)     

Phytochrome-controlled extension growth of Avena sativa L. seedlings

The effects of continuous red and far-red light and of brief light pulses on the growth kinetics of the mesocotyl, coleoptile, and primary leaf of intact oat (Avena sativa L.) seedlings were investigated. Mesocotyl lengthening is strongly inhibited, even by very small amounts of P_fr, the far-red light absorbing form of phytochrome (e.g., by [P_fr]0.1% of total phytochrome, established by a 756-nm light pulse). Coleoptile growth is at first promoted by P_fr, but apparently inhibited later. This inhibition is correlated in time with the rupturing of the coleoptile tip by the primary leaf, the growth of which is also promoted by phytochrome. The growth responses of all three seedling organs are fully reversible by far-red light. The apparent lack of photoreversibility observed by some previous investigators of the mesocotyl inhibition can be explained by an extremely high sensitivity to P_fr. Experiments with different seedling parts failed to demonstrate any further obvious interorgan relationship in the light-mediated growth responses of the mesocotyl and coleoptile. The organspecific growth kinetics, don't appear to be influenced by P_fr destruction. Following an irradiation, the growth responses are quantitatively determined by the level of P_fr established at the onset of darkness rather than by the actual P_fr level present during the growth period.Abbreviation P_fr far-red light absorbing form of phytochrome     

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.     

Variation in dynamics of phytochrome A in Arabidopsis ecotypes and mutants

Phytochromes are photoreceptors in plants which can exist in two different conformations: the red light‐absorbing form (P_r) and the far‐red light‐absorbing form (P_fr), depending on the light quality. The P_fr form is the physiologically active conformation. To attenuate the P_fr 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 P_fr. Dark reversion is the light‐independent conversion of physiologically active P_fr into inactive P_r. 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.     

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])     

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     

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     

Pattern formation underlying phytochrome-mediated anthocyanin synthesis in the cotyledons ofSinapis alba L.

The ability to respond to phytochrome (P_fr, 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 P_fr in the epidermal cells, with regard to the initial action of P_fr (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 P_r, P_fr red and far-red absorbing forms of phytochrome - HS Hoagland's nutrient solution     

Investigations on the role of ethylene in phytochrome-mediated photomorphogenesis

The etiolating, intact mustard (Sinapis alba L.) seedling exhibits a distinct temporal pattern of ethylene production. Light, operating through phytochrome, increases the rate of ethylene production without changing the pattern. Ethylene production of the isolated plant parts (segments), added together, exceed the production of the intact system even if the wound effect is taken into account. There is no significant light effect on ethylene production of the segments. Phytochrome-mediated anthocyanin synthesis in the cotyledons is inhibited by ethylene. The responsiveness towards ethylene of the anthocyanin producing metabolic chain is decreased by phytochrome. As anthocyanin synthesis is only partly inhibited under saturating ethylene concentrations in the atmosphere around the seedlings (100 l l^–1), a twofactor analysis becomes feasible. This analysis leads to the result that phytochrome and ethylene show multiplicative behavior, meaning that phytochrome and ethylene act on the same metabolic sequence (leading to anthocyanin) but independently of each other, and at different sites. Therefore, the hypothesis that ethylene mediates the action of phytochrome in anthocyanin synthesis and photomorphogenesis in general appears to be inapplicable.Abbreviations P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome, i.e. [P_r]+[P_fr]     

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.     

The in vivo properties of Amaranthus phytochrome

Summary Phytochrome has been measured in etiolated seedling of Amaranthus caudatus. The phytochrome content increases from the time of germination until 72 hr from sowing, after which it remains constant at 27.5x10^-3 (OD) units per 200 seedlings. After a saturating dose of red light P _fr decays in the dark to a form not detectable photometrically. There is no evidence for the process of dark reversion of P _fr to P _fr found in other dicotyledons. Even in the presence of azide, a selective inhibitor of decay, the process of dark reversion is not observed. The decay of P _fr has been investigated at different temperatures and follows first order decay kinetics throughout. Over the temperature range 15–30° the Q ₁₀ of decay remained constant at 4.3.The photostationary states of phytochrome (P _fr /P _total )maintained by mixed red/far-red light have been measured in both seedlings and partially purified protein extracts, with good agreement. The rate of phytochrome decay can be manipulated by changing the P _fr /P _total ratio. The lag period before a decay curve becomes exponential is characteristic of a particular P _fr /P _total ratio and represents the time for attainment of the photostationary state. The effect of energy on decay has been investigated under red and blue light. The rate of phytochrome decay is dependent on the P _fr /P _total ratio and only becomes energy dependent when the light intensity is so low that the photostationary state is never attained.The process of apparent phytochrome synthesis has been found in Amaranthus. After reducing the phytochrome to a low level by red light treatment a rate of apparent synthesis of 1.35×10^-4 (OD) units per hr per 200 seedlings was observed, levelling off at 29% of the original phytochrome level.Under white tungsten lights of high intensity there is a deviation from the expected first order decay kinetics. The nature of this low rate of decay cannot be explained at the present time.     

Correlation between far-red absorbing phytochrome and response in phytochrome-mediated anthocyanin synthesis

Abstract Mustard seedlings were light treated at 24 h after sowing (25°C) to induce phytochrome-mediated anthocyanin synthesis in cotyledons and hypocotylar hook. All light treatments were performed within the range of the reciprocity law. The in situ photoconversion kinetics of phytochrome (P_r→ P_fr) were measured under the same light treatment. It was found that between 0.4 and 1.0 relative P_fr level the amount of anthocyanin extracted from the organs at 52 h after sowing was linearily correlated with the amount of P_fr produced at 24 h in cotyledons and hypocotylar hook. It is concluded that an explanation of the fluence response function for red light mediated anthocyanin synthesis in the mustard seedling does not require the concept of active vs. bulk phytochrome.     

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.     

Differential reactivity of the red-and far-red-absorbing forms of phytochrome to [14C] N-ethyl maleimide

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.     

Nachweis der Phytochrom-induzierten de novo-Synthese von Phenylalaninammoniumlyase (PAL,E.C.4.3.1.5) in Keimlingen von Sinapis alba L. durch Dichtemarkierung mit Deuterium

Summary Using the in vivo density labeling technique with deuterium oxide it is confirmed that during phytochrome mediated photomorphogenesis in mustard seedlings a true de novo synthesis of phenylalanine ammonia-lyase is induced by active phytochrome (P _fr).     

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.     

Phytochrome in seeds of Amaranthus caudatus

Summary Dry seeds of Amaranthus caudatus show little or no photoreversible absorption changes, attributable to phytochrome. During imbibition phytochrome appears in two phases, one immediately after sowing and the second after about 8 hr. Experiments at different temperatures and under continuous illumination with red, far-red and blue light suggest that there are two pools of phytochrome. The first phase in the appearance of phytochrome could be due to the change in optical properties of the sample on hydration or to rehydration of inactive phytochrome, or both. The second phase probably represents phytochrome synthesis. It is absent at 0° and precedes the water uptake associated with germination by some 10 hr. This second pool of phytochrome does not accumulate in red and blue illuminated seeds indicating that the rate of P _fr decay is more rapid than the rate of phytochrome synthesis. The difference spectra of phytochrome in both 2 hr imbibed seeds and 72 hr old seedlings show peaks of absorption at 663 and 735 nm. The presence of P _fr in dark imbibed seeds and the process of inverse reversion of P _r to P _fr in darkness have been demonstrated. The results are discussed in relation to previous hypotheses for the mechanism of photocontrol of Amaranthus seed germination.     

Evidence that a mustard seedling responds to the amount of Pfr and not to the Pfr/Ptotratio

Abstract Phytochrome-mediated anthocyanin synthesis of the mustard seedling (Sinapis alba L.) was investigated. Light pre-treated and dark-grown seedlings differing in responsiveness and level of phytochrome (P_tot) were compared. The data obtained support the traditional view that a seedling measures the amount of P_fr. The alternative view that a plant measures the P_fr/P_tot ratio does not seem to be compatible with the data obtained with the mustard seedling.