Pfr Destruction Research Articles

Dynamic properties of endogenous phytochrome A in Arabidopsis seedlings.

The dynamic behavior of phytochrome A (phyA) in seedlings of the model plant Arabidopsis was examined by in vivo spectroscopy and by western and northern blotting. Rapid accumulation of phyA was observed, reaching a steady state after 3 d. Both red and far-red light initiated a rapid destruction of the far-red-light-absorbing form of phytochrome (Pfr); the apparent half-life was only 4-fold longer in far-red than in red light. Furthermore, the Pfr-induced destruction of the red-light-absorbing form of phytochrome (Pr) of phyA occurred in darkness with a rate identical to that of Pfr destruction. A 2-fold decrease in mRNA abundance was observed after irradiation, irrespective of the applied light quality. However, reaccumulation occurred rapidly after far-red but slowly after red irradiation, indicating different modes of regulation of phytochrome expression after light-dark transitions depending on the light quality of the preceding irradiation. The wavelength dependency of the destruction rates was distinct from that of mustard, a close relative of Arabidopsis, and was explained on the basis of Pfr-induced Pr destruction and a simple kinetic two-step model. No dark reversion was detectable in the destruction kinetics after a red pulse. From these data we conclude that Arabidopsis phyA differs significantly in several aspects from other dicot phytochromes.

KCN Blocks the Inhibiting Effect of Red Light Night-Break onthe Flowering of Pharbitis nil

Summary The seedlings of Pharbitis nil cultivated under noninductive conditions of continuous white light weresubjected to generative induction applying one 16-h-Iong period of inductive night. During the eight hour the night was interrupted with 1 min of red light pulse which completely inhibited the flowering. Treating the plants with KeN blocked the inhibiting effect of red light. Because KeN lowers considerably the rate of destruction of labile Pfr(PfrI) in some plant systems, it seems probable that flower induction in Pharbitis could appear in the presence of a substantial level of PfrI. It suggests that labile phytochrome PfrI is not involved in the control of night-break inhibition of flowering.

The kinetics of type 1 phytochrome in green, light-grown wheat (Triticum aestivum L.)

The kinetics of type 1 phytochrome were investigated in green, light-grown wheat. Phytochrome was measured by a quantitative sandwich enzyme-linked immunosorbent assay using monoclonal antibodies. The assay was capable of detecting down to 150 pg of phytochrome. In red light, rapid first-order destruction of the far-red-light-absorbing form of phytochrome (Pfr) with a half-life of 15 min was observed. Following white light terminated by red, phytochrome synthesis was delayed in darkness by about 15 h compared to plants given a terminal far-red treatment. Synthesis of the red-light-absorbing form of phytochrome (Pr) was zero-order in these experiments. Phytochrome synthesis in far-red light was approximately equal to synthesis in darkness in wheat although net destruction occurred in light-grown Avena sativa tissues in continuous far-red light, as has been reported for other monocotyledons. In wheat, destruction of Pfr apparently did not occur below a certain threshold level of Pfr or Pfr/total phytochrome. These results are consistent with an involvement of type 1 phytochrome in the photoperiodic control of flowering in wheat and other long-day plants.

Light‐ and sucrose‐dependent gene expression in photomixotrophic cell suspension cultures and protoplasts of rape (Brassica napus L.)

Light‐dependent gene expression was analysed in photomixotrophic cell suspension cultures of rape (Brassica napus L.) growing in media containing either 2.0% or 0.6% sucrose. During growth in darkness phytochrome type I and NADPH‐protochlorophyllide oxidoreductase (Pchlide reductase) accumulated in both cell culture lines to a similar extent. Illumination with continuous white, blue or red light, but not with far‐red light, resulted in disappearance of both chromoproteins within 24 h in both cell cultures. Further analysis showed that the phytochrome system of rape cell cultures reacts in a similar way to that of re‐etiolated dicotyledonous plants, showing rapid Pfr destruction and rapid Pfr dark reversion. In contrast, the light‐dependent expression of genes encoding the major chlorophyll a‐ and b‐binding protein (CAB) and the re‐accumulation of chlorophyll were found to be strongly dependent on sucrose concentration in culture media. Whereas cells grown in darkness in medium containing 2.0% sucrose showed, after exposure to continuous white light, a very weak re‐induction of CAB mRNA, CAB protein and chlorophyll accumulation, the cells in medium containing 0.6% sucrose reacted very strongly. It was also possible to demonstrate that phytochrome (by high irradiance response, HIR, and by low fluence response, LF) and the blue/UV‐A receptor are involved in the light‐dependent gene expression of CAB. Similar to complete cells, protoplasts derived from the two different cell cultures showed an almost identical sucrose concentration‐dependent and light‐quality‐dependent regulation of CAB mRNA accumulation. As the dark‐grown photomixotrophic cells and protoplasts reflect some typical photoregulatory characteristics known from dark‐grown plants it is supposed that this system will be an excellent tool for studying biochemical and molecular biological aspects of light‐dependent signal transduction in cells of higher plants.

Spectrophotometric characterization of phytochromes in dimorphic cocklebur seeds in relation to capacity for germination

Phytochrome was measured spectrophotometrically in different tissues of the upper (positively photoblastic) and lower (negatively photoblastic) seeds of the cocklebur (Xanthium pennsylvanicum Wallr.). Axial parts of the seeds, in particular parts of the radicle, contained high levels of phytochrome, while cotyledonary parts contained only low levels. These results were consistent with the distribution of the light‐sensitive areas of the seeds that were associated with germination. Phytochrome levels in both types of dimorphic seeds increased gradually with increasing duration of dark imbibition for 4–8 h, then the rates of increase in levels of phytochrome accelerated. In both types of seed, some phytochrome was measurable even before imbibition. In the lower seeds, up to 20% of the phytochrome was occasionally observed as Pfr in samples imbibed in darkness for a short time (up to 12 h). A slight blue shift of the peak of PT in the difference spectrum of phytochrome was observed in the case of lower seeds imbibed for 0–2 h. These results suggest that, to some extent, the lower axes contain dehydrated Pfr or intermediate(s) in the photoconversion of phytochrome.The dark reactions of Pfr were also examined in excised axes of both types of dimorphic seed after they had been pre‐imbibed for 16 h in darkness. Dark destruction of Pfr was observed in both types of seed. In addition, net increases in levels of Pr were observed in the dark controls and in the samples irradiated with red light after the level of Pfr diminished. No ‘inverse’ dark reversion from Pr to Pfr was detected. Thus, after 16 h of imbibition, there were no differences in terms of properties of phytochrome between the two types of seed, and the different responses to light of upper and lower seeds might depend mainly on a difference in the physiological state of the two types of seed rather than the properties of phytochrome.

PHYTOCHROME‐MEDIATED FERN‐SPORE GERMINATION: INHIBITION BY ELEVATED TEMPERATURES*

Abstract— The inhibition of light‐induced fern‐spore germination by elevated temperatures is only effective if this temperature treatment is given during coupling of the far‐red light‐absorbing form of phytochrome (Pfr) to the transduction chain. There is no evidence so far for enhanced dark destruction of Pfr as a main factor of this temperature effect.

Developmental Physiology Apparent Dark Decay of Phytochrome Pfr in Fern Spores

AbstractGermination of spores of Dryopteris fllix‐mas has been induced by two pulses of saturating red light, separated by a dark period of about 8 to 24 h. By chosing different wavelengths, different Pfr/Ptot levels could be established. Thus, by a “null method” the second pulse could be used as a “test pulse”, determining the actual Pfr level remaining from the “start pulse”, and thus providing information about an apparent Pfr decay. It cannot be decided yet whether this apparent Pfr decay results from dark destruction or dark reversion.The apparent Pfr decay depends, as expected, on the temperature, being accelerated with increasing temperatures. Moreover, the later after sowing that the decay is tested, the faster it proceeds; a tentative interpretion is that newly synthesized Pr undergoes faster decay after phototransformation than that phytochrome pool present in the resting spores. A third factor that influences the apparent Pfr decay is the Pfr/Ptot level established by the first pulse (start pulse). The lower this level, the slower the decay kinetics. This could be due to phytochrome biosynthesis partly compensating for Pfr destruction, and the relative contribution of this biosynthesis to the total effect increases with lower Pfr levels. Spores of D. paleacea yield virtually the same results.Whatever the real basis of the observed Pfr decay, i.e. destruction, reversion, or a combination of these reactions with biosynthesis, it can be concluded that modification of this Pfr decay by various factors is the basis of the effect of those factors on light‐induced germination.

Intracellular localisation of phytochrome and ubiquitin in red-light-irradiated oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome and ubiquitin in irradiated oat coleoptiles was analysed by electron microscopy. We applied indirect immunolabeling with polyclonal antibodies against phytochrome from etiolated oat seedlings or polyclonal antibodies against ubiquitin from rabbit reticulocytes, together with a goldcoupled second antibody, on serial ultrathin sections of resin-embedded material. Immediately after a 5-min pulse of red light-converting phytochrome from the red-absorbing (Pr) to the far-redabsorbing (Pfr) form-the label for phytochrome was found to be sequestered in electron-dense areas. For up to 2 h after irradiation, the size of these areas increased with increasing dark periods. The ubiquitin label was found in the same electrondense areas only after a dark period of 30 min. A 5 min pulse of far-red light, which reverts Pfr to Pr, given immediately after the red light did not cause the electron-dense structures to disappear; moreover, they contained the phytochrome label immediately after the far-red pulse. In contrast, after the reverting far-red light pulse, ubiquitin could only be visualised in the electron-dense areas after prolonged dark periods (i.e. 60 min). The relevance of these data to light-induced phytochrome pelletability and to the destruction of both Pr and Pfr is discussed.

PHYTOCHROME IN ETIOPLASTS IN RELATION TO CHLOROPHYLL ACCUMULATION*

Abstract— De‐etiolation of maize seedlings reduces their sensitivity for red light potentiation of rapid chlorophyll accumulation in white light. An earlier proposal (Raven and Spruit, 1973) attributes this to migration of the far‐red absorbing form of phytochrome (Pfr) to receptors essential for chlorophyll synthesis, thereby increasing the local Pfr/total phytochrome (Ptot)ratio. We have studied etioplasts as possible loci for such P(r receptors.The level of spectrophotometric phytochrome in purified etioplasts isolated from red preirradiated maize seedlings was higher than that of dark grown plants. The difference was marginally significant, however. We argue that migration of a fraction of cytoplasmic Pfr to the etioplasts, too small to be spectrophotometically demonstrable, could still meet the requirements of the model.Dark destruction of bulk spectrophotometric Pfr following saturating red irradiation of seedlings is not paralleled by a decrease of etioplast phytochrome. the latter remaining essentially constant over long periods. On the other hand, the potentiating effect of red light in intact seedlings is still partially reversible by far red light even after 24 h of darkness when destruction of bulk Pfr is complete. Since this demonstrates persistent presence of Pfr active in potentiation, we propose that at least part of this Pfr is associated with the etioplasts.

A Comparison of Seed and Seedling Phytochrome inAvena sativaL. using Monoclonal Antibodies

Hilton, J. R. and Thomas, B. 1985. A comparison of seed and seedling phytochrome in Avena sativa L. using monoclonal antibodies.—J. exp. Bot. 36: 1937-1946. The kinetics of phytochrome accumulation during imbibition and germination of seeds of Avena sativa L. cv. Saladin has been determined in vivo by spectrophotometry and in extracts by using Enzyme-Linked Immunosorbent Assay (ELISA). In vivo measurements show two phases of phytochrome increase. The first occurs during the initial 2 h of imbibition and is associated with the hydration of the seed proteins; the second larger increase begins after 16 h, due probably to de novo synthesis. An ELISA using monoclonal antibodies purified from dark grown Avena seedlings detected only the second increase in phytochrome content. Mixing experiments indicate that the inability to detect phytochrome by ELISA during the first 16 h is not due to the presence of inhibitors in the extracts. It is concluded that pre-existent seed phytochrome is antigenically dissimilar to seedling phytochrome. These two pools of phytochrome are stable and unstable respectively with regard to Pfr destruction.

Thoughts on the possible role of phytochrome destruction in phytochrome controlled responses

Abstract. Fluence‐response curves for low‐energy phytochrome responses show two steps at red light exposures c. two orders of magnitude apart. This Feature of the fluence‐response relationship can be interpreted as the consequence of the following processes: Induction of a photoresponse by a very low level of Pfr. Activation of a Pfr‐destroying enzyme above a threshold level of [Pfr]. Dependency of the rate of Pfr destruction on [Pfr] once the threshold level is reached. In this way, adaptation of the phytochrome control system to a broad range of light doses could be realized.

Comparative studies of the phytochrome system in cell cultures from different plants

Phytochrome contents have been assayed in vivo in cell suspension cultures of Petroselinum hortense, Daucus carota and Glycine max. After transferring the cells to fresh medium phytochrome increased in parallel with the increase in cell number, whereas the amount of phytochrome per cell remained constant. The rate of phytochrome reaccumulation after pretreatment with 15 h red light was very similar in all three systems (2.8-3.6 Δ(ΔE) 10(-5)/h). Dark reversion and a fast and slow Pfr destruction were observed in all systems. The rate constants of these reactions varied strongly between the systems. The phytochrome systems of the cell cultures were compared with those of etiolated and light-grown seedlings and it was concluded that the cell suspension cultures of Petroselinum hortense and Daucus carota behaved similarly to light-grown seedlings. In contrast, those of Glycine max behaved similarly to a dark grown seedling.

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 Pfr, the far-red light absorbing form of phytochrome (e.g., by [Pfr]≈0.1% of total phytochrome, established by a 756-nm light pulse). Coleoptile growth is at first promoted by Pfr, 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 Pfr. 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 Pfr destruction. Following an irradiation, the growth responses are quantitatively determined by the level of Pfr established at the onset of darkness rather than by the actual Pfr level present during the growth period.

PHYTOCHROME DESTRUCTION IN DARK ‐ AND LIGHT‐GROWN AMARANTHUS CAUDATUS SEEDLINGS

Abstract— Slow destruction of the far‐red‐absorbing form of phytochrome (Pfr), which has been observed in light‐grown oat and maize, occurs in light‐ and dark‐grown Amaranthus, Pharbitis, and Brassica seedlings as well. Destruction of Pfr in these seedlings shows two phases: if a high level of Pfr is produced in dark‐grown seedlings, the destruction is fast in the beginning and then slows after a low Pfr level has been reached. Slow Pfr destruction is predominant in light‐grown tissue.

Characterization of the Destruction of Phytochrome in the Red-absorbing Form

Both the red-absorbing (Pr) and far red-absorbing (Pfr) forms of phytochrome undergo destruction, defined as the loss of photoreversibly detectable chromoprotein following actinic irradiation of dark-grown tissue, in 4-day-old etiolated oat seedlings. Pr and Pfr destruction follow the same time course, exhibit the same time delay after actinic irradiation when the plants are grown in sealed containers, result in a loss of antigenically detectable phytochrome, as determined by radial immunodiffusion assay, equal to the loss of spectrophotometrically detectable phytochrome, and have the same sensitivity to 2-mercaptoethanol and azide. We suggest that Pr destruction is a consequence of the same mechanism that is responsible for Pfr destruction.

VARIATION IN THE RATES OF SYNTHESIS AND DEGRADATION OF PHYTOCHROME IN COTYLEDONS OF CUCURBITA PEPO L. DURING SEEDLING DEVELOPMENT

Abstract— The time courses for Pr appearance, Pr disappearance and Pfr destruction have been analysed in cotyledons of Cucurbita pepo L. after different preirradiation programs. In etiolated seedlings the rate of Pr appearance is low in young seedlings reaching a maximum in 3.5–5 day old seedlings then decreasing rapidly with increasing age. The rate of Pfr destruction is very low in young seedlings, increases rapidly up to the 4th day and then remains almost constant. The disappearance of Pr becomes significant for seedlings older than 45 days. These reactions seem not to be influenced by short preirradiations. However, after prolonged preirradiation, a degree of control of P, appearance and/or disappearance by the “internal clock appears to be operative.

Absence of Pfr Destruction in the Modulation of Phenylalanine Ammonia-Lyase Synthesis of Mustard Cotyledons

The relationship between the amount of active phytochrome (Pfr) produced by 5-minute light pulses and the rate of subsequent enzyme accumulation (phenylalanine ammonia-lyase, EC 4.3.1.5) of mustard (Sinapis alba L.) cotyledons was investigated. The response rapidly adjusts to changes of the Pfr level produced by light pulses of different wavelengths. Regardless of total phytochrome levels in the cotyledons, response adjustments to new photostationary states (phi(lambda)) are correlated with phi(alpha) values. On the other hand, the kinetics of enzyme accumulation shows no influence of Pfr destruction as determined spectrophotometrically (tau((1/2)) = 45 min) in the same organ (see Schäfer et al. 1973 Photochem Photobiol 18: 331-334). It is concluded that the phytochrome molecules involved in the regulation of this response by light pulses comprise a small fraction of the total phytochrome of the cotyledons. In contrast to bulk phytochrome this fraction appears to be not subject to Pfr destruction.

PHOTOCONTROL OF PHYTOCHROME DESTRUCTION AND BINDING IN DICOTYLEDONOUS vs MONOCOTYLE‐DONOUS SEEDLINGS. THE INFLUENCE OF WAVELENGTH AND IRRADIANCE

Abstract— The irradiance and wavelength dependence of phytochrome destruction in vivo was analysed in etiolated cotyledons of Cucurbita pepo L. and etiolated seedlings of Amaranthus caudatus L. In contrast to grass seedlings, the rate of Ptot destruction could only be saturated by light sources that establish relatively high Pfr levels (about 50% of total phytochrome, corresponding to the photostationary state established by 693 nm light). To explain the irradiance dependence of Ptot destruction in dicots at irradiances above 0.1 Wm‐2, where the light reaction is at least one order of magnitude faster than Pfr destruction, we suggest there is a fast intercalary dark reaction between photoreaction and destruction. This dark reaction is probably—as in grass seedlings—the binding of Pfr to a receptor site. We conclude that the differences between dicots and grass seedlings with respect to the phytochrome system are of a quantitative rather than a qualitative nature.

An attempt to localize the threshold reaction in phytochrome-mediated control of lipoxygenase synthesis in the mustard seedling.

Abstract—Synthesis* of the enzyme lipoxygenase (LOG)† in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by phytochrome (Pfr) through a threshold (all‐or‐none) mechanism. The data of the present paper confirm the previous assumption (Oelze‐Karow and Mohr, 1973) that the primary reaction of Pfr (Pfr+ X → PfrX ⇄ PfrX‘) is the site of the highly cooperative threshold reaction. Suppression of LOG synthesis depends on the presence of PfrX’. However, PfrX‘ is only stable above the threshold level of Pfr. If the level of Pfr is below the threshold, PfrX is stable, and no suppression of LOG synthesis occurs. As long as the level of Pfr remains below the threshold, no destruction of Pfr takes place. Destruction of Pfr occurs only as long as [Pfr]†is above the threshold level. Thus the simplest formulation of the actual threshold reaction in the LOG response is PfrX⇄frX’ state at [Pfr] below threshold no Pfr destruction LOG synthesis suppressed state at [Pfr] above threshold Pfr destruction(1kd LOG synthesis unimpaired The reversible threshold reaction is thus an integral part of the “primary reaction” of Pfr occurring at the “matrix” specific for the LOG response. The data and conclusions on the LOG response are consistent with an “open phytochrome‐receptor model” recently advanced by E. Schäfer (1975). The data are not consistent with the concept that a rapid dark reversion (Pfr→Pr) exists in dicotyledonous seedlings and that the degree of Pfr dark reversion strongly depends on the initial photostationary state, φ†, established by a saturating light pulse.

Etude du phytochrome des graines de Pinus nigra Arn par spectrophotom�trie bichromatique in vivo

Phytochrome photoconversions Pr→Pfr and Pfr→Pr can be measured by differential spectrophotometry in “dry” seeds (6% water content) of Pinus nigra Arn. A red light irradiation given before imbibition induces germination when the seeds are subsequently wetted and kept in darkness. In continuous darkness the phytochrome content shows a drastic increase at the beginning of moistening. The detectable pigment is entirely in the Pr form. The normal Pfr→Pr dark reversion is observed. Pfr destruction does not take place.