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.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - HIR high-irradiance response - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - Ptot total phytochrome (Pr + Pfr) - R red light The authors wish to thank Prof. Daphne Vince-Prue (University of Reading) for many helpful discussions regarding this work. Hugh Carr-Smith was supported by a Science and Engineering Research Council studentship and Chris Plumpton by an Agricultural and Food Research Council (AFRC) studentship. B. Thomas and G. Butcher were supported by the AFRC.     

Phytochrome control of its own accumulation and leaf expansion in tentoxin- and norflurazon-treated mung bean seedlings

Primary leaves of 4-day-old, dark-grown mung bean [ Vigna radiata (L.) Wilczek cv. Berken] seedlings were exposed to 24 h of white light (200 μmol m⁻² s⁻¹) which was terminated by a 15 min, phytochrome-saturating red or far-red light exposure. Phytochrome content (in vivo and in vitro) and leaf area were monitored during the subsequent dark period. Red light treatments resulted in lower phytochrome content and greater leaf expansion than did far-red treatments. Phytochrome accumulation and leaf expansion were less in norflurazon- (no carotenoids and very low Chl) than in tentoxin- (very low Chl) treated leaves. After 3 days of darkness, leaf expansion was about 25% greater and phytochrome content was about 50% less in red- than in far-red-treated leaves of all treatments. These effects generally took longer to develop in norflurazon- than in tentoxin-treated tissues. Norflurazon-treated tissues exposed to long white light periods apparently do not as accurately reflect phytochrome-controlled photomorphogenic events of green tissues as do tentoxin-treated tissues of mung bean seedlings.     

Destruction and possible de novo synthesis of phytochrome in subcellular fractions of laminae from Avena sativa L.

Phytochrome was determined in etiolated laminae of Avena sativaL. either without pretreatment or after 5 min of red irradiation followed by different periods of darkness (0–24 h). At given intervals laminae were homogenized and phytochrome was determined spectrophotometrically in the total homogenate and in purified etioplasts and mitochondria. Enhanced specific activity of phytochrome was found in all fractions after the irradiation in comparison to dark controls. Phytochrome destruction was observed in all fractions at the beginning of the subsequent dark period. Whereas the homogenate and the mitochondrial fraction showed a continuous destruction so that phytochrome reached a level far below that in etiolated plants, the phytochrome level in the plastid fraction reacheda minimum at 2 h with a subsequent increase beyond the dark level. This increase was most pronounced between 4 and 8 h after the red irradiation. The results are discussed in terms of the destruction and possible de novo synthesis of phytochrome that may be different in mitochondria and plastids.Abbreviations P_tot total phytochrome - P_r red absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - ER endoplasmic reticulum     

A photoperiod-insensitive barley line contains a light-labile phytochrome B

Barley (Hordeum vulgare L.) is a long-day plant whose flowering is enhanced when the photoperiod is supplemented with far-red light, and this promotion is mediated by phytochrome. A chemically mutagenized dwarf cultivar of barley was selected for early flowering time (barley maturity daylength response [BMDR]-1) and was made isogenic with the cultivar Shabet (BMDR-8) by backcrossing. BMDR-1 was found to contain higher levels of both phytochrome A and phytochrome B in the dark on immunoblots with monoclonal antibodies from oat (Avena sativa L.) that are specific to different members of the phytochrome gene family. Phytochrome A was light labile in both BMDR-1 and BMDR-8, decreasing to very low levels after 4 d of growth in the light. Phytochrome B was light stable in BMDR-8, being equal in both light and darkness. However, phytochrome B became light labile in BMDR-1 and this destabilization of phytochrome B appeared to make BMDR-1 insensitive to photoperiod. In addition, both the mutant and the wild type lacked any significant promotion of flowering in response to a pulse of far-red light given at the end of day, and the end-of-day, far-red inhibition of tillering is normal in both, suggesting that phytochrome B is not involved with these responses in barley.     

Phytochrome in the Fern, Adiantum capillus-veneris L.: Spectrophotometric Detection in Vivo and Partial Purification

Spectrophotometric studies of fern phytochrome were performedusing dark-grown leaves of Adiantum. The absorbance differencespectrum between the red- and far-red-light irradiated sampleshowed a photoreversible absorbance change in the far-red region,with a maximum located at 728–730 nm. The concentrationof phytochrome was highest at the leaf tips and decreased graduallyalong the leaf axis. As in the case of angiosperm phytochrome,the level of fern phytochrome decreased under continuous whitelight, and the level increased again when deetiolated tissuewas transferred back to the dark. When the fern tissue was exposedto a pulse of red light, the dark reversion of P_FR to P_R tookplace with almost no destruction of P_FR. Phytochrome could beextracted from light-grown young leaves of the fern with a slightlyalkaline, aqueous buffer that contained 1 M NaCl. The differencespectrum of the partially purified phytochrome from fern wassimilar to that of partially degraded phytochrome from angio-sperms.A polyclonal antibody raised against phytochrome from etiolatedrye seedlings immuno-stained (albeit weakly) a 110-kDa polypeptideafter fractionation by SDS-polyacrylamide gel electrophoresisof the preparation of fern phytochrome. The band was very probablyfern phytochrome since it emitted zinc-induced fluorescence. (Received July 12, 1990; Accepted October 5, 1990)     

Far-red mediated polyribosome formation in radish cotyledons

The total ribosome content of radish cotyledons increases during the first 2–3 days of germination both in darkness and under far-red light irradiation; ribonuclease activity is not under phytochrome control during this period. Changes in ribonuclease activity interfere with the analysis of the polyribosomal population. A maximal ratio of polysomes to monosomes is observed 12 h after the onset of far-red light and then it decreases. A 12 h far-red irradiation stimulates the in vivo incorporation of amino acids into proteins. This stimulation persists when seedlings are transferred for 4 h to the dark.     

Spectral properties and chromophore rotation of phytochrome bound to substituted Sepharose

Brushite purified phytochrome from Avena sativa L. cv. Sol II was bound to phenyl Sepharose, octyl Sepharose, CNBr-activated Sepharose and to anti-phytochrome immunoglobulins immobilized on Sepharose. The spectral properties of phytochrome bound to anti-phytochrome immunoglobulins and to phenyl Sepharose were similar to phytochrome in solution. Phytochrome bound to CNBr-activated Sepharose or to octyl Sepharose showed reduced P_fr formation after red irradiation. The reversal to P_r with far-red light was only partial but a further increase at 667 nm took place slowly in the dark. A peak at 657 nm was seen in the difference spectrum between CNBr-activated Sepharose-bound phytochrome kept in darkness and the identical sample immediately after a far-red irradiation.
The change in linear dichroism at 660 nm and 730 nm, induced by plane polarized red or far-red light, was measured. It was computed that the long-wavelength transition moment of phytochrome had an average rotation angle of 31.5° or 180°–31.5°. The substrate used for immobilization had a limited effect on the rotation angle. Phytochrome immobilized on CNBr-activated Sepharose gave an angle of 27.8° and phytochrome immobilized on phenyl Sepharose gave an angle of 32.6°.     

APICAL GROWTH OF PROTONEMATA IN ADIANTUM CAPILLUSVENERIS. I. RED FAR-RED REVERSIBLE EFFECT ON GROWTH CESSATION IN THE DARK

Apical growth of individual protonemata in Adiantum capillus-veneris was microphotographically observed before, during and after light treatment. When single-celled protonemata precultured under continuous red light were transferred to darkness, the apical growth continued for the next 24 hr at a rate somewhat slower than that under continuous red light, but the rate significantly decreased thereafter and growth ceased within 72 hr in the dark. The growth in the dark was strongly inhibited by a brief irradiation with far-red light given immediately before the dark period, and the effect of far-red light was fully reversed by subsequent red light. This reversibility was repeatedly observed, suggesting the involvement of a phytochrome system.
The intracellular localization of the phytochrome system in the protonemata was studied, using a narrow-beam irradiator. The results showed that the photoreceptive sites of far-red light are not localized in any particular region of the cell.     

The function of phytochrome A

Knowledge of the photoperceptive function of phytochrome A has improved substantially thanks to the availability of mutants lacking phytochrome A and transgenic plants transformed with the PHYA gene in sense or anti-sense orientation. In imbibed seeds, phytochrome A mediates very-low-fluence responses. In etiolated seedlings, phytochrome A mediates very-low-fluence responses, high-irradiance responses under continuous far-red light, responsivity amplification to phytochrome B and red-light enhancement of the phototropic response to blue light. In light-grown seedings, phytochrome A modulates the extent of response to reductions in red/far-red ratio perceived by phytochrome B, perceives daylength extensions and night interruptions affecting flowering, and perceives light treatments resetting endogenous rhythms. Under natural radiation these abilities are manifested during seed germination and seedling de-etiolation under dense canopies or extremely low light fluences, and during early neighbour detection, but other processes await experimental evaluation. Phytochrome A affects growth and development throughout the whole life cycle of angiosperms.     

Control of Growth and Reproduction in Cultivated and Wild Rice by Light Quality and Dark Period

One cultivated and two wild rice varieties have been subjectedto variation in photoperiod and light quality by daily exposureof the seedlings at the four-leaf stage to 8 h of natural daylightfollowed by white incandescent, red, green or blue light for2,4 or 8 h in a temperature and humidity-controlled growth chamber.In some cases far-red irradiation was applied after white orred for 1 and 2 h. The treatments caused marked differencesin growth and reproduction between the cultivated and wild rices.The cultivar Dudkalmi showed extensive tillering after far-redexposure. Earliest flowering was observed with a 16-h dark periodboth in the cultivated and wild rices. Failure of floweringwith and 8-h day and 8-h artificial light of different wavelengthscould be overcome by red or far-red of 1-h duration. The lightquality interacted differently with the dark period in the accelerationof flowering in the three varieties. In another experiment theeffects of interruption of the dark period by a light periodof 2 h after from 4–12 h of darkness in a 24-h cycle werestudied in the two wild rice varieties. Light of different wavelengthsinterposed in the dark period caused variation in tiller numberand stem length in comparison to an uninterrupted dark periodof 16 h. The effect at the beginning of the dark period wasearlier flowering; flowering was delayed by interruption at4 h and inhibited after 8 h but accelerated after a 10- to 12-hdark period. The results are discussed in the light of the significanceof the dark period and light quality in regulating hormone balanceand phytochrome reactions.     

Integral association of phytochrome with a membranous fraction fromAvena shoots: in vivo characterization and physiological significance

Phytochrome in the far-red light absorbing form (Pfr) was observed to disappear in vivo more rapidly from the non-cation-requiring pelletable phytochrome population than from the supernantant phytochrome population of oat seedlings given an increasing dark incubation after red irradiation. The amount of pelletable phytochrome in the red light absorbing form (Pr) remained relatively stable while supernatant Pr was lost. These observations indicated that supernant Pfr was subject to loss during the incubation, while pelletable Pfr was subject to both dark reversion and loss.During the incubation, the ability of far-red irradiation to reverse the red-induced increase in phytochrome pelletability was lost, with kinetics similar to those of the loss of pelletable Pfr.Far-red reversibility of the red-induced increase in coleoptile elongation correlated with the change intotal Pfr in both supernatant and pelletable phytochrome populations, but with the change in the ratio of Pfr to total phytochrome only in the pelletable phytochrome population.The possible significance of these results is discussed with reference to the action of phytochrome in the photocontrol of physiological growth responses.Abbreviations Pfr phytochrome in the far-red light absorbing form - Pr phytochrome in the red absorbing form - Ptot total phytochrome     

Photocontrol of stem elongation in light-grown plants of Fuchsia hybrida

Stems of the caulescent long-day plant, Fuchsia hybrida cv Lord Byron, showed 2 types of response to light. In one, internode length was increased by far-red irradiation given at the end of an 8 h photoperiod: the response was no greater with prolonged exposure and was less when the start of far-red was delayed. The effect of far-red was reversible by a subsequent exposure to red light. Internode length was inversely proportional to the P_fr/P ratio established before entry to darkness and there was no evidence for loss of P_fr during a 16 h dark period. The inhibitory effect of P_fr acted at a relatively late stage of internode growth. With the development of successive internodes a second response appeared in which stems lengthened following prolonged daily exposures to red or far-red light, or mixtures of the two, or to brief breaks with red or white light. In these later internodes, a short exposure to far-red near the middle of the night was not reversible by red because red alone promoted elongation at this time. Internode length increased with increase in the daily duration of light and, when light was given throughout an otherwise dark period of 16 h, with increase in illuminance to a saturation value of 200 lx from tungsten lamps. Elongation increased as a linear function of decrease in photostationary state of phytochrome down to P_fr/P0.3; however, internodes were shorter in far-red light than in 25% red/red+far-red. It was concluded that stem length is a net response to two modes of phytochrome action. An inductive effect of P_fr inhibits a late stage in internode expansion, and a phytochrome reaction which operates only in light (and may involve pigment cycling) promotes an early stage of internode development. Stem elongation is thus a function both of the daily duration of light and its red/red+far-red content. The outgrowth of axillary buds was controlled by the first type of phytochrome action only.Abbreviations and symbols FR far red light - R red light - P phytochrome - P_fr phytochrome in the far-red light absorbing form - SD 8 h short days - LDP long-day plant - SDP short-day plant     

Twilight effect: initiating dark measurement in photoperiodism of xanthium

Six experiments studied the effects of low levels of red and far-red light upon the initiation of measurement of the dark period in the photoperiodic induction of flowering in Xanthium strumarium L. (cocklebur), a short-day plant, and compared effects with those of comparable light treatments applied for 2 hours during the middle of a 16-hour inductive dark period. Red light, or red plus far-red, at levels that inhibit flowering when applied during the middle of the inductive dark period, either had no effect on the initiation of dark measurement (i.e., were perceived as darkness), or they delayed the initiation of dark measurement by various times up to the full interval of exposure (2 hours). Far-red light alone had virtually no effect either at the beginning or in the middle of the dark period. These results confirm that time measurement in the photoperiodic response of short-day Xanthium plants is not simply the time required for metabolic dark conversion of phytochrome. Results also suggest that the pigment system (phytochrome?) and/or responses to it may be significantly different as they function during twilight (initiation of dark measurement), and as they function during a light break several hours later. Possible mechanisms by which cocklebur plants detect the change from light to darkness are discussed.Comparing experimental results with spectral light measurements during twilight and with measurements of light from the full moon led to two conclusions: First, light levels pass from values perceived by the plant as full light to values perceived as complete darkness in only about 5.5 to 11.5 minutes, although twilight as perceived by the human eye lasts well over 30 minutes. Second, cocklebur plants probably do not respond to light from the full moon, even when most sensitive, 7 to 9 hours after the beginning of darkness.     

Phytochrome Control of Turion Formation in Spirodela polyrhiza L. Schleiden

Turion yield in Spirodela polyrhiza, strain SJ, is increasedby increasing the daily light period. This effect is more pronouncedin autotrophic than in mixotrophic conditions. Night-break irradiation(15 mins) increased turion yield by 150 % under the conditionsof an 8-h daily light period. Besides the effect of night-breakirradiation, end-of-day far-red irradiation decreased turionyield with increasing photoperiod, whereas end-of-day red irradiationwas without any effect. This demonstrates the promoting effectof the P_fr form of phytochrome on formation of light-grown turions. Formation of dark-grown turions was increased by about 240%by a single red light pulse and was reversed by an immediatelyapplied far-red light pulse. Consequently, under heterotrophicconditions phytochrome modulates the turion formation process. Spirodela polyrhiza L. Schleiden, duckweed, Lemnaceae, photomorphogenesis, phytochrome, turion     

Evidence of phytochrome involvement in the entrainment of the circadian rhythm of carbon dioxide metabolism in Bryophyllum

The rhythm of carbon dioxide output in Bryophyllum leaves was entrained on exposure to 0.25 h of white light every 24 h. Entrainment also occurred on similar exposure to monochromatic radiation in spectral bands centred at 660 nm and, to a lesser extent, at 730 nm, but a band centred at 450 nm was without effect. A skeleton irradiation programme comprising two 0.25-h exposures to white light per 24 h also entrained the rhythm when the intervening dark periods were either 7.5 h and 16 h, or 10.5 h and 13 h. The rhythm disappeared when the two exposures were separated by 11.5-h and 12-h dark periods. Regular 0.25-h exposures to red light separated by 11.75-h periods of darkness also resulted in loss of the rhythm. Red/far-red reversibility was observed in irradiation schedules having either one or two exposures to red light daily. In the latter case, far-red reversal of the effects of one of the exposures to red light resulted in entrainment of the rhythm by the other, instead of abolition of the rhythm. The occurrence of distinct red/far-red reversibility suggests strongly that phytochrome is the pigment involved in entrainment of this rhythm by cycles of light and darkness.Abbreviation LD light-dark rhythm     

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

For dark-grown seedlings of Pharbitis nil capacity to flower in response to a single inductive dark period was established by 24 h white, far-red (FR) or ruby-red (BCJ) light and by a skeleton photoperiod of 10 min red (R)-24 h dark-10 min R. FR alone was ineffective without a brief terminal (R) irradiation, confirming that the form of phytochrome immediately prior to darkness is a crucial factor for flowering in Pharbitis. The magnitude of the flowering response was significantly greater after 24 h FR or white light (WL) (at 18° C and 27° C) than after two brief skeleton R irradiations, but the increased flowering response was not attributable to photosynthetic CO₂ uptake because this could not be detected in seedlings exposed to 24 h WL at 18° C. Photophosphorylation could have contributed to the increased flowering response as photosystem I fluorescence was detectable in plants exposed to FR, BCJ, or WL, but there were large differences between flowering response and photosystem I capacity as indicated by fluorescence. We conclude that phytochrome plays a major role in photoresponses regulating flowering. There was no simple correlation between developmental changes, such as cotyledon expansion and chlorophyll formation during the 24-h irradiation period, and the capacity to flower in response to a following inductive dark period. Changes in plastid ultrastructure were considerable in light from fluorescent lamps and there was complete breakdown of the prolamellar body with or without lamellar stacking at 27 or 18° C, respectively, but plastid reorganization was minimal in FR-irradiated seedlings.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing from of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps