Response of tissue with different phytochrome contents to various initial photostationary States

Pretreatment of etiolated pea plants with red light and with red combined with far-red light produced morphologically similar plants having 4-fold differences in spectrophotometrically detectable phytochrome. Stem segments from the variously pretreated plants respond in the same way to different percentage conversions of phytochrome to P_FR. These results suggest that the P_FR./P_R ratio, rather than the concentration of P_FR, governs pea stem segment elongation. However, the ratio hypothesis does not explain contradictions between spectrophotometric and physiological assays previously obtained with this tissue, nor does it explain similar contradictions obtained in other systems. The only hypothesis consistent with the data to date is that of the existence of bulk and active phytochrome fractions, with the latter present in insufficient quantities to be spectrophotometrically detectable.     

Relationships between phytochrome state and photosensitive growth of Avena coleoptile segments

Using various photostationary state light sources to obtain reproducible phytochrome conversion of from 5 to 88% P_FR, assayed by 2 wavelength in vivo spectrophotometry, relationships between initial percent P_FR and elongation of apical Avena coleoptile segments over the succeeding 20 hours in darkness were studied. With material grown in total darkness, all P_FR levels promote elongation, and maximal promotion requires roughly 50% P_FR. The promotion caused by an initial 5 minute red (88% P_FR) treatment at hour 0 is partially reversible at hour 5 by sources forming less than 48% P_FR, but totally irreversible at hour 8, though less than 50% of the growth has been accomplished by this time. Direct photometric assays at hour 5 indicate a phytochrome state of roughly 45% P_FR, consistent with the reversal data. At hour 8, however, 11 to 22% of the phytochrome still assays as P_FR, an inconsistency suggesting simply that the elongation process has proceeded beyond photochemical control. Thus, in contrast with results previously reported for Pisum and Phaseolus, there is no contradiction between photometric and physiological assays of phytochrome state in Avena coleoptile segments.

Attempts to expand this study by using segments from seedlings pretreated with red light showed that such pretreatment as little as 1 to 2 hours before drastically reduces subsequent elongation and photoresponse on the medium employed. This decline in growth potential can be halted at any time before its completion by either excision of the segment or far-red treatment of the intact seedling.

     

Isolation of phytochrome from the alga mesotaenium and liverwort sphaerocarpos

Phytochrome has been isolated from the green alga Mesotaenium and the liverwort Sphaerocarpos. The Mesotaenium pigment had absorption peaks at 649 and 710 nm for the P_R and P_FR forms, respectively. Corresponding difference spectrum maxima for the Sphaerocarpos pigment were at 655 and 720 nm. While the absorption maxima differ, the reversibility and efficiency with which red and far-red light transform the Mesotaenium pigment are very similar to that reported for phytochrome isolated from etiolated seedlings of higher plants. Methods are described which allow efficient separation of phytochrome from highly pigmented light-grown material.     

Phytochrome in Pharbitis nil during and after de-etiolation

Phytochrome (P) was measured by in vivo spectrophotometry in the cotyledons of Pharbitis nil Choisy cv. Violet. In etiolated plants exposure to red light (R) results in a rapid fall in P content by destruction of the far-red absorbing form of phytochrome (Pfr). In continuous R or white light the P content falls as a result of destruction to a stable 3% of that originally present. In Norflurazon-treated white light de-etiolated plants, Pfr undergoes reversion in darkness to the red absorbing form of phytochrome (Pr) with a half life of 4 h at 19°C, while total P remains constant. Synthesis of Pr after de-etiolation occurs at a slow rate and is enhanced by terminating the de-etiolation light treatment with far-red light. The results are discussed in relation to the P control of flowering.     

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     

Phytochrome-mediated effects of near-ultraviolet radiation in the induction of flowering in etiolated Lemna paucicostata T-101, a short-day plant

Induction of flowering of etiolated Lemna paucicostata Hegelm. T-101, a short-day plant, was inhibited by far-red (FR) or blue light (BL) applied at the beginning of a 72-h inductive dark period which was followed by two short days. In either case the inhibition was reversed by a subsequent exposure of the plants to near-ultraviolet radiation (NUV), with a peak of effectiveness near 380 nm. Inhibition by BL or FR and its reversion by NUV are repeatable, i.e., NUV is acting in these photoresponses like red light although with much lower effectiveness. Thus, it is considered that NUV acts through phytochrome and no specific BL and NUV photoreceptor is involved in photocontrol of floral induction on this plant.Abbreviations BL blue light - FR far-red light - NUV near ultraviolet radiation - P red-absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - R red light     

Phototaxis in a dinoflagellate: Action spectra as evidence for a two-pigment system

Summary Action spectra were determined in the UV region of the spectrum for the first phase of the phototactic response (stop response) and for the phytochrome pigment associated with this response in the dinoflagellate Gyrodinium dorsum Kofoid. Differences between these action spectra indicate the participation of two pigments in phototaxis. Following R (620 nm) irradiation of the phytochrome, the stop response maxima occur at 470 and 280-nm; after FR irradiation they shift to 490 and 300–310 nm. These maxima suggest that the photoreceptor pigment for phototaxis is a carotenoprotein. The action spectrum shift following the different phytochrome conversions may represent a trans to cis isomer change by the carotenoid. The absorption maximum of P_R in the UV appears to be at 320 nm, which is consistent with the shift of the R absorption maximum to shorter wavelengths (620 nm) as compared to higher plants. The P_FR absorption maximum appears as a broad band between 360 and 390 nm. Comparison of P_R to P_FR conversions by different intensities of 620-nm and 320-nm light indicates that at lower intensities the logarithm of the threshold for the stop response is inversely proportional to the logarithm of the intensity of the sensitizing light. The ratio of response activation by R and UV light is about 4:1.Abbreviations FR far-red - R red - P_FR far-red-absorbing form of phytochrome - P_R red-absorbing form of phytochrome - UV ultraviolet     

Red/far-red modulation in vitro of enzyme activity in a membrane fraction from Phaseolus aureus

In a membrane fraction isolated from hypocotyls of Phaseolus aureus Roxb. the activity of a number of enzymes was regulated by red and far-red irradiation in vitro, provided that the tissue received a brief red light treatment before extraction. Other enzymes showed no photoregulation. There were two types of photocontrol, neither of which could be detected in the solute fraction, nor in extracts from completely etiolated material. One (Type I) was a red/far-red reversible regulation of the rate of enzyme activity, depending on the light given (in vivo or in vitro) before the assay was begun. The second (Type II) was a promotion of enzyme activity by red or far-red light given during the assay. The action spectra for type II responses do not coincide with either the phytochrome absorption or difference spectra. However, the effectiveness of red and far-red was correlated with the P_fr/P ratio present at the beginning of the assay, such that far-red was more efficient at high P_fr/P and red at low P_fr/P ratios. All enzymes that were regulated involved ATP. In samples that showed enzyme regulation, small changes in fluorescence yield of tryptophan and the covalent probe Fluram (Roche) accompanied the photoconversion of phytochrome, but no fluorescence changes could be measured after briefly incubating the membrane fraction with ATP. The results indicate that light may affect the interaction of ATP with the membrane fraction.Abbreviations F far-red light - P_r and P_fr phytochrome in the red and far-red absorbing forms - P_tot total phytochrome - R red light - RNP ribonucleoprotein     

Differences between the Surface Properties of the PR and PFR Forms of Native Pea Phytochrome, and Their Application to a Simplified Procedure for Purification of the Phytochrome

The ability of phytochrome from etiolated pea shoots (Pisumsativum L. cv. Alaska) to bind to various chromatographic adsorbentsand its mobility during non-denaturing electrophoresis wereexamined with phytochrome in either the red light-absorbingform (PR) or the far-red light-absorbing form (P_FR). Preferentialbinding of P_FR to modified hydrophilic polyvinyl resins, suchas butyl Toyopearl, phenyl Toyopearl, Blue Toyopearl (CibacronBlue F3G-A conjugated) and Red Toyopearl (Procion Red HE-3Bconjugated), was observed. A simplified method for purificationof native phytochrome was developed based on the propertiesof P_R and P_FR. P_FR bound preferentially to the hydrophobic adsorbents,to indicate that the surface of P_FR is more hydrophobic thanthat of P_R. A difference in net surface charges between P_R andP_FR was detected by an analysis based on the different mobilitiesof the two forms during non-denaturing polyacrylamide gel electrophoresisin gels prepared with various concentrations of polyacrylamide.The apparent molecular weights of P_R and P_FR, estimated fromthe analysis, were 378 and 419 kilodaltons, respectively. Thedifference suggests that a significant change in molecular shapeoccurs during the photoconversion. The differences in surfaceproperties of P_R and P_FR are discussed. (Received April 20, 1991; Accepted August 26, 1991)     

Phytochrome changes correlated to mesocotyl inhibition in etiolated Avena seedlings

The elongation of etiolated Avena mesocotyls is inhibited by red light (660 mμ). Immediately after exposing mesocotyl sections to varying doses of red light the ensuing concentrations of phytochrome in the far-red absorbing form (P₇₃₀) were measured. The extent of mesocotyl inhibition observed 5 days later is proportional to the logarithm of P₇₃₀ concentration in mesocotyl tissue at the time of red light exposure.

The inhibition of mesocotyl growth by red light can be reversed partially by subsequent exposure to far-red light (730 mμ). Increasing doses of far-red light result in decreasing concentrations of P₇₃₀ as compared with the original P₇₃₀ level due to the preceding red light exposure. The reduced mesocotyl inhibition of seedings which had been exposed to red and far-red light is proportional to the logarithm of P₇₃₀ concentration remaining in the tissue at the end of the two light exposures.

This indicates that the same correlation exists between P₇₃₀ concentration and growth response whether the seedlings had been exposed to red light only or to red followed by far-red light.

     

Stable concentrations of phytochrome in pisum under continuous illumination with red light

In vivo spectrophotometry showed that the phytochrome concentration in pea epicotyl hooks decreased at a constant rate for 4 hours when the tissue was exposed to continuous red light. Thereafter the rate slowed progressively so that a steady concentration of phytochrome was approached at hour 7. Returning the plants to darkness resulted in an increase in phytochrome due to the apparent synthesis of P_R. A closely similar pattern of changes was found in the amount of phytochrome extracted from the tissue. The establishment of the stable concentration was inhibited by 2,4-dichlorophenoxyacetic acid and did not occur in segments which had been incubated for longer than 24 hours, but was observed when segment growth was inhibited by mannitol. The results may be explained by an equilibrium between P_FR destruction and apparent P_R synthesis.     

Phytochrome A, phytochrome B and other phytochrome(s) regulate ATHB-2 gene expression in etiolated and green Arabidopsis plants

The expression of the Arabidopsis ATHB-2 gene is light-regulated both in seedlings and in adult plants. The gene is expressed at high levels in rapidly elongating etiolated seedlings and is down-regulated by a pulse of red light (R) through the action of a phytochrome other than phytochrome A or B, or by a pulse of far-red light (FR) through the action of phytochrome A. In green plants, the expression of the ATHB-2 gene is rapidly and strongly enhanced by lowering the R:FR ratio perceived by a phytochrome other than A or B. Returning the plant to a high R:FR ratio results in an equally rapid decrease of the ATHB-2 mRNA. Consistently, plants overproducing ATHB-2 show developmental phenotypes characteristic of plants grown in low R:FR: elongated petioles, reduced leaf area, early flowering, and reduced number of rosette leaves. Taken together, the data strongly suggest a direct involvement of ATHB-2 in light-regulated growth phenomena throughout Arabidopsis development.     

Phytochrome A affects stem growth, anthocyanin synthesis, sucrose-phosphate-synthase activity and neighbour detection in sunlight-grown potato

Phytochrome action in fully de-etiolated sunlight-grown potato (Solanum tuberosum L.) was studied by comparing wild-type (WT) plants and transgenic plants with either a sense or an anti-sense phytochrome A (phyA) construction. Radial stem growth, anthocyanin levels, and sucrose-phosphate-synthase activity were directly related to the levels of phyA (severely reduced in transgenics with anti-sense phyA, normal in WT and increased in transgenic with sense phyA). In contrast, longitudinal stem growth was inversely related to the levels of phyA. Phytochrome A influenced stem-extension growth responses to red/far-red ratios perceived by stable phytochrome[s]. First, far-red light reflected by non-shading neighbours promoted stem growth in WT plants but transgenic plants with either increased or reduced phyA levels failed to respond to this light signal. Second, plants with low phyA levels also showed impaired sensitivity to reductions in end-of-day red/far-red ratios. In addition, phyA appears to perceive changes in irradiance reaching the stem: lowering the amount of red plus far-red light reaching the stem promoted stem growth in WT plants. This effect was exaggerated in phyA overexpressors and absent in phyA underexpressors. Thus, phyA is active in fully de-etiolated, sunlight-grown plants. Received: 4 October 1997 / Accepted: 24 October 1997     

Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.Abbreviations FR far-red light - R red light - WL white light - WL + FR white light supplemented with FR - HIR high-irradiance response - PAR photosynthetically active radiation - Pr, Pfr R- and FR-absorbing forms of phytochrome - Ptot total phytochrome - phyA (PhyA) gene (encoded protein) for phytochrome - WT wild type This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.M.; J.R. Cherry and R.D. Vierstra, (Department of Horticulture, University of Wisconsin-Madison, USA) are thanked for the provision of the transgenic tobacco line.     

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     

Genetic interactions in plant photomorphogenesis

Phytochrome and a blue light receptor mediate a developmental switch from etiolated growth to the photosynthetically competent 'de-etiolated' program. The analysis of Arabidopsis mutants deficient in photomorphogenetic responses (e.g. hy, blu) has identified several elements that mediate the red/far-red and blue light responses. Mutants that appear de-etiolated in the absence of light (e.g. det1, det2, cop1) implicate negatively-acting elements that integrate red and blue light signals. Phenocopy of the de-etiolated mutants by cytokinin implicates a role for this hormone in promoting seedling photomorphogenesis. Epistasis analyses support a pathway in which DET1 and DET2 are downstream effectors of phytochrome function.     

Phytochrome modulation of ATPase activity in a membrane fraction from Phaseolus

Membrane-bound phytochrome and ATPase (ATP phosphohydrolase EC 3.6.1.3.) activity extracted from hypocotyl hooks of etiolated Phaseolus aureus Roxb. were both separated from solute proteins by gel filtration on Sepharose C1-2B. The amount of phytochrome detected in the membrane fraction was very small and was not significantly increased by red irradiation (in vivo or in vitro). Membrane-bound ATPase activity was modulated in vitro by the phytochrome in the membrane fraction, being lower after red light than after far-red light. This effect was potentiated by a preliminary light reaction which occurred only in vivo and, in continuous red light, required 60 to 90 s at 25°C. Thus a two minute, in vivo, red irradiation reduced membrane-bound ATPase activity to about half that of the etiolated state. Subsequently bound-ATPase activity was determined by the form of phytochrome (P_r or P_fr) irrespective of whether established in vivo or in vitro. These results indicate that binding or release (of enzyme, cofactors or inhibitors) is not involved in phytochrome modulation of enzyme activity in the membrane fraction.Abbreviations R red light - F far red light - P_r inactive form of phytochrome (_max=660 nm) - P_fr active form of phytochrome (_max=730 nm) - MOPS N-morpholino-3-propansulphonic acid     

Reversible phytochrome regulation influenced the severity of ozone-induced visible foliar injuries in Trifolium subterraneum L.

In Trifolium subterraneum, oxidative stress caused by ozone has been shown to result in more severe visible foliar injuries when plants were kept in dim broadband white light during the night (i.e. a long photoperiod) compared to darkness during the night (a short photoperiod). As phytochrome signalling is involved in photoperiod sensing, the effect of night-time red and far-red illumination on the ozone-induced response was studied. T. subterraneum plants were treated with ozone enriched air (70?ppb) for either 1?h for a single day or 6?h for three consecutive days. After the first ozone exposure, plants were separated into six night-time light regimes during the two subsequent nights (10?h?day, 14?h night): (1) darkness, (2) far-red light (FR), (3) a short night-break of red followed by far-red light during an otherwise dark night (R FR), (4) a short night-break of red, far-red and finally red light during an otherwise dark night (R FR R), (5) dim white light (L) and (6) red light (R). The treatments L and R resulted in significantly more severe ozone-induced visible foliar injuries relative to D and FR treatments, indicating a phytochrome-mediated response. The night-breaks resulted in a photoreversible and significantly different ozone response depending on the light quality of the last light interval (R FR or R FR R), supporting a photoreversible (between P_r and P_fr) phytochrome signalling response. Thus, in T. subterraneum, the outcome of oxidative stress due to ozone appears to depend on the photoperiod mediated by the night-time conformation of phytochrome.     

Photomorphogenesis in Sinningia speciosa, cv. Queen Victoria I. Characterization of Phytochrome Control

The morphological development of Sinningia speciosa plants that were exposed to supplementary far red light was very different from that of plants receiving dark nights. After several nights of such irradiation, stems and petioles were elongated, petioles were angulated, leaf blade expansion was inhibited, plants were chlorotic and the accumulation of shoot dry weight was retarded.

Red reversibility of the morphological changes potentiated by far red light indicated control by the phytochrome system. A high P_FR level during the last half of the night inhibited stem elongation and promoted leaf blade expansion, but both of these processes were hardly affected by the P_FR level during the first half of the night. Thus sensitivity to P_FR was cyclic.

The interpretation of our experiments was complicated by quantitative morphological differences resulting from long, as compared to short, far red irradiations.