A monoclonal antibody specific for the red-absorbing form of phytochrome

Characterisation of a new monoclonal antibody (mAb), designated LAS 41, directed against 124-kilodalton (kDa) etiolated-oat (Avena sativa L.) phytochrome, indicates that it recognises an epitope unique to the red-light-absorbing form, Pr. In a solid-phase enzyme-linked immunosorbent assay (ELISA), LAS 41 exhibits a seven- to eight-fold higher affinity for Pr than for the far-red-light-absorbing form of phytochrome, Pfr. In addition, in immunoprecipitation assays LAS 41 effectively precipitates 100% of phytochrome presented as Pr but only precipitates a maximum of 24.5% of phytochrome presented as Pfr. These values are indicative of binding exclusively to Pr. Peptide-mapping studies show that LAS 41 recognises and epitope located within a region 6–10 kDa from the aminoterminus of the phytochrome molecule. Since binding of LAS 41 to Pr induces alterations in the spectral properties of Pr, this indicates that at least part of the 4 kDa domain to which the antibody binds is essential for protein-chromophore interaction. Subsequent photoconversion of LAS 41-Pr complexes produces native Pfr spectra, with concomitant production of free antibody and antigen, as shown by a modified ELISA. The specificity of LAS 41 for Pr has facilitated the purification of Pfr which is free of contaminating Pr. This has enabled direct determination of the mole fraction of Pfr established by red light to be 0.874.Abbreviations ELISA enzyme-linked immunsorbent assay - kDa kilodalton - mAb monoclonal antibody - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - (A) difference in absorbance (A ₆₆₅ ^Pr –A ₇₃₀ ^Pr )-(A ₆₆₅ ^Pfr –A ₇₃₀ ^Pfr ) - Ar/Afr spectral change ratio (SCR) - max mole fraction of Pfr following saturating red light     

Phytochrome-mediated polarotropism of Adiantum capillus-veneris L. protonemata as analyzed by microbeam irradiation with polarized light

Summary Perception of polarized light inducing phytochrome-mediated polarotropism in protonemata of the fern Adiantum capillus-veneris L. was analyzed using brief microbeam irradiation with polarized red (R) or far-red light (FR). The polarotropic response inducible by irradiation of the subapical 10–30-m part with polarized R vibrating parallel to the cell axis was nullified by subsequently giving R at the apical 0–2.5-m region. This inhibitory effect of R showed an action dichroism, that is, polarized R vibrating normal to the cell axis was effective but the parallel-vibrating R was not. On the other hand, FR irradiation of the extreme tip after irradiation of the whole cell with depolarized R effectively induced a tropic response. This FR effect also showed action dichroism, with parallel-vibrating polarized FR being more effective than FR vibrating normal to the cell axis. When the apical-dome region and the adjacent subapical 10–20-m region were sequentially irradiated with polarized R vibrating obliquely in different directions, polarotropism took place depending on the vibrating direction of the light given to the apical-dome region. Obliquely vibrating polarized FR given to the apical dome after irradiation of the whole cell with depolarized R also induced polarotropism. Thus, the difference in amount (or percent) of the far-redabsorbing form of phytochrome (Pfr) between the extreme tip and the subapical region appears to be crucial in regulating the direction of apical growth; the difference in Pfr level between the two sides of the protonemal apex may occur mainly at the apical dome. Furthermore, the transition moments of the red-absorbing form of phytochrome (Pr) and Pfr seem to be aligned parallel and normal, respectively, to the cell surface at the periphery of the apical hemisphere.Abbreviations FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light     

Proteolysis alters the spectral properties of 124 kdalton phytochrome from Avena

Native phytochrome from Avena sativa L. is homogeneous with a monomeric molecular weight of 124 kdalton; 6–10 kdalton larger than the heterogeneous 120 kdalton preparations previously considered to be undegraded (Vierstra and Quail, 1982, Proc. Natl. Acad. Sci. USA, 79: 5272–5276). The phototransformation difference spectrum (Pr-Pfr) of 124 kdalton phytochrome measured in crude extracts has a minimum in the farred region at 730 nm, the same as that observed in vivo. These spectral properties contrast with those of 120 kdalton phytochrome purified by column immunoaffinity chromatography where the difference minimum is at 724 nm. When 124 kdalton phytochrome is incubated as Pr in crude extracts, the difference minimum shifts progressively to shorter wavelengths (from 730 to 722 nm) concomitant with the proteolytic degradation of the chromoprotein to the mixture of 118 and 114 kdalton species that comprise 120 kdalton phytochrome preparations. These two effects are inhibited in concert by the serine protease inhibitor, phenylmethylsulfonylfluoride, and or maintenance of the phytochrome in the Pfr form. These results provide further evidence that 124 kdalton phytochrome is the native molecule in Avena and indicate that the peptide segments removed by proteolysis of the Pr form are important to the pigment's spectral integrity. The present data thus resolve the previously unsettled question of why the Pfr form of 120 kdalton phytochrome isolated by various procedures from Avena has been found to absorb at shorter wavelengths than that observed in vivo. Previous spectral studies with 120 kdalton phytochrome preparations are open to reexamination.Abbreviations, symbols PMSF phenylmethylsulfonylfluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - Ig immunoglobulin - A_minimum, A_maximum phototransformation difference spectrum (Pr-Pfr) minimum and maximum - A_r/A_fr ratio spectral change ratio     

Avena sativa L. contains three phytochromes,only one of which is abundant in etiolated tissue

Phytochrome from leaves of light-grown oat (Avena sativa L. cv. Garry) plants is characterized with newly generated monoclonal antibodies (MAbs) directed to it. The results indicate that there are at least two phytochromes in green oat leaves, each of which differs from the phytochrome that is most abundant in etiolated oat tissue. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with reference to 124-kilodalton (kDa) phytochrome from etiolated oats, the two phytochromes from green oats have monomer sizes of 123 of 125 kDa. Immunoblot analysis of SDS, sample buffer extracts of lyophilized, green oat leaves indicates that neither the 125-kDa nor the 123-kDa polypeptide is a degradation product arising after tissue homogenization. Of the two, the 123-kDa phytochrome appears to be the predominant species in light-grown oat leaves. During SDS-PAGE in the presence of 1 mM Zn²⁺, 123-kDa phytochrome undergoes a mobility shift corresponding to an apparent mass increase of 2 kDa. In contrast, the electrophoretic mobility of 125-kDa phytochrome is unaffected by added Zn²⁺. Some MAbs that recognize 123-kDa phytochrome fail to recognize 125-kDa phytochrome and vice versa, indicating that these two phytochromes are not only immunochemically distinct from 124-kDa phytochrome, but also from each other. It is evident, therefore, that there are at least three phytochromes in an oat plant: 124-kDa phytochrome, which is most abundant in etiolated tissue, plus 123-and 125-kDa phytochromes, which predominate in light-grown tissue.Abbreviations Da Dalton - HA hydroxyapatite - MAb monoclonal antibody - PAb polyclonal antibody preparation - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). We thank Dr. Alan Jones, Department of Biology, University of North Carolina, Chapel Hill, USA, for kindly providing rabbit antiserum 4032, and Mrs. Donna Tucker and Mrs. Danielle Neal for their technical assistance.     

Phytochrome structure: Peptide fragments from the amino-terminal domain involved in protein-chromophore interactions

We have undertaken a study of the structure of the amino-terminal domain of the phytochrome polypeptide purified from Avena sativa L. Amino-acid sequencing was used to indentify arginine 52 as the precise location of a conformation-specific cleavage of phytochrome by subtilisin. The location of the epitopes for a class of monoclonal antibodies designated type 2 has been shown to be located between approx. 10 and 20 kilodaltons (kDa) from the amino terminus. These two new spatial markers, in addition to the chromophore and another epitope recognized by type 1 monoclonal antibodies and located within 6 kDa from the amino terminus, have been used to map the locations of several new protease-accessible sites along the polypeptide. After extensive digestion of phytochrome with subtilisin, a stable spectrally-active group of peptides remains. Within this group is a 16-kDa chromopeptide which, either alone or as part of an assemblage of peptides, elutes from a size-exclusion column under nondenaturing conditions at a volume consistent with a molecular mass of 35–40 kDa. This group of peptides has an absorbance spectrum similar to the red-absorbing form of phytochrome (Pr) and is red/far-red photoreversible between this and a photobleached form. These data indicate that this group of peptides still retains the principal structural requisites for Pr-chromophore-protein interactions and for photoreversibility, but not for Pfr (far-red-absorbing phytochrome)-chromophore-protein interactions. It is uncertain if these structural requisites reside exclusively on the 16-kDa chromopeptide or result from an assemblage of these peptides. However, we have excluded any role for an adjacent 14-kDa fragment (approximately residues 50 to 200) in the observed spectral properties since it can be selectively removed without any effect on the photoreversibility.Abbreviations Da dalton - M_r relative molecular mass - Pr, Pfr red and far-red-absorbing forms of phytochrome, respectively - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis This work was presented, in part, at the XVI Yamada Conference on Phytochrome and Plant Photomorphogenesis, Okazaki, Japan, October 1986     

The levels of two distinct species of phytochrome are regulated differently during germination in Avena sativa L.

The abundance and molecular mass of phytochrome in germinating embryos of A. sativa (oat) grown in light or darkness have been monitored using immunoblot and spectrophotometric assays. Immunoblot analysis shows that imbibed but quiescent embryos have two immunochemically distinct species of phytochrome with monomeric molecular masses of 124 and 118 kDa (kdalton). The 118-kDa species has the properties of the 118-kDa phytochrome extracted from fully green oat tissue (J.G. Tokuhisa, S.M. Daniels, P.H. Quail, 1985, Planta 164, 321–332), whereas the 124-kDa polypeptide appears similar to the well-characterized photoreceptor of etiolated tissue. The capacity of antibodies directed against etiolated-oat phytochrome to immunoprecipitate the 124-kDa species but not the 118-kDa species has been exploited to quantitate the levels of each separately over a 72-h time course of germination and seedling development. The abundance of the 124-kDa molecule increases at least 200-fold in etiolated seedlings over 72 h whereas in light-grown seedlings the level of this molecule is relatively constant. In contrast, the amount of the 118-kDa species increases only twofold in both dark- and light-grown seedlings over the same period of time. These data indicate that whereas the abundance of 124-kDa phytochrome is regulated at the protein level by the well-documented, differential stability of the red- and far-red-absorbing forms in vivo, the 118-kDa molecule is present at a low constitutive level, presumably reflecting no such difference in the stability of the two spectral forms.Abbreviations ELISA enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - Pfr, Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Spectrophotometric phytochrome measurements in light-grown Avena sativa L.

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

Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in light-grown Avena sativa L.

A method is described for the extraction of phytochrome from chlorophyllous shoots of Avena sativa L. Poly(ethyleneimine) and salt fractionation are used to reduce chlorophyll and to increase the phytochrome concentration sufficiently to permit spectral and immunochemical analyses. The phototransformation difference spectrum of this phytochrome is distinct from that of phytochrome from etiolated shoots in that the maximum in the red region of the difference spectrum is shifted about 15 nm to a shorter wavelength. Immunochemical probing of electroblotted proteins (Western blotting), using a method sensitive to 50 pg, demonstrates the presence of two polypeptides in green tissue that bind antiphytochrome antibodies: a predominant species with a relative molecular mass (M_r) of 118000 and a lesser-abundant 124000-M_r polypeptide. Under nondenaturing conditions all of the 124000-M_r species is immunoprecipitable, but the 118000-M_r species remains in the supernatant. Peptide mapping and immunochemical analysis with monoclonal antibodies show that the 118000-M_r species has structural features that differ from etiolated-oat phytochrome. Mixing experiments show that these structural differences are intrinsic to the molecular species from these two tissues rather than being the result of post-homogenization modifications or interfering substances in the green-tissue extracts. Together the data indicate that the phytochrome that predominates in green-tissue has a polypeptide distinct from the well-characterized molecule from etiolated tissue.Abbreviations and symbols Ig immunoglobulin - M_r relative molecular mass - Pfr, Pr far-red-absorbing and red-absorbing forms of phytochrome respectively - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - _max ^R , _max ^FR maxima of the phototransformation difference spectrum in the red and far-red region     

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

Carotenoid accumulation in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by light. Besides the stimulatory function of phytochrome in carotenogenesis the experiments reveal the significance of chlorophyll accumulation for the accumulation of larger amounts of acrotenoids. A specific blue light effect was not found. The data suggest that light exerts its control over carotenoid biogenesis through two separate mechanisms: A phytochrome regulation of enzyme levels before a postulated pool of free carotenoids, and a regulation by chlorophyll draining the pool by complex-formation.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR high irradiance reaction (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - P_fof total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_fof], wavelength dependent photoequilibrium of the phytochrome system - red red light - fr far-red light     

Phytochrome-mediated induction of phenylalanine ammonia-lyase in the cotyledons of tomato (Lycopersicon esculentum Mill.) plants

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) induction in cotyledons from 96-h dark-grown Lycopersicon esculentum Mill. was studied in response to continuous light and hourly light pulses (blue, red, far red). The increases of PAL promoted by blue and red pulses are reversed completely by immediately following 758 nm irradiations. The response to continuous red light could be substituted for by hourly 6-min red light pulses. The effect of continuous red treatments is mainly due to a multiple induction effect of phytochrome. In contrast to red light, hourly light pulses with far red and blue, light can only partially substitute for continuous irradiation. The continuous blue response could be due to a combination of a multiple induction response and of a high irradiance response of phytochrome. The continuous far red response, could represent a high irradiance response of phytochrome. Dichromatic irradiations indicate that phytochrome is the photoreceptor controlling the light response (PAL) in tomato seedlings.Abbreviations Norflurazon NF-4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3 (2H) pyridazinone - PAL phenylalanine ammonia-lyase - phytochrome photoequilibrium P_fr/P_tot - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome: P_r+P_fr     

Native phytochrome: immunoblot analysis of relative molecular mass and in-vitro proteolytic degradation for several plant species

The relative molecular mass (M_r) of the native phytochrome monomer from etiolated Cucurbita pepo L., Pisum sativum L., Secale cereale L. and Zea mays L. seedlings has been determined using immunoblotting to visualize the chromoprotein in crude extracts subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single phytochrome band is observed for each plant species when the molecule is extracted under conditions previously demonstrated to inhibit the proteolysis of native Avena sativa L. phytochrome. A comparison among plant species indicates that the M_r of native phytochrome is variable: Zea mays=127000; Secale=Avena=124000; Pisum=121000; Cucurbita=120000. The in-vitro phototransformation difference spectrum for native phytochrome from each species is similar to that observed in vivo in each case and is indistinguishable from that described for native Avena phytochrome. The difference minima between the red- and far-red-absorbing forms of the pigment (Pr-Pfr) are all at 730 nm and the spectral change ratios (A_r/A_fr) are near unity. When incubated in crude extracts, phytochrome from all four species is susceptible to Pr-specific limited proteolysis in a manner qualitatively similar to that observed for Avena phytochrome, albeit with slower rates and with the production of different M_r degradation products. Further examination of the in-vitro proteolysis of Avena phytochrome by endogeneous proteases has identified several additional phytochrome degradation products and permitted construction of a peptide map of the molecule. The results indicate that both the 6000- and 4000-M_r polypeptide segments cleaved by Pr-specific proteolysis are located at the NH₂-terminus of the chromoprotein and are adjacent to a 64000-M_r polypeptide that contains the chromophore.Abbreviations and symbols A_minimum phototransformation difference spectrum (Pr-Pfr) minimum - Ig immunoglobulin - Mops 3-(N-morpholino)propanesulfonic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

Absence of fluence rate dependency of phytochrome modulation of stem extension in light-grown Sinapis alba L.

In background white light, supplementary far-red (_max 700 nm) is an order of magnitude less effective than supplementary far-red (_max 739 nm) in the stimulation of stem extension in Sinapis alba. The relationship between phytochrome photoequilibrium and extension rate increase for the two supplementary far-red treatments is, however, very similar. This evidence indicates that phytochrome cycling is not involved in the phytochrome control of stem extension in light-grown Sinapis alba and that the response to supplementary far-red light is not fluence rate (irradiance) dependent.Abbreviations Pfr far-red absorbing form of phytochrome - the phytochrome photoequilibrium (Pfr/Ptotal)     

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     

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

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

Light quantity and quality interactions in the control of elongation growth in light-grown Chenopodium rubrum L. seedlings

The spectral control of hypocotyl elongation in light-grown Chenopodium rubrum L. seedlings has been studied. The results showed that although the seedlings responded to changes in the quantity of combined red and far-red radiation, they were also very sensitive to changes in the quantity of blue radiation reaching the plant. Altering the proportion of red: far-red radiation in broad waveband white light caused marked differences in hypocotyl extension. Comparison of the responses of green and chlorophyll-free seedlings indicated no qualitative difference in the response to any of the light sources used, although photosynthetically incompetent plants were more sensitive to all wavelengths. Blue light was found to act primarily of a photoreceptor which is different from phytochrome. It is concluded that hypocotyl extension rate in vegetation shade is photoregulated by the quantity of blue light and the proportion of red: far-red radiation. In neutral shade, such as that caused by stones or overlying soil, hypocotyl extension appears to be regulated primarily by the quantity of light in the blue waveband and secondarily by the quantity of light in the red and far-red wavebands.Abbreviations B blue - FR far-red - k ₁, k ₂ rate constants for photoconverison of Pr to Pfr and Pfr to Pr, respective - k ₁/k ₁ +k ₂= phytochrome photoequilibrium - k ₁ +k ₂= phytochrome cycling rate - Pr=R absorbing form of phytochrome - Pfr=FR absorbing form of phytochrome - P_tot Pr+Pfr - PAR photosynthetically active radiation = 400–700 nm - R red - WL white light     

Photoregulation of germination in seed of transgenic lines of tobacco and Arabidopsis which express an introduced cDNA encoding phytochrome A or phytochrome B

Photoinduction and photoinhibition of germination in seed from a homozygous tobacco (Nicotiana tabacum L.) line containing an introduced oat phyA cDNA (encoding phytochrome A) is compared with that of isogenic wild-type (WT) tobacco. Under continuous irradiation by a light source with a low redfar-red (RFR) ratio the transgenic tobacco seed appeared to be less susceptible to photoinhibition of germination compared with WT seed. However, induction of germination following a short pulse by R (666 nm) was not enhanced in the genotype transformed by oat phyA cDNA compared with the WT; neither did germination of the transgenic tobacco seed show an increased sensitivity to saturating pulses of light of longer wavelengths (666–730 nm). In seeds of transgenic Arabidopsis thaliana (L.) Heynh. which contained an introduced phytochrome-B-encoding cDNA, levels of dark germination were enhanced, consistent with mediation of response by phytochrome B-P_fr. The germination behaviour of Arabidopsis genotypes wich contained an introduced cDNA encoding phytochrome A, however, did not significantly differ from that of the WT.Abbreviations ABO seed transformed with Arabidopsis phyB - cDNA; CaMV cauliflower mosaic virus - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_tot total phytochrome - P_fr/P_tot phytochrome photoequilibrium - R red light - RBO seed transformed with rice phyB cDNA - RFR quantum ratio of red and far-red light - WL white light - WL + FR whitelight supplemented with far-red light - WT wild type The authors wish to thank R.D. Vierstra (Department of Horticulture, University of Wisconsin-Madison, USA) for providing the transgenic tobacco line, and M.T. Boylan, D. Wagner and P.H. Quail (U.C. Berkeley/USDA Plant Gene Expression Center, Albany, Calif. USA) for providing the transgenic Arabidopsis lines. The work presented in this paper was funded by grants from the Agricultural and Food Research Council (H.S., A.C.M., G.C.W.).     

Coaction of three factors controlling chlorophyll and anthocyanin synthesis

In a three-factor analysis the rate of chlorophyll a (Chl) accumulation in excised mustard cotyledons was studied as a function of kinetin, light (operating through phytochrome, P _fr) and an excision factor. It was found that the three factors operate additively provided that the P _fr level is high enough. When the P _fr level is below approximately 1 per cent (<0.01) the effectiveness of the excision factor decreases while the effect of kinetin remains additive. The observed additivity is explained by a model where the three factors operate independently through a common intermediate (presumably 5-aminolevulinate) in the biosynthetic chain leading to Chl. With regard to the coaction of the excision factor and phytochrome it is concluded that the production of the excision factor requires the operation of phytochrome (even though saturated at a low P _fr level) while the action of the excision factor is independent of phytochrome. This conclusion was confirmed by experiments in which the rate of light-mediated anthocyanin synthesis was measured in excised mustard cotyledons. The effect of excision in the case of anthocyanin formation differs kinetically from the effect of excision on Chl formation.Abbreviations Chl chlorophyll(ide) a - P _fr far-red absorbing form of phytochrome - P _fr/P _tot ratio at photoequilibrium - RL red light - FR far-red light - GL green light - RG9 light long wavelength far-red light - WL white light     

The effect of light pretreatments on phytochrome-mediated induction of anthocyanin and of phenylalanine ammonia-lyase

Induction by light of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and of anthocyanin in cotyledons of the mustard (Sinapis alba L.) seedling is strongly affected by a light pretreatment which operates through phytochrome. If PAL or anthocyanin is induced by a light pulse, the effectiveness of phytochrome (P_fr) is strongly increased by a light pretreatment; however, if the increase of the PAL level or synthesis of anthocyanin is elicited by continuous far-red light (operating via phytochrome in the High Irradiance Response), effectiveness of light is strongly reduced by the same light pretreatment. This reduction of effectiveness is correlated with a decrease of total phytochrome (P_tot) caused by the light pretreatment. It is argued that the observations are compatible only with the open phytochrome-receptor model as suggested by Schäfer (J. Mathem. Biol. 2, 41–56, 1975). The peaks of the time courses of the PAL levels under continous far-red light are located at 48 h after sowing and do not depend on the original level of phytochrome. The decrease of the PAL levels beyond 48 h after sowing takes place independently of phytochrome and of the actual level of PAL.Abbreviations P_r red absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - P_tot total phytochrome (P_r+P_fr) - {ie369-1} [P_fr] /[P_tot], photoequilibrium of phytochrome at wavelength - HIR High Irradiance Response - PAL phenylalanine ammonialyase (EC 4.3.1.5)     

The loss of phytochrome photoreversibility in vitro

Killer, a substance extracted from stem tissue of etiolated pea seedlings (Pisum sativum L. v. Alaska), interacts specifically with the far-red-absorbing form of phytochrome (Pfr) in vitro in a temperature-independent, rapid, stoichiometric fashion to cause a loss of phytochrome photoreversibility. The chromatographic, solubility, and spectral properties of partially purified fractions indicate that Killer is a cyclic, unsaturated molecule containing ionizible hydroxyl groups; its molecular weight is unknown, although probably low. Possible mechanisms by which the Killer-phytochrome interaction results in the loss of photoreversibility are discussed.I=Fox, 1975     

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