Partial characterization of the interaction between phytochrome and a particulate fraction of maize coleoptiles

Extraction of red light-irradiated maize coleoptiles with Mg⁺⁺-containingbuffers enhances the pelletability of phytochrome. The enhancedphytochrome pelletability is taken to represent in vivo interactionof phytochrome with its binding site. The bonds involved inthe interaction appear to be ionic. This is shown by the requirementof Mg⁺⁺ for the enhanced pelletability and for the maintenanceof phytochrome repelletability during subsequent fractionation.Also, the phytochrome-binding site complex is responsive tothe disruptive effect of high ionic strength buffers, but itisrelatively insensitive to pH changes. Due to the ionic natureof the interaction, the complex can withstand membrane dissolutionby the non-ionic detergent Triton X 100. Though the molecularbasis for the persistent re-pelletability of phytochrome afterdetergent treatment remains to be determined, data obtainedindicate that the phenomenon is not due to denaturation of thephytochrome molecule per se. (Received May 25, 1976; )     

Particle-bound phytochrome: Differential pigment release by surfactants, ribonuclease and phospholipase C

Surfactants and hydrolytic enzymes were used to probe the natureof the constituents) to which phytochrome binds in paniculatefractions from red-irradiated Cucurbita. [¹⁴C]-choline and [³H]-uridinepre-labelled tissue was used to monitor the release of phospholipidsand RNA by these agents. Ribonuclease (RNase) digestion of 20,000xgpellets eliminates both the phytochrome and ribonucleoprotein(RNP) which cosediment at 31S. Little [¹⁴C]-choline occurs inthe 31S fraction and the amount is not changed by RNase digestion.This is further evidence that phytochrome binds directly tothe RNP in the 31S fraction rather than to any membranous materialpresent. The distribution profile of the RNA in a second ( =‘heavy’)phytochrome fraction does not correlate with that of the pigment.This suggests that the phytochrome in this fraction is not boundto RNP. The RNA is of ribosomal origin but much less degradedthan that of the 31S RNP and is resistant to RNase digestion.Phospholipase C releases>80% of the [¹⁴C]-choline from the‘heavy’ fraction without freeing phytochrome. Thisindicates that the pigment does not bind to the polar head groupsof the membrane phospholipids present. Low concentrations ofdeoxycholate dissociate phytochrome from this fraction withoutreleasing substantial quantities of integral membrane proteinsor phospholipids. Some RNP is dislodged by the surfactant butthe phytochrome and RNP are not released as a complex. The datasuggest that the pigment in the ‘heavy’ fractionmay be loosely bound to a protein constituent rather than toRNP or polar phospholipids. ¹This work was done while on sabbatical leave from the WeizmannInstitute of Science, Rehovot, Israel. (Received April 1, 1976; )     

Non-Specific Association of Phytochrome to Nuclei during Isolation from Dark-Grown Pea (Pisum sativum cv. Alaska) Plumules.

Although phytochrome is known to regulate gene expression inplants, the location of the phytochrome molecules involved inthe response remains unclear. Thus the aim of this investigationwas to test the possibility that nuclei contain phytochrome.Nuclei were isolated from dark-grown pea (Pisum sativum cv.Alaska) plumules in the dark and the nuclear proteins were resolvedon SDS polyacrylamide gels and transferred to nitrocelluloseby western blotting. A significant amount of phytochrome wasdetected in the nuclear proteins by immunostaining using monoclonaland polyclonal anti-phytochrome antibodies. Most of the phytochromeassociated with the nuclei could not be removed by treatmentwith high salt and low magnesium, indicating that the bindingwas rather stable. However, the isolation of nuclei in the presenceof exogenously added [¹²⁵I]phytochrome of P_R form in the darksuggested that significant amount of the phytochrome detectedin the nuclei was derived from contamination by the solublefraction during isolation. It is an open question as to whether,besides this contaminated phytochrome, isolated nuclei endogenouslycontain very small amount of phytochrome. ⁴Present Address: Plant Molecular Biology and Biochemistry ResearchGroup, Departments of Biochemistry and Botany, University ofGlasgow, Glasgow G12 8QQ, U.K. (Received February 8, 1988; Accepted July 27, 1988)     

Characterization of the Phytochrome-containing Particles Obtained by Glutaraldehyde Pre-fixation of Maize Coleptiles

Almost all of the particulate phytochrome of maize coleoptiles,obtained by cross-linking phytochrome to its putative receptormembrane by glutaraldehyde within the intact cells, sedimentedduring relatively low-speed centrifugation (10 000-20 000 g).The sediment contained morphologically identifiable mitochondria,some microsomes, plasma membranes, and membranes whose originis not yet known. Although its density was very similar to thatof mitochondria the phytochrome-containing material sedimentedseparately from mitochondria and microsomes but roughly in parallelwith an oligomycin-insensitive ATP-hydrolysing activity. Enrichmentof the phytochrome-containing material was obtained by subfractionationof the 20 000 g sediment. This enrichment of phytochrome coincidedwith an enrichment of certain vesicles, which, on the basisof their staining properties, are taken to be plasma membranevesicles, in the subfraction. The evidence is held to supportthe view that phytochrome attaches to and interacts with theplasma membrane upon photo transformation to the Pfr1 form.     

The Distribution of a Phytochrome-Like Protein in the Fern Psilotum nudum.; An Immunoblotting Analysis of an Early Ancestor of all Vascular Plants

The distribution of a 125 kg . mol^?1 protein recognized by a monoclonal antibody raised against phytochrome of maize was analyzed in the sporophyte of the fern Psilotum nudum. Highest amounts (up to 5 μg per fresh weight) of this protein were found in the tips of expanding shoots. Green sporangia as well as the pale tips of the rhizome contained this 125 kg . mol^?1 protein, too. In the brown parts of the rhizome it was more rarely contained. Unlike phytochrome from etiolated higher plants, the Psilotum protein appeared to be scarcely degraded by the illuminated plants. In this respect the protein of Psilotum seems to resemble the small fraction of phytochrome contained in green and illuminated higher plants. Moreover, after illuminating the Psilotum rhizome for 3 d, higher amounts of this protein were detected therein as before.     

Effects of lanthanum on rhythmic and nyctinastic leaflet movements in Albizzia lophantha

The involvement of extracellular calcium in rhythmic and nyctinasticmovement oi Albizzia lophantha Benth. leaflets has been studiedby testing the effect of LaCl₃ and its interaction with thephytochrome control of these movements. A 2h pulse of LaCl₃(10–50 mM) promotes a loss of rhythmicity, leaving leafletsin an open position, and also overrides the phase shift causedby phytochrome. A 2 h pulse of LaCl₃ (1 mM) decreases the amplitudeof rhythmic oscillations but does not promote arhythmicity normodify the phase shift caused by red light. The red light pulseabolishes the damping effect of 1 mM La³⁺. LaCl₃ inhibits nyctinasticclosure and decreases the phytochrome control of nyctinasticclosure. A subsequent supply of CaCl₂ (10 to 100 mM) does notreverse La³⁺ (10 mM) inhibition of closure. Light-induced openingis independent of LaCl, but rhythmic opening in darkness showsdifferent responses to La³⁺ depending on the time at which La³⁺is applied. Data suggest that extracellular calcium is requiredfor the closure mechanism and for the expression of rhythmicmovement. It could also be involved in the phytochrome transductionpathway and/or in the linking steps between phytochrome andthe circadian clock. Key words: Albizzia lophantha, calcium, circadian rhythm, lanthanum, phytochrome     

Phytochrome regulation of peroxidase activity in maize V. Role of RNA and protein synthesis

The phytochrome mediated enhancement of peroxidase activityin maize leaves was repressed by inhibitors of cytoplasmic proteinsynthesis, whereas inhibitors of RNA and organelle protein synthesiswere ineffective. Continuous far-red light had no effect onthe DNA level in the leaves, but it increased the RNA levelafter a lag of 2 hr. Under continuous far-red light the totalcontent of polyribosomes also increased after a lag of 2 hr.Isolated polyribosomes from far-red grown plants showed an enhancedrate of the in vitro incorporation of amino acids into proteinsas compared to dark grown plants. These results indicate thatthe phytochrome regulation of peroxidase activity occurs atthe translational level. ¹Present address: School of Life Sciences, University of Hyderabad,Hyderabad-500001, India (Received July 25, 1979; )     

Phytochrome Control of Its Own Synthesis in Pisum sativum

An analysis of phytochrome synthesis in Pisum seedlings by measuringthe activity of polysomal polyadenylated RNA (poly-A⁺-RNA) codingfor phytochrome apoprotein showed phytochrome control of itsown synthesis; brief red-light irradiation of pea seedlingsinhibited the activity of the RNA, and the red-light effectwas red/far-red reversible. ⁴ Permanent address: Biology Department, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received August 13, 1984; Accepted September 17, 1984)     

Further characterization of the in vitro binding of phytochrome to a membrane fraction enriched for mitochondria

This study employs ¹²⁵I-labeled phytochrome (¹²⁵I-P) from oats to quantitate the binding of phytochrome to a membrane fraction from oats that is highly enriched for mitochondria, and it examines several parameters that influence this attachment. The binding of ¹²⁵I-Pfr to the mitochondrial fraction of unirradiated oat seedlings is significantly higher than that of ¹²⁵I-Pr. However, ¹²⁵I-Pfr and ¹²⁵I-Pr bind in equal quantities to mitochondrial preparations isolated from light-exposed seedlings. Maximum ¹²⁵I-Pfr binding to membranes from light-exposed plants occurs within 30 seconds and is optimized in a reaction buffer containing 5 millimolar MgCl₂ at pH 6.8. Scatchard plots of the binding data for Pfr indicate a single high-affinity site with an affinity constant of 1.79 × 10¹¹ per molar. When optimal binding conditions are used, over 20% of the ¹²⁵I-P added is bound and a stoichiometry of about 100 molecules per mitochondrion is attained. When the specificity of binding is tested using competition experiments with a 15-fold excess of unlabeled phytochrome, ¹²⁵I-Pfr shows no specific binding to rat liver mitochondria.     

Isolation of cDNA for Pea Phytochrome Using an Expression Vector

Partially purified phytochrome mRNA was obtained from etiolatedpea epicotyls by polyribosome immunoprecipitation or by sizefractionation of total poly(A)⁺RNA, and used for the synthesisof double-stranded complementary DNA (cDNA). cDNA librarieswere constructed using an Escherichia coli expression vector,pUC9, and screened for phytochrome cDNA by colony immunologicalassay. Nine colonies were found to produce a 27 kDa polypeptidethat was reactive to both polyclonal and monoclonal antipeaphytochrome antibodies. The plasmids from these colonies containedcDNA inserts of 1.2 or 2.0 kbp. Hybridization-arrest translationassay verified that the cDNA clones contained a sequence codingfor phytochrome polypeptide. RNA blot hybridization analysisindicated that the cDNA hybridized to a 4.1 kb poly(A)⁺RNA indark-grown pea. (Received March 22, 1986; Accepted June 13, 1986)     

Effect of Phytochrome on Uptake and Efflux of Gibberellin

The effect of red light on uptake and efflux of gibberellinby etiolated pea stem sections has been determined by the techniqueof compartmental analysis. Exposure of segments to red lightinhibited uptake and efflux of [³H]-GA₁ by the cytoplasmic compartmentwhile efflux from the vacuolar compartment was not significantlyaffected. It is suggested that phytochrome may alter the permeabilityproperties of the plasma membrane and/or the binding of GA₁,as, a means of controlling the basipetal transport of gibberellin. (Received November 19, 1984; Accepted March 2, 1985)     

Effects of divalent cations and EDTA on spectral properties of phytochrome in particulate fractions from etiolated pea epicotyls

The photoreversible absorbance change of phytochrome in suspensionsof a 20,000xg particulate fraction (20kP) prepared from a 1,000xgsupernatant (1kS) of etiolated pea epicotyl extracts decreasedremarkably in the presence of 5 mM Cu²⁺, Zn²⁺ and Co²⁺, butremained unchanged in 5 mM Ca²⁺, Mg²⁺, Fe²⁺ or Mn²⁺. This spectraldistortion of phytochrome was more evident in soluble preparationsand in suspensions of pellets prepared from red light (R)-irradiatedtissues than it was in suspensions of pellets prepared in thedark from etiolated tissues that received no actinic irradiation. When Cu²⁺ was added to the red-light-absorbing form of phytochrome(Pr) in resuspended pellets prepared from R-irradiated tissues,the distortion of its difference spectrum took place after irradiationwith the first actinic R. In contrast, when Cu²⁺ was added tothe far-red-light-absorbing form of phytochrome (Pfr) in thesame resuspended pellet, no distortion was seen, unless thePfr in the pellet was first photoconverted to Pr and then photoconvertedback to Pfr. Spectral distortion of Pr remained small during dark incubationat 25°C when suspensions of 20kPs were prepared and incubatedwith a buffer containing EDTA, whether the 20kP was preparedfrom nonirradiated tissue or from R-irradiated tissues. But,when EDTA was added to a suspension of 20kP prepared from 1kS,after the 1kS was irradiated with R in the presence of 10 mMCaCl₂, the spectral distortion of Pr in 20kP occurred instantaneously. (Received April 14, 1980; )     

Subcellular localization of the red-absorbing form of phytochrome by immunocytochemistry

Summary An immunocytochemical technique was used to localize the red-absorbing form of phytochrome at the light- or electron-microscope level in etiolated barley (Hordeum vulgare L.) coleoptile tip, rice (Oryza sativa L.) coleoptilar node, maize (Zea mays L.) coleoptile tip, rye (Secale cereale L.) coleoptile tip and coleoptilar node, and oat (Avena sativa L.) root cap. Staining for phytochrome in the cells was found to be generally distributed throughout the cytoplasm. In addition, barley also showed staining around the periphery of vesicles, and rice showed staining in numerous discrete regions in the cytoplasm. Electron-microscopic localization studies of the nodal region of rye and the root cap of oat indicate staining associated with the nuclear membrane and with the interior of mitochondria and amyloplasts as well as general staining like that observed with the light microscope. Cells of the coleoptile tip of maize were unusual in having heavy staining associated with amyloplasts only.Abbreviations DAB 3,3-diaminobenzidine - PAP peroxidase-antiperoxidase complex - Pr red-absorbing form of phytochrome - Pfr far-red-absorbing form of phytochrome     

Effects of Blue Light Pretreatments on Internode Extension Growth in Mustard Seedlings after the Transition to Darkness: Analysis of the Interaction with Phytochrome

The effects of blue light (B) pretreatments on internode extensiongrowth and their possible interaction with phytochrome mediatedresponses were examined in Sinapis alba seedlings grown for11 d under 280 µmol m^–2 s^–1 of continuousblue-deficient light from low pressure sodium lamps (SOX). SupplementaryB (16 µmol m^–2 s^–1) caused no detectable inhibitionof the first internode growth rate under continuous SOX, butgrowth rate was inhibited after transfer to darkness. This effect,and the growth promotion caused by far-red bend-of-day' lightpulses were additive. The addition of B at 16 µmol m^–2s^–1 during 11 d, or only during the first 9 or 10 d orthe latest 0.75, 1 or 2 d of the SOX pretreatment caused approximatelythe same extent of inhibition after the transition to darkness.A single hour of supplementary B before darkness caused morethan 50% of the maximum inhibition. However, 24 h of lower fluencerates of B (4 or 7 µmol m^–2 s^–1) were ineffective.Covering the internode during the supplementary B period didnot prevent the response to B after the transition to darkness.Far-red light given simultaneously with B (instead of the SOXbackground) reduced the inhibitory effect of B. Above a given threshold fluence rate, B perceived mainly inthe leaves inhibits extension growth in subsequent darkness,provided that high phytochrome photo-equilibria are presentduring the irradiation with B. Once triggered, this effect doesnot interact significantly with the ‘end-of-day’phytochrome effect. Key words: Blue light, extension growth, phytochrome     

The Deduced Amino Acid Sequence of Phytochrome from Adiantum Includes Consensus Motifs Present in Phytochrome B from Seed Plants

A cDNA for the phytochrome of the fern Adiantum capillus-venerisL. was cloned and sequenced. The deduced phytochrome is 50{smalltilde}55% identical to phytochromes of seed plants, and 68%identical to Selaginella phytochrome. Regions resemble thosein previously characterized phytochromes from ferns, lower plantsand seed plants. ³Present address: Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka,Tsukuba-shi, Ibaraki, 305 Japan ⁴Present address: Plant Growth Regulation Laboratory, The Instituteof Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi,Saitama, 351-01 Japan ⁵Present address: Advanced Research Laboratory, Hitachi, Ltd.,Hatoyama, Saitama, 350-03 Japan     

The spontaneous growth response of maize coleoptile segments and the change in tissue 8

Decapitated segments from maize (Zea mays L.) coleoptiles orientedvertically in an upright position show a strong spontaneousgrowth response (SGR) 3 h after decapitation. The latent periodof the SGR is markedly reduced when these segments are orientedin an inverted position. Coleoptile segments with intact tipsexhibit a weak and transient SGR in the vertical upright orientation.However, in the inverted orientation, these segments show atypical SGR. The data are inconsistent with the current hypothesisthat the SGR is caused by a time-dependent increase in tissuesensitivity to auxin. The parallel increase in membrane potentialdifference and growth rate during the time-course of the SGRindicates a possible role for PM H⁺-ATPase in the establishmentof the SGR in maize coleoptile segments. Key words: Auxin, spontaneous growth response, membrane potential, plasma membrane H⁺-ATPase, Zea mays L.     

Synthesis of Translatable mRNA for Phytochrome during Imbibition in Embryonic Axes of Pisum sativum L.

The activity of translatable mRNA for phytochrome was measuredin excised embryonic axes of Pisum sativum L. during imbibitionboth in the dark and under continuous irradiation with whitelight. When measured in cell-free protein synthesis systemsof both rabbit reticulocyte lysate and wheat germ extract, theactivity of translatable mRNA for phytochrome was not detectedin dry quiescent axes but increased rapidly after imbibitionin the dark. After 24 h imbibition, the level of translatablemRNA for phytochrome, in terms of the incorporation of [³⁵S]methioninein the wheat germ system, was ca. 0.0034% of total translatablemRNA. In the presence of 0.5 µg ml^–1 -amanitin,the appearance of translatable mRNA for phytochrome was inhibitedby 60%, while 2 µg ml^–1 -amanitin was almost completelyinhibitory. This indicates that the synthesis of translatablemRNA for phytochrome in embryonic axes begins upon imbibition. When the axes were imbibed under continuous white light, theactivity of phytochrome mRNA increased as rapidly during thefirst 3 h as in the dark. After this time, the activity wasmarkedly lower than in the dark. Nevertheless, during the 24h of imbibition, activity in the light was always found to bemore than half of that in the dark. These results indicate thatin germinating pea axes the level of translatable mRNA for phytochromeis partially repressed by light. (Received June 5, 1985; Accepted September 2, 1985)     

Glucose Induced H+ Influx and Transient Currents in Excised Roots: particularly those of Zea mays

The application of D-glucose to solutions bathing excised maize,wheat, pea and bean roots causes a rapid depolarization of theelectrical potentials between the cut tops of the roots andthe bathing solutions. Similar effects are observed for theplasma membrane potentials of maize lateral roots. A flow cell apparatus was used to demonstrate qualitative andquantitative relations between glucose induced H⁺ influx andthe transient decrease in current through the root. The currentchanges appear to be due entirely to H⁺ fluxes. Current andH⁺ fluxes are strongly influenced by external pH, the optimumpH for glucose induced current change being about 4.0. A similarpH optimum was found for 3-O-methyl-D-glucopyranoside but 1-O-methyl--D-glucopyranosidedid not significantly affect the trans-root potential at anypH, suggesting a significant role for the anomeric hydroxylgroup of glucose. Compounds which depolarize the trans-root potential also inhibitthe glucose induced depolarization. Surface -SH groups are probablynot involved in the glucose/H⁺ cotransport. Eadie-Hofstee plots relating the depolarization of trans-rootpotential to the concentrations of D-glucose or 3-O-methyl-D-glucopyranosidehave shown that K_m values increase with increasing monosaccharideconcentration and are very similar to reported values of 3-O-methyl-D-glucopyranosideuptake in maize root segments. K_m values for a similar rangeof D-glucose concentrations do not vary significantly with pHor with membrane depolarization due to a 10-fold increase ofKCl concentration. However, V_max is lowered by an increase inexternal pH or a decrease in trans-root potential. It appearsthat both proton and electrical gradients can affect glucoseinduced H⁺ influx. The auxin herbicide, 2, 4-dichlorophenoxyethanoic acid (0.01mM) stimulates the glucose induced depolarizations in a mannerconsistent with an increase in cytoplasmic pH. This is discussedin relation to the reported action of indole-3-acetic acid andfusicoccin on maize root tissue.