Differential Exposure of Tryptophan Residues in the Red and Far-Red Light Absorbing Forms of Phytochrome, as Revealed by Chemical Modification

The effects of the chemical modification of tryptophan residuesin native pea (Pisum sativum L.) phytochrome by 2-hydroxy-5-nitrobenzylbromide (HNB-Br) were examined. Such treatment had no effecton the spectral properties or on the pattern of tryptic digestionof phytochrome, which indicated that no major conformationalchange in phytochrome had occurred. Amino acid analysis of theHNB-Br-treated phytochrome indicated that the number of modifiedTrp residues after the treatment was dependent on the light-absorbingform. The values were three for P_R and five for P_FR (out ofa total of ten) per monomer. The results indicate that two additionalTrp residues are exposed on the molecular surface of P_FR whenthe photoconversion of P_R to P_FR occurs. The amino acid analysisof a 58-kDa tryptic fragment of phytochrome (a mixture of peptides,residues 63–583 and 66–587) showed that one Trpresidue in the fragment from P_R and two in that from P_FR (outof six) were modified by HNB-Br. In the 56-kDa fragment (a mixtureof peptides, residues 598–1121 and 603–1124), therewere two modified Trp residues in P_R and three in P_FR (out offour). The Trp residue in a 36-kDa fragment (residues 66–383),which includes the tetrapyrrolic chromophore, was not modifiedin the either case. These results indicate that new exposedsites that are generated by the photoconversion of P_R to P_FRare in the region between Trp–456 and Trp–567 andin that between Trp–644 and Trp–787. (Received February 25, 1993; Accepted August 16, 1993)     

Effect of Neutral Salts on Spectral Characteristics of Undegraded Phytochrome Partially Purified from Etiolated Pea Shoots

Spectral characteristics of partially purified undegraded peaphytochrome were investigated in different ionic conditions.At the red-light-induced photostationary state in low ionicstrength buffer phytochrome had reduced absorbance in its far-redpeak as reported previously. Elevation of the ionic strengthof the buffer reversibly increased the absorbance in the far-redregion at the photostationary state. It was found that the effectof increase of ionic strength was strengthened secondarily bychaotropicity of salts. It was confirmed that phytochrome preparations of low ionicstrength contained a photosensitive component(s) other thanthe red-light-absorbing form (P_R) and farred- light-absorbingform (P_FR) during photochemical transformation, as well as duringthe first several min in the dark after phototransformation.At high ionic strength, phytochrome became a two-component systemcomposed of only P_R and P_FR at the redlight-induced photostationarystate though a significant accumulation of another component(s)occurred during phototransformations. Increasing ionic strengthalso enhanced A723 of phytochrome at the red-light-induced photostationarystate. The effect could result from either an increased molefraction of P_FR at the photostationary state induced by redlight, or a change in the extinction coefficients of P_FR. ¹ Present address: Division of Biological Regulation, NationalInstitute for Basic Biology, Myodaijicho, Okazaki 444, Japan (Received March 18, 1981; Accepted August 3, 1981)     

Identification of Surface-Exposed Parts of Red-Light- and Far-Red-Light- Absorbing Forms of Native Pea Phytochrome by Limited Proteolysis

Peptide fragments were obtained by limited proteolysis withtrypsin and Staphylococcus aureus V 8 protease from either theP_R or the P_FR form of 121-kDa phytochrome purified from etiolatedpea (Pisum sativum L.) shoots. Patterns of bands after polyacrylamidegel electrophoresis in the presence of SDS of the digests weredifferent, with some bands appearing preferentially when thedigestions were carried out with the P_R or the P_FR form. Amino-terminalsequences of the fragments were analyzed to determine the exactlocations of the amino-termini of the fragments within the aminoacid sequence of the apoprotein of pea phytochrome. The aminoacid compositions of some of the sequenced fragments were determinedin order to confirm the carboxy-terminal amino acids. Threecleavage regions were identified as kinetically favored sitesof cleavage of P_FR (Arg-746 to Lys-752, around Glu-877 and aroundArg-1010), whereas only one was identified for P_R (Glu-38 toArg-62). Regions of Glu-255, Arg-383, Arg-583 to Glu-620 andLys-1093 to Glu-1115 were also identified as potential sitesof proteolytic cleavage in both forms of the phytochrome. Othercleavage sites, the specificities of which have not yet beendetermined, are Glu-404, Glu-695 and Lys-1045. Surface-exposed parts of phytochrome in the P_R and P_FR formsare discussed. (Received June 13, 1992; Accepted October 27, 1992)     

Electrostatic Binding of Proteins and Phytochrome to Differently Charged Liposomes

The sign and magnitude of the surface charge of liposomes containingelectrostatically neutral lecithin and cholesterol was alteredby incremental additions of dicetyl phosphate or stearylamine.Such liposomes instantaneously bound authentic proteins at 0°Conly when they had electrostatically opposite charges; 1 M NaClinhibited the binding. The amount of protein bound was dependentupon the concentration of protein and the charge of liposomes.Phytochrome in a crude extract of etiolated pea (Pisum sativumcv. Alaska) shoots could bind equally well to liposomes witheither positive or negative charges irrespective of P_R and P_FRboth of which showed no spectral distortion. Both P_R and P_FRof purified pea phytochrome bound entirely to positively chargedliposomes but partially to negatively charged ones. In thisassociation both P_R and P_FR became pelletable at similar rates.Absorption spectra of liposome-bound P_R showed a small blueshift and then a crucial spectral distortion after red-lightirradiation. (Received October 22, 1980; Accepted January 22, 1981)     

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)     

Purification and spectral characterization of a 121-kilodalton phytochrome from etiolated pea (Pisum sativum L.) seedlings

Procedures for the purification of native phytochrome from etiolatedpea seedlings without the use of immuno-purification techniquesare described. Phytochrome (in the P_FR form) was purified bypolyethyleneglycol fractionation, adsorption to pentyl agaroseand batch elution, chromatography on DEAE-Sepharose, adsorptionto phenyl Toyopearl and batch elution, and chromatography onRed Toyopearl. The resulting phytochrome had specific absorbanceratios (SAR = A₆₆₆/A₂₈₀ of P_R) that ranged from 0.55 to 0.6.The subsequent chromatography on Sephacryl S-300 yielded verypure phytochrome with a SAR of 0.98. P_R and P_FR peaks in thedifference spectrum of the phytochrome were centered at 665and 730 nm, respectively. The spectral change ratio (Ar/Afr)of the difference spectrum was unchanged after the chromatographyon phenyl Toyopearl, and the value was 1.05–1.08, indicatingthat the spectral properties of this preparation were intact.The absorption spectra indicated that the peak absorbance ofP_FR was at 728–730 nm and that of P_R was at 666–667nm. These peak positions were essentially same as those obtainedwith the undegraded oat phytochrome. Incubation of the samplepurified on Sephacryl S-300 at 25?C for 5 h in either the P_Ror P_FR form did not result in degradation of the molecule. Therate of dark reversion of P_FR observed with the purified peaphytochrome was similar to that observed in vivo. The additionof dithionite had no effect on the reversion rate. ²Present address: Fuji-Gotenba, Research Lab. of Chugai PharmaceuticalCo. Ltd., Gotenba, Shizuoka, 412 Japan (Received February 22, 1990; Accepted May 28, 1990)     

Phototransformation of the Red-Light-Absorbing Form to the Far-Red-Light-Absorbing Form of Phytochrome in Pea Epicotyl Tissue Measured by a Multichannel Transient Spectrum Analyser

Phototransformation of the red-light-absorbing form (P_R) tothe far-red-light-absorbing form (P_FR) of phytochrome in 7-day-oldetiolated pea epicotyl hook segments was examined at 0.5C aftera red laser flash excitation using a multichannel transientspectra analyser with electrically gated photomultiplier. Effectsof a red laser pulse on the induction of phototransformationfrom P_R to P_FR were saturated at Ca. 15 mJ for flash wavelengthsof both 640 and 655 nm. The amount of P_FR induced by a saturatinglaser pulse was ca. 50% of that obtained at the photostationaryequilibrium. A difference spectrum measured 15 µs afterthe flash showed an absorbance increase at 697 nm and a decreaseat 663 nm. A difference spectrum determined 200 ms after theflash showed no such major absorbance increase. Kinetic analysisof the rapid absorbance decrease at 700 and 710 nm gave onesimple first-order reaction component having a rate constantof 2,500 s^–1. Kinetics of P_FR appearance measured by absorbanceincrease at 750 nm was resolved into three first-order reactionshaving rate constants of 5, 1.8 and 0.4 s^–1. The secondflash light of 710 nm given 2 µs and 2 ms after the firstred flash irradiation on P_R resulted in the formation of P_Rrather than P_FR. (Received February 8, 1985; Accepted April 11, 1985)     

A Re-evaluation of the Mole Fraction of PFR at the Red-light-induced Photostationary State of Undegraded Rye Phytochrome

Photochemical properties were determined for undegraded phytochromepurified from lyophilized rye seedlings (Secale cereale cv.Cougar). The preparation was shown to be a two-component systemduring phototransformations and dark transformation from red-light-inducedphotostationary state. The mole fraction of P_FR at the 665-nm-light-inducedphotostationary state was 0.84. The ratio of the quantum yieldof photoconversion from P_R to P_FR to that from P_FR to P_R was1.53. ¹ Present address: Division of Biological Regulation, NationalInstitute for Basic Biology, Myodaijicho, Okazaki 444, Japan (Received March 18, 1981; Accepted August 3, 1981)     

Low Temperature Spectrophotometry on Phototransformation of PR in Pelletable Phytochrome of Pea

Phototransformation of P_R to P_FR in a 1,000–7,000 x gpelletable fraction (1–7 KP), which was extracted fromdark-grown pea shoots that had been irradiated by red then far-redlight, was studied by low temperature spectrophotometry. Redlight irradiation of P_R in 1–7 KP at –160°Cinduced an absorption increase at 695–696 nm with a concomitant,small decrease of P_R absorption at 670 nm. These changes werepartially photoreversed when the sample was irradiated subsequentlywith 700-nm light. At –55°C, red light irradiationof P_R resulted mainly in bleaching and consequently in a reductionof the P_R peak, accompanied by minor absorbance increases around695 nm. The intermediates formed at –165°C by 660-nmlight irradiation partly reverted back to P_R or formed a bleachedintermediate (probably the same bleached intermediate describedabove) in the dark, when the pellets were warmed to –60°C.The bleached intermediate was transformed to P_FR in the darkat –10°C or above. These characteristics of P_R transformation observed in the pelletablephytochrome were essentially the same as those observed in invivo or soluble phytochrome. (Received December 24, 1982; Accepted July 28, 1983)     

Effect of Monoclonal Antibodies on the in Vitro PFR Dark Reversion of Pea Phytochrome

Extraction as P_FR and immunoaffinity chromatography yieldeda pea phytochrome sample with polypeptide size of 121 kdalton,the same as in a crude extract which was immediately heatedin SDS. A difference spectrum was almost the same as that observedin etiolated pea epicotyls except that A₆₆₆/A₇₃₀ of 1.20 wassignificantly larger. At 10C dark reversion from P_FR occurred,with the decrease in A₇₂₈ being almost equal to the increasein A₆₆₇. The kinetics could be resolved into three first-ordercomponents, the major, slow component accounting for more than90% of the absorbance changes. In the presence of monoclonalanti-pea phytochrome antibodies mAP-1, 3 or 5, which bind awayfrom the chromophore, and mAP-7, which binds near the chromophore,the rate of the major component was reduced at either one orboth wavelengths. None of these antibodies affected the absorptionspectra of phytochrome. In the presence of mAP-9, which is suggestedto bind near the amino-terminus, the absorption at the red-light-inducedphotostationary state was reduced and the rate of dark reversionwas increased, resembling partially degraded phytochrome of114 kdalton, but with no evidence of proteolysis. ¹ Permanent address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan.     

Spectral Properties of Chromophore-Containing Fragments Prepared from Pea Phytochrome by Limited Proteolysis

Five chromophore-containing fragments were prepared from peaphytochrome in PR form (monomer mol wt 114,000) by limited proteolysiswith trypsin, thermolysin or chymotrypsin, and their absorptionand circular dichroism (CD) spectra were determined. The fragmentsof mol wt 62,100 and 56,400 showed photoreversible transformationbetween P_R and P_FR like phytochrome. The smaller fragments ofmol wt 40,300, 39,000 and 33,000 showed an absorption maximumat 657–660 nm (P₆₆₀) which was transformed to a bleachedform (P_BL) after a brief red-light exposure. The phototransformationbetween P₆₆₀ and P_BL was repeatedly reversible. Both P₆₆₀ andP_BL showed a negative CD band in red region like P_R, in contrastwith P_FR which has a positive band in far-red region. The natureof a chromophore domain of phytochrome and spectral propertiesof P_BL are discussed. ¹This study is dedicated to the late Professor J. Ashida. ²Permanent address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received August 7, 1982; Accepted March 26, 1983)     

The Role of Phytochrome in Anthocyanin Synthesis in Seedlings of Vigna radiata (L.) Wilczek

Previously, it has been demonstrated that the red light-inducedanthocyanin accumulation in mung bean seedlings is mediatedby phytochrome [Dumortier and Vendrig Z. Pflanzenphysiol. 87:313 (1978)]. In this paper the importance of phytochrome forthe accumulation of anthocyanins in seedlings of mung beanswas studied in non-irradiated seedlings and in seedlings irradiatedwith 5 min R. A short FR-irradiation given early after sowing reduced theamount of anthocyanins which were normally found in non-irradiatedseedlings. This indicates that P_FR may be important for at leastpart of the anthocyanin synthesis in the dark. As for the redlight-mediated anthocyanin accumulation, irradiation appearedto be most effective when given to seedlings at the age of 36–48hr. Although the seedlings were sensitive to red light irradiationbefore that time, they were not able to synthesize anthocyaninsuntil they had reached the age of 36 hr. Complete escape ofred/far-red reversibility occurred only when far-red was given12 hr after red, although partial escape could be observed witha shorter time-interval. Furthermore, the time-course of anthocyaninaccumulation after a two-fold R-irradiation was compared withthe effect of a single R-exposure. From the results could beconcluded that the pattern of anthocyanin accumulation is dependenton the time during which P_FR is present in the seedlings. Theseexperiments also indicate that P_FR not only plays a role inthe synthesis of anthocyanins but probably also in their degradation. The results of our study show that phytochrome is importantfor anthocyanin accumulation in non-irradiated mung bean seedlingsas well as in R-irradiated, and that it probably is also involvedin the degradation of the pigment. (Received January 18, 1982; Accepted April 30, 1982)     

Action Spectra Confirm Two Separate Actions of Phytochrome in the Induction of Flowering in Lemna paucicostata 441

Action spectra studies have shown that in the short day plant(SDP) Lemna paucicostat441 there are at least two actions ofphytochrome in the induction of flowering. At the beginningof the dark period far-red light inhibited flowering, and theaction spectrum corresponded to the absorption spectrum of P_FR,while at the middle of the inductive dark period both red andfar-red light were inhibitory. The action spectrum for the redlight corresponded to that of PR absorption, but there was activityin the region beyond 720 nm which exactly coincided with theabsorption by P_FR observed at the beginning of the dark period,indicating that at the middle of the dark period there was absorptionby both P_R and P_FR. The difference in quantum efficiency betweenthe red and far-red light effects was about 60-fold. These resultsare consistent with there being a stable pool of P_FR necessaryfor the induction of flowering and another pool of phytochromein a different cellular environment which participates in thenight-break reaction as P_R. ¹ Present address: School of Applied Biology, Faculty of Science,Lancashire Polytechnic, Preston PR1 2TQ, U.K. ² 2 Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabemachi, Tsukuba, Ibaraki305, Japan. ³ Present address: Division of Plant Biological Regulation,The Riken Institute for Frontier Research Program, Hirosawa,Wako-shi, 351-01, Japan. (Received December 13, 1986; Accepted July 17, 1987)     

Some Properties of Phytochrome Isolated From Dark-grown Oat Seedlings (Avena sativa L.)

Phytochrome was partially purified from etiolated seedlings of Avena sativa L. Several properties of the red-absorbing (P_R) and far-red absorbing (P_FR) forms of the pigment were compared. The 2 forms could not be shown to differ with respect to their sedimentation velocity in sucrose density gradients, elution volume from Sephadex G-200 columns, binding properties on calcium phosphate, or electrophoretic mobility. P_FR, however, was more labile than P_R during precipitation with 50% ammonium sulfate. Sephadex G-200 elution diagrams obtained with fresh phytochrome preparations revealed 2 components of different molecular weights, 1 roughly 180,000, and 1 roughly 80,000. Native phytochrome had an absorption spectrum in vivo showing an absorption maximum for P_R of 667 nm. Both the large and small forms of phytochrome mentioned above can be maintained with an absorption maximum for P_R of 667 nm. However, allowing them to remain for several hours as P_FR, even at 4°, shifted this peak to 660 nm. The protein conformational change during phytochrome transformation may be quite small, though the various comparative techniques used do not strictly rule out a fairly large one. The need for maintaining the pigment as P_R during all steps of purification, but particularly during ammonium sulfate precipitation is underscored.     

Binding Proteins to Phytochrome A in Etiolated Pea Seedlings

In order to detect and characterize a putative receptor(s) fora signal from PhyA, proteins that bind to purified pea PhyAwere searched for in the crude extract of etiolated pea seedlingswith affinity chromatography. PhyA was coupled to the columnsubstrate either in P_R form (P_R column) or in red-irradiatedform (P_FR column). The coupled PhyA of both columns retainsits spectral reversibility between P_R and P_FR, although theirpeptide mapping by trypsin digestion suggests that the C-terminalhalf of PhyA in the P_FR column is partially fixed in P_FR structure.15 polypeptides were detected reproducibly in the elution fromthe P_FR column by silver-staining of SDS-PAGE. These 15 polypeptidesmay form two complexes judging from their elution profiles.Of the 15 polypeptides, the 6 major polypeptides have approximatemol wt of 80, 55, 53, 46, 40 and 35 kDa. On the other hand,only a trace amount of protein, which mainly consists of the46 kDa species, was eluted from P_R column, indicating the presenceof P_FR-specific BPs in the crude extract of etiolated pea seedlings.Of the 6 major polypeptides, the 40 kDa species binds to thePhyA in a photoreversible manner. (Received June 19, 1998; Accepted December 19, 1998)     

Differences in Photoresponse and Phytochrome Spectrophotometry Between Etiolated and De-etiolated Pea Stem Tissue

Morphologically similar pea plants having a 4-fold difference in spectrophoto-metrically detectable phytochrome can be produced by pretreatment of etiolated plants with red light (R) or with red and far-red light combined (RF). A search for response differences which could be ascribed to differences in phytochrome content has resulted only in the establishment of differences due to de-etiolation. Segments of etiolated plants differ from those of plants de-etiolated by R and RF pretreatments in 2 ways. Segments from etiolated plants appear to respond rapidly to the far-red absorbing form of phytochrome (P_FR), while segments from de-etiolated plants do not respond rapidly to P_FR. This statement is based upon 2 observations: (i) the red light induced growth inhibition in segments from etiolated plants rapidly escapes reversibility by far-red light, while with segments from R or RF pretreated plants, the red light effect is fully reversed by subsequent far-red light for up to 2 hr; and (ii) segments from etiolated plants were inhibited to a greater degree than were segments from RF pretreated plants when various photostationary state levels of P_FR were maintained for 30 or 90 min and then removed by photoconversion to P_R. The in vivo nonphotochemical transformation curves of the phytochrome of etiolated and RF pretreated plants appear to differ in 2 related respects: (i) the amount of phytochrome destroyed in de-etiolated tissue is greater than that in etiolated tissue, perhaps as a result of the fact that (ii) the rate and extent of apparent reversion of P_FR to P_R in etiolated tissue is about twice that in de-etiolated tissue.     

Dichroic Orientation of Phytochrome Intermediates in the Pathway from PR to PFR as Analyzed by Double Laser Flash Irradiations in Polarotropism of Adiantum Protonemata

The polarotropic response in protonemata of the fern Adiantumis regulated by phytochrome (Kadota et al. 1984); P_R and P_FRhave been shown to be dichroically oriented parallel and normalto the cell surface, respectively (Kadota et al. 1982). Thischange in the dichroic orientation of phytochrome during photoconversionwas analyzed by a newly-built, polarization plane-rotatabledouble laser flash irradiator. A polarotropic response was effectivelyinduced with a flash of polarized red (640 nm) light (6xl0^–7s) having the vibration plane of the electrical vector parallelto the protonemal cell axis. When a flash of polarized far-red(710 nm) light (6xl0^–7s) was given 30 sec after the redflash, the red flash-induced response was reversed by a far-redflash vibrating normal to the cell axis but not by one vibratingparallel. However, when given 2 µs or 2 ms after the redflash, the polarotropic response was not reversed by a polarizedfar-red flash vibrating normal to the cell axis but was reversedby a parallel-vibrating flash. These results suggest that theorientation of phototransformation intermediates existing 2µs or 2 ms after a red flash is still parallel to thecell surface, and that the change in the orientation of phytochromemolecules occurs between 2 ms and 30 s after the red flash. (Received February 3, 1986; Accepted April 23, 1986)     

Phototransformation of the Far-red Light-absorbing Form of Large Pea Phytochrome by Laser Flash Excitation

Phototransformation of the far-red light absorbing form (P_FR)of large pea phytochrome to the red-light absorbing form (P_R)was examined at 2?C after a 715 nm laser flash excitation usinga custom-built multichannel transient spectra analyzer. Themaximum amount of phototransformation intermediates was producedby a pulse of about 50 mJ, which resulted in ca. 65% of P_R obtainedat the photostationary equilibrium. Some flash-induced intermediateswere assumed to return to P_FR in the dark. A difference spectrummeasured at 10 µsec after the flash showed an absorbanceincrease at 651 nm and a decrease at 724 nm. When the samplewas left in darkness after the flash light irradiation, absorbancein the red and far-red region gradually increased, but thatin the green region rapidly decreased. The decay curve of intermediatesmeasured at 554 nm could be resolved into three reaction componentshaving rate constants of 2,500, 590 and 48 sec^–1, respectively.Difference spectra also indicated that a small but significantincrease in absorbance between 370 and 380 nm and a decreasearound 415 nm took place 10–310 µsec after a flash. (Received February 13, 1982; Accepted April 21, 1982)     

Regulation of NADH-Glutamate Dehydrogenase Activity by Phytochrome, Calcium and Calmodulin in Zea mays

Regulation by the active form of phytochrome (P_FR) and the effectof Ca²⁺ and calmodulin was examined with glutamate dehydrogenase(GDH) of Zea mays. A brief irradiation (5 min) to 5 day oldplants with red light resulted in 5-6 fold increase in GDH activity.This effect was nullified when red light was followed immediatelyby far-red light. The photoreversibility showed that P_FR regulatesGDH activity in vivo. To the enzyme extract obtained after EGTAtreatment, when Ca²⁺ was added in vitro, GDH was activated by6 fold. The maximum response by Ca²⁺ was obtained at 80 µM.Both P_AR and Ca²⁺ effects were found to be age dependent. Theenzyme activity was inhibited by compound 48/80 in partiallypurified extracts and the effect was reversed by calmodulin.The purified GDH, however, was not activated directly by calmodulin;it required the presence of another protein factor which wasseparated by gel permeation column by HPLC. Neither anticalmodulindrugs nor addition of calmodulin had any effect on nitrate re-ductaseactivity. (Received July 13, 1988; Accepted October 31, 1988)     

Intracellular Localization and Dichroic Orientation of Phytochrome in Plasma Membrane and/or Ectoplasm of a Centrifuged Protonema of Fern Adiantum capillus-veneris L.

Protonemata of the fern Adiantum capillus-veneris grown undercontinuous red light for 6 days were kept in darkness for 15h and subsequently centrifuged 3 times in different directions,so that oil droplets and other cytoplasm were removed from theapical region of the protonemata. Electron micrographs clearlydemonstrated that cell wall, plasma membrane, ectoplasm andmicrotubules remained in the apical and subapical regions afterthe centrifugal treatments. A brief local exposure of the flankof the subapical region of the centrifuged protonemata to amicrobeam of red light effectively induced a phototropic responsetoward the irradiated side, suggesting that phytochrome is locatedin the ectoplasm and/or plasma membrane. When the flank of thecentrifuged protonema was irradiated with linearly polarizedred or far-red light, red light with an electrical vector parallelto the cell surface was more effective than that perpendicularto the cell surface. The direction of the electrical vectorof far-red light for reversion of the preirradiated red lighteffect, however, was opposite. These results suggest that differentdichroic orientations of P_R and P_FR exist in the plasma membraneor ectoplasm. (Received May 26, 1983; Accepted September 1, 1983)