Changes in the Content of Phytochrome I and II Apoproteins in Embryonic Axes of Pea Seeds during Imbibition

The contents of phytochrome I and II in crude extracts fromembryonic axes of Pisum sativum cv. Alaska seeds were immunochemicallydetermined using purified pea phytochrome I and II as standards.We have produced and used three different types of mouse monoclonalanti-pea phytochrome antibodies (mAP) such as one reacting preferentiallywith phytochrome I, one with phytochrome II, and one with bothI and II. Phytochrome II was separated from I in the samplesusing immobilized column chromatography with mAPl. The amountsof two phytochrome species were quantitatively measured withwestern blotting and ELISA. Ca. 0.2 µg /axis of phytochromeI and ca. 0.05 µg /axis of phytochrome II were detectedby ELISA after imbibition for 12 h in the dark, though smallamounts of both were detected in dry axes. Ca. 0.05 µg/axis each of phytochrome I and II were detected by ELISA afterimbibition for 12 h in the light, and the results were confirmedby western blotting. This study showed that phytochrome II isnot green-tissue-specific, being also found in dark-imbibedembryonic axes, and that although light significantly lowersthe content of phytochrome I in the axis, it does not significantlyaffect that of phytochrome II. (Received June 10, 1987; Accepted August 27, 1987)     

Volume changes in activated human neutrophils: The role of Na+/H+ exchange

The apparent volume of neutrophils, as measured electronically with the Coulter counter, has been reported to increase upon treatment with chemotactic factors. The occurrence of a volume change was confirmed by forward angle light scattering and by isotopic measurements of intracellular water space in cells treated with 12-O-tetradecanoylphorbol 13, acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP). Cell swelling was associated with an increase in the osmotic content of the cells, determined from Boyle-van't Hoff plots, and with an increase in Na⁺ content, measured by flame photometry. The volume change was inhibited by replacement of extracellular Na⁺ with K⁺ or N-methyl-D-glucamine⁺, or by addition of amiloride. Swelling was also inhibited by the 5-N-substituted analogs of amiloride, which are potent specific inhibitors of the Na⁺/H⁺ antiport. This pathway is activated in neutrophils by both TPA and FMLP. Activation of Na⁺/H⁺ exchange, determined as a Na⁺-dependent and amiloride-sensitive cytoplasmic alkalinization, was also found when neutrophils were treated with hypertonic solutions. The hypertonic activation of the antiport was similarly followed by cell swelling, detectable by electronic sizing. The results indicate that activation of Na⁺/H⁺ exchange can lead to significant cell swelling in neutrophils.     

Effect of pH on Absorption Spectra of Pea 114 and 121 Kilodalton Phytochromes during and after Red-Light Irradiation

Absorption spectra of pea 114 and 121 kDa phytochromes weremeasured at pH 6.8, 7.8 and 8.8 using a custom-made transientmultichannel spectrum analyzer. The absorption spectra of 114kDa phytochrome as P_R and P_FR were least affected by mediumpH. The absorption spectra at photostationary state under redlight, however, were different under the three different pHconditions, and were different from those obtained 55 s afterred-light irradiation, owing to rapid pH-dependent absorbanceincrease in both red and far-red regions in the dark. In contrast,the absorption spectra of 121 kDa phytochrome were significantlyless affected by medium pH. The absorption spectra measuredat the photostationary state showed a lower P_FR peak at higherpH. The absorption spectra obtained 55 s after the irradiationwere similar under the three pH conditions since the rapid absorbanceincrease in the far-red region in the dark was small. Possibleaccumulation of 114 kDa phytochrome population(s) with low absorbanceat red-light-induced photostationary state at pH 8.8, and theprotective role of the 7 kDa polypeptide at the amino terminusagainst the pH effect in 121 kDa phytochrome are discussed. (Received February 1, 1986; Accepted April 1, 1986)     

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 DNA synthesis on induction of preneoplastic and neoplastic lesions in rat liver by a single dose of methylazoxymethanol acetate

A single intravenous injection of methylazoxymethanol (MAM) acetate in doses of either 20 or 35 mg/kg body weight to male Sprague-Dawley rats induced altered liver cell foci and later, liver neoplasms in a dose related manner. Sequential observations in the rats given 35 mg/kg and thereafter fed an iron-loading diet revealed that the number of iron-excluding foci/cm2 increased with time. Partial hepatectomy (PH) before the high dose of MAM acetate resulted in 100% lethality while hepatectomy before the low dose carcinogen exposure lead to a higher incidence of neoplasms than in rats that received carcinogen alone. PH after either high or low dose carcinogen exposure did not result in a greater occurrence of liver neoplasms.     

Studies on the Photoreceptors for the Promotion and Inhibition of Flowering in Dark-Grown Seedlings of Pharbitis nil Choisy

Three-day-old etiolated seedlings of Pharbitis nil were exposedto red light for 10 min and sprayed with N⁶-benzyladenine beforetransfer to a 48-h inductive dark period, after which they weregrown under continuous white light. A second red irradiationpromoted flowering when given at the 5 and 24th hour of theinductive dark period but inhibited flowering at the 10 and15th hour. Far-red light inhibited flowering when given at anytime during the first 24 h of the dark period. Red/far-red reversibilitywas clearly observed at the 0, 5, 10 and 24th hour, but notat the 15th hour when both red and far-red lights completelyinhibited flowering. The action spectrum for the inhibition of flowering at the 15thhour of the inductive dark period had a sharply defined peakat 660 nm and closely resembled the absorption spectrum of theP_R form of phytochrome. The photoreceptors involved in thesephotoreactions are discussed. (Received June 10, 1983; Accepted July 6, 1983)     

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 structure of the phytoplankton community in the subsurface chlorophyll maxima in the western North Pacific Ocean

The phytoplankton taxonomic composition of the subsurface chlorophyllmaximum layer (SCM) was compared with that of the surface layerin July, September and November 1979 in the western North PacificOcean. Besides the use of chemical fixatives for preservablephytoplankton, serial dilution culture method was employed fornon-preservable flagellates and monads, i.e., fragile forms.The SCM was characterized by a high species diversity of preservablephytoplankton and by the numerical dominance of fragile forms.Among the fragile forms, Micromonas and Ochromonas were dominant.This dominance is consistent with their viability at low lightintensity. Cluster analysis revealed that, for blue-green algae,coccolithophorids, silicoflagellates and diatoms, a marked differenceexisted in species assemblages between the SCM and the surfacebut there was no distinct difference in dinoflagellate assemblages.The difference between both layers is discussed, as relatedto the mechanisms of formation of the SCM. ¹Present address: Nodai Research Institute, Tokyo Universityof Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156,Japan.     

Apical Growth of Protonemata in Adiantum capillus-veneris IV. Phytochrome-Mediated Induction in Non-Growing Cells

In non-growing two-celled protonemata of Adiantum capillus-veneris,apical growth was induced most effectively by red light irradiation;half of the samples were induced to grow by 660 nm light ofca. 1.5 J m^–2 and the maximum number by ca. 70 J m^–2.The reciprocity law was valid in this photoinduction. The growthresumption became detectable 6 hr after the light irradiationand reached a plateau within 24 hr irrespective of given fluences.When non-growing samples were irradiated with red light of 4.6W m^–2 for 4 sec or shorter, the effect was fully reversedby a subsequent irradiation with far-red light to the far-redlight control level. But, when the red light was given for 16sec or longer, photoreversibility became partial. An interveningdark period of 2 min between red and far-red light did not significantlyinfluence the photoreversibility so that the escape reactionin the dark may not be attributed to the above-mentioned lossof photoreversibility. By means of a local irradiation with a narrow red light beam(10 µm in width), the apical cell was found to be photosensitivefor the growth induction, but basal cell was not. Photoreceptivesite was not localized in any particular region of the apicalcell, but was rather dispersed in the entire apical cell. (Received January 26, 1981; Accepted March 10, 1981)     

Phytochrome action in Oryza sativa L.

Summary In-vivo phytochrome determinations in totally etiolated rice seedlings with a dual-wavelength spectrophotometer showed that on a fresh weight basis phytochrome concentration was highest in the coleoptile apex (0.175 of mean) ( O.D.) g^-1 (fresh weight). The age of the seedlings had little effect on the pattern of phytochrome distribution in the coleoptiles.The extent of growth inhibition observed 2 days after the irradiations was proportional to the logarithm of P _fr amount in the coleoptiles at the time of initial exposure to either red or blue light. Ultraviolet irradiation, however, did not induce either reversible growth inhibition or optically detectable phytochrome changes in vivo.After the conversion of P _r to P _fr bya brief red irradiation, non-photochemical transformation of phytochrome was observed in intact coleoptile tissues. Most of the optically measurable P _fr disappeared within 6 hours at 27°, when the total ( O.D.) decreased to about one fifth of the original level. The optical data did not agree with the fact that 50% of the initial physiological reversibility was still observed 9 hours later. No significant difference in dark transformation rate was seen between intact and excised coleoptile tissues.Abbreviations P _r red light absorbing form of phytochrome - P _fr far-red light absorbing form of phytochrome - ( O.D.) the change in the optical density difference reading at two wavelengths, following irradiation of the sample with actinic sources of red and far-red light - UV ultraviolet light