Structural modifications and δ-aminolevulinate dehydratase synthesis/transport in cotyledons of far-red irradiated radish

δ-Aminolevulinate dehydratase (ALAD) is synthesized in the cytoplasm and subsequently transported into the plastids in cotyledons of tar-red irradiated radish ( Raphanus sativus L. ev. Longue Rave Saumonée). The intra-cellular localization of ALAD activity was determined under far-red irradiation and darkness. After 60 h far-red, ALAD was essentially eytoplasmic; 96 h far-red corresponded to a peak in total ALAD activity, as well as the onset of massive ALAD transfer into the plastids, and after 120 h a maximum enzyme activity was located in the plastids. Under dark conditions, essentially no transport of ALAD was noted.
Structural modifications of plastid-eytoplasmic relationships were analysed at the three developmental periods under dark and far-red conditions. 60 h far-red induced a change in the "elasticity" of plastid envelopes leading to envelope proliferations and amoeboid shaped plastids. After 96 h tar-red, membrane proliferations formed a highly evaginated plastid periphery. It is suggested that these membranes form a transport network between the two compartments. Also at 96 h, numerous vesicles, which appear to originate from the endoplasmic reticulum (ER), surround the plastid profile. On the basis of the structural observations, it is proposed that ALAD would be synthesized within the ER and transported toward the plastids via ER extensions, which take the form of smooth vesicles. Our hypothesis is that the transition vesicles and proliferations of the outer envelope membrane undergo a fusion process emptying the content of the vesicle into the intermembrane space of the envelope.     

Capacity for chlorophyll synthesis in heat-bleached 70S ribosome-deficient rye leaves

The leaves of young rye plants (Secale cereale L.) grown at 32° were deficient in chlorophyll and in chloroplastic rRNA as compared to those grown at 22°, which developed normally. Both chlorophyll accumulation and the formation of plastidic rRNA were largely restored at 32° when the plants were transfered several times for 1 h per day to 22°. In the chlorotic 32°-grown rye leaves the in vivo activity of -aminolevulinate synthetase was very low. Aminolevulinate dehydratase however, exhibited high activity in extracts from 32°-grown leaves and was localized in the plastid fraction isolated from the chlorotic leaf tissue. After application of -aminolevulinic acid to chlorotic parts of leaves growing at 32°, protochlorophyll(ide) was formed and accumulated in the dark. In the light, the protochlorophyll(ide) was photooxidized at 32°. The results suggest a cytoplasmic site of synthesis for the series of enzymes converting -aminolevulinate to protochlorophyll(ide). It is concluded that an inhibition of -aminolevulinate synthetase and the photooxidation of protochlorophyll(ide) or chlorophyll are responsible for the chlorosis of the leaves at 32°.Abbreviations ALA -aminolevulinic acid - ALAD -aminolevulinate dehydratase - ALAS -aminolevulinate synthetase     

Density-labelling evidence for the phytochrome-mediated activation of phenylalanine ammonia-lyase in mustard cotyledons

When dark-grown mustard seedlings are irradiated with far-red light the level of phenylalanine ammonia-lyase (EC 4.3.1.5) activity increases. After ²H₂ O treatment phynlalanine amonia-lyase from seedlings irradiated with far-red light is density-labelled to a lesser extent than enzyme from dark-grown tissue. Theoretical arguments are advanced and data presented which show that this result cannot be explained in terms of an increase in de novo synthesis of phenylalanine ammonia-lyase and that the increase most likely involves activation of existing enzyme.     

eid1: A New Arabidopsis Mutant Hypersensitive in Phytochrome A–Dependent High-Irradiance Responses

To identify specific mutants for components of phytochrome A (phyA) signaling in Arabidopsis, we established a light program consisting of multiple treatments with alternating red and far-red light. In wild-type seedlings, irradiation with multiple red light pulses can reduce the amount of phyA, which in turn decreases the high-irradiance responses (HIRs) mediated by the subsequent treatments with far-red light. Our mutants were able to avoid this red light–dependent reduction of the HIR. Here, we describe eid1, a new recessive mutant with increased sensitivity to far-red light. The eid1 mutation maps to the top of chromosome 4. The mutants showed no change in phenotype in darkness or under continuous white light, but they exhibited an increased sensitivity to red light and an increased persistence of HIR during prolonged dark phases after multiple short pulses of far-red light. The eid1 seedlings accumulated normal amounts of phytochrome and showed no alterations in the degradation or de novo synthesis of phyA. The expression of the Eid1 phenotype requires the presence of phyA. Our data provide evidence that EID1 is a negatively acting component in the phyA-dependent HIR-signaling pathway.     

Influence de l'intensité et de la longueur d'onde de la lumière sur le rendement des radicaux libres dans les semences de tomate et de carotte

Influence of light intensity and wavelength on the yield of free radicals in seeds of tomato and carrot.
Seeds of tomato ( Lycopersicum esculentum L. cv. Gribovsky-1180) and carrot ( Daucus carota L. cv. Santanet) were irradiated by continuous light of different wavelengths (white, blue, green, red, far-red and He-Ne laser) and different power density. Free radical output was measured using Electron Spin Resonance spectra (ESR). The dependence of the content of free radicals (FR) on the light power density was established. The yield of FR in the seeds irradiated with monochromatic light of the same power density was inversely proportional to the wavelength. The ESR spectra showed maximum absolute number of FR for white light. Successive irradiations gave cumulative effects on the output of free radicals in both directions: increase by the action of white light and decrease by the action of red and far-red light. The contact of irradiated seeds with steam decreased the amount of FR.     

Le rôle du phytochrome dans la germination des graines de Nemophila insignis (L.)

Summary After a long exposure to far-red light (20 hrs, 715 mn) a short red irradiation does not induce germination again. After shorter exposure to far-red (10 hrs) the reversible red far-red reaction takes place. The germination in complete darkness is probably due to the presence of P_fr ¹ in dry seeds.The photoinhibition of the germination by far-red light depends on the level of the photostationary state (P₇₃₀/P_Total) and on the quantum flux density.

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Equipe de recherche associée au C.N.R.S. »Phytochrome et perméabilité membranaire«     

eid1: a new Arabidopsis mutant hypersensitive in phytochrome A-dependent high-irradiance responses

To identify specific mutants for components of phytochrome A (phyA) signaling in Arabidopsis, we established a light program consisting of multiple treatments with alternating red and far-red light. In wild-type seedlings, irradiation with multiple red light pulses can reduce the amount of phyA, which in turn decreases the high-irradiance responses (HIRs) mediated by the subsequent treatments with far-red light. Our mutants were able to avoid this red light-dependent reduction of the HIR. Here, we describe eid1, a new recessive mutant with increased sensitivity to far-red light. The eid1 mutation maps to the top of chromosome 4. The mutants showed no change in phenotype in darkness or under continuous white light, but they exhibited an increased sensitivity to red light and an increased persistence of HIR during prolonged dark phases after multiple short pulses of far-red light. The eid1 seedlings accumulated normal amounts of phytochrome and showed no alterations in the degradation or de novo synthesis of phyA. The expression of the Eid1 phenotype requires the presence of phyA. Our data provide evidence that EID1 is a negatively acting component in the phyA-dependent HIR-signaling pathway.     

δ-Aminolevulinate dehydratase from Raphanus sativus cotyledons

Phytochrome induces δ-aminolevulinate dehydratase (ALAD) activity in radish seedling cotyledons under continuous far red light. Analysis of the enzymatic activity in etioplasts vs total activity shows a constant ALAD level in these organelles (10 %) in etiolated seedlings. In far red irradiated seedlings, the percentage of enzyme detected into etioplasts increases up to 45 % of the total. Comparative kinetic studies of ALAD activity detected in the cytoplasm and the etioplasts indicate an increase in both compartments with a maximum value reached respectively at 96 and 120 hr from sowing. Treatment with cycloheximide shows a very fast abolition of cytoplasmic ALAD activity which is always correlated to an etioplast decrease with a time shift of ca 24 hr. Erythromycin acts only on the cytoplasmic level of ALAD, and only for far red irradiated seedlings, with an increase of activity twice the level detected in untreated ones. This unexpected effect is discussed.     

Phytochrome-induced synthesis of ribonuclease de novo in lupin hypocotyl sections

1. Density-labelling with 99 atoms% of (2)H(2)O distinguished pre-existing from newly synthesized ribonuclease molecules in sections of developing hypocotyl tissue. 2. Activity profiles of enzyme extracted from the fraction pelletable at 100000g showed heterogeneity after isopycnic centrifugation in CsCl gradients. 3. Measurement of density shifts of the entire heterogeneous band shows that ribonuclease protein is synthesized de novo in both continuous far-red light and darkness. 4. A twofold increase in enzyme activity after irradiation was accompanied by band-broadening and a significantly faster rate of labelling than in darkness. 5. The conclusion is drawn from the experimental evidence and theoretical arguments presented that phytochrome regulates the synthesis of new enzyme molecules against a background of continuous (dark-rate) synthesis and degradation. 6. Further information has been deposited as Supplementary Publication SUP 50033 (3 pages) at the British Library Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.     

Amino-acid biosynthesis in the cotyledons of Sinapis alba L. in darkness and far-red light studied by deuterium labelling and mass spectrometry

The incorporation of deuterium from deuterium oxide into the free amino acids of the cotyledons of Sinapis alba L. was studied by gas chromatography-mass spectrometry and was similar, both qualitatively and quantitatively, after incubation of the seedlings in darkness or far-red light. The results support studies which show that phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is synthesised de novo, rather than activated, in response to far-red light.Abbreviations GC-MS Gas chromatography-mass spectrometry - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - HFB n-propyl heptafluorobutyryl n-propyl     

Kinetic parameters of the inhibition of red blood cell aminolevulinic acid dehydratase by triethyl lead and its reversal by dithiothreitol and zinc

In chronic or acute exposure to triethyl lead, a de novo synthesis of aminolevulinic acid dehydratase (δ-ALAD) in bone marrow and an increased activity in circulating red blood cells can be demonstrated by activating the enzyme with dithiothreitol (DTT) and zinc. We determined the median inhibitory concentration and the apparent inhibition constant for triethyl lead on δ-ALAD. After dosing with triethyl lead, in vivo inhibition of ALAD only occurred at the high dose, but activation analysis in vitro showed increased ALAD activity to be present at all dose levels in a dose-dependent fashion. The use of an activation assay for red blood cell ALAD may have value as a bio-effects monitor of exposure to organic lead.     

Effects of 6-Benzylaminopurine and Potassium on {delta}-Aminolevulinate Dehydratase Activity of Excised Cucumber Cotyledons

Changes in -aminolevulinate dehydratase (ALAD) activity werefollowed in 7-day excised cotyledons maintained on water ortreated with 6-benzylaminopurine (BA 1 µg.ml^–1),KCl (10 mM) or a combination of BA + KCl. Cotyledons were incubatedfor 3 days in the dark or in the light, or preincubated for18 h in the dark before illumination. Cycloheximide treatments(10µg.ml^–1) were performed to differenciate betweenALAD biosynthesis or activation. The excision entailed an increase in ALAD activity. Then a decreaseappeared after a 24-h incubation on water in the dark. It wasmore precocious in the light, but was delayed when the cotyledonswere illuminated after a dark preincubation. BA had little effect on ALAD activity in the dark. In the light,BA completely prevented the decrease in ALAD activity observedin the control, and enhanced this activity when a dark preincubationpreceded illumination. KCl had a slight stimulating effect inthe dark, but was uneffective in the light. The combinationBA + KCl was always devoid of stimulating effect. Stimulatingeffects of BA or KCl seemed to be due to an increase in ALADbiosynthesis. No treatment markedly affected ALAD exportationfrom cytoplasm to plastids. These results are discussed in relation with cotyledon greening. (Received November 25, 1986; Accepted February 5, 1987)     

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Influence de la Concentration Bacterienne sur la Croissance de Colpidium campylum*

RÉSUMÉ. L'étude de la phase de latence et du début de la phase exponentielle de croissance dans des cultures monoxéniques de Colpidium campylum a été réalisée en examinant l'influence de la concentration en bactéries à la fois sur le nombre d'individus, leur volume moyen et le biovolume total de la population de Ciliés. Il est possible de définir plusieurs phénomènes liés à l'abondance du substrat, en particulier une concentration critique conditionnant la maintenance de la population et une diminution du taux d'assimilation consécutive à une suralimentation. A partir des résultants obtenus, la notion de volume minimum nécessaire à la division et l'interaction entre l'énergie nécessaire à la synthèse de matière vivante et celle liée à la nutrition, sont discutées afin d'expliquer l'évolution des cultures. SYNOPSIS. In a study of the stationary and the early exponential growth phases of the monoxenic cultures of Colpidium campylum, the influence of the bacterial concentration upon the number of ciliates, their mean volume, and the biomass of the total ciliate population was examined. It is possible to identify several phenomena linked to the abundance of the substrate, particularly a critical concentration affecting the maintenance of the ciliate population, and a decrease of the assimilation rate following overfeeding. On the basis of the results obtained, an estimate of the minimum volume necessary for division as well as the interaction between the energy required for the synthesis of living matter and that derived from nutrition are discussed, to explain the development of the cultures.     

Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves

The effects of red and far-red light on the enhancement of in vitro nitrate reductase activity and on nitrate accumulation in etiolated excised maize leaves were examined. Illumination for 5 min with red light followed by a 4-h dark period caused a marked increase in nitrate reductase activity, whereas a 5-min illumination with far-red light had no effect on the enzyme activity. The effect of red light was completely reversed by a subsequent illumination with the same period of far-red light. Continuous far-red light also enhanced nitrate reductase activity. Both photoreversibility by red and far-red light and the operation of high intensity reaction under continuous far-red light indicated that the induction of nitrate reductase was mediated by phytochrome. Though nitrate accumulation was slightly enhanced by red and continuous far-red light treatments by 17% and 26% respectively, this is unlikely to account for the entire increase of nitrate reductase activity. The far-red light treatments given in water, to leaves preincubated in nitrate, enhanced nitrate reductase activity considerably over the dark control. The presence of a lag phase and inhibition of increase in enzyme activity under continuous far-red light-by tungstate and inhibitors of RNA synthesis and protein synthesis-rules out the possibility of activation of nitrate reductase and suggests de novo synthesis of the enzyme affected by phytochrome.     

Regulation of fetal lung disaturated phosphatidylcholine synthesis by de novo palmitate supply

Lung surfactant disaturated phosphatidylcholine (PC) is highly dependent on the supply of palmitate as a source of fatty acid. The purpose of this study was to investigate the importance of de novo fatty acid synthesis in the regulation of disaturated PC production during late prenatal lung development. Choline incorporation into disaturated PC and the rate of de novo fatty acid synthesis was determined by the relative incorporation of [14C]choline and 3H2O, respectively, in 20-day-old fetal rat lung explants and in 18-day-old explants which were cultured 2 days. Addition of exogenous palmitate (0.15 mM) increased (26%) choline incorporation into disaturated PC but did not inhibit de novo fatty acid synthesis, as classically seen in other lipogenic tissue. Even in the presence of exogenous palmitate, de novo synthesis accounted for 87% of the acyl groups for disaturated PC. Inhibition of fatty acid synthesis by agaric acid or levo-hydroxycitrate decreased the rate of choline incorporation into disaturated PC. When explants were subjected to both exogenous palmitate and 60% inhibition of de novo synthesis, disaturated PC synthesis was below control values and 75% of disaturated PC acyl moieties were still provided by de novo synthesis. These data show that surfactant disaturated PC synthesis is highly dependent on the supply of palmitate from de novo fatty acid synthesis.