Regulation of Protochlorophyll(ide) Levels in Dark-Grown Non-Dividing Euglena 3. Proplastid Structure during Loss and Regeneration of Protochlorophyll(ide)

Cells of Euglena gracilis Klebs var. bacillaris Cori growingin darkness on a complete medium have small undifferentiatedproplastids. On transfer to an incomplete (resting) medium indarkness, the cells cease division within 72 h. During thistime the proplastid expands and several prothylakoids and prolamellarbodies develop even though phototransformable protochlorophyll(ide)[PT-Pchl(ide)] is decreasing. As PT-Pchl(ide) decreases furtherand reaches a stable plateau after 4–5 more days in darkness,the proplastid structure becomes highly reduced. Forty minutesof light plus a one h dark period, or addition of glutamateor malate for 7 h does not change the proplastid structure significantlyeven though PT-Pchl(ide) returns to the level found in growingcells. Upon prolonged incubation in darkness after light treatment(72 h) an expanded proplastid containing prothylakoids, prolamellarbodies and membrane whorls with mitochondria in close associationis seen; most of the cellular paramylum is lost during thisperiod leaving cavities in the cytoplasm. Without light, prolongedincubation in darkness (72 h) with malate leads to accumulationof cellular paramylum but no change in proplastid structurewhile prolonged treatment with glutamate (72 h) allows the formationof a few prothylakoids but no prolamellar bodies. ¹Supported by Grants GM 14595 from the National Institutes ofHealth. ²Permanent address: Department of Microbiology, Tokyo MedicalCollege, 6-1-1 Shinjuku, Tokyo 160, Japan. ³Abraham and Etta Goodman Professor of Biology. (Received July 23, 1983; Accepted September 22, 1983)     

Relation between Paramylum Content and the Length of the Lag Period of Chlorophyll Synthesis during Greening of Dark-grown Euglena gracilis

Euglena cells, strains Z and bacillaris, were grown in the dark under various nutritional deficiencies. After 3 days of nondivision, cells were transferred to the light, and the following parameters were measured: the paramylum content at the time of illumination (zero time), the rate of paramylum consumption during the first 10 hours of greening, and the length of the lag phase of chlorophyll synthesis. Similar results were obtained with both strains and can be summarized as follows. (a) The use of various nutritional deficiencies allows the control, to a certain extent, of the amount of paramylum present at zero time. (b) The rate of paramylum consumption is proportional to the cellular paramylum content for values in excess of 50 picograms/cell. (c) The length of the lag phase increases rapidly when the cellular content of paramylum decreases below 50 picograms. This period can be greatly diminished by the addition of an exogenous organíc carbon source. (d) The amount of paramylum (rate of paramylum consumption × length of lag phase) consumed during the lag phase is around 5 to 10 picograms/cell for cells which contain less than 50 picograms of paramylum/cell. It increases when the cellular paramylum content increases, this increment being more rapid for bacillaris than for Z cells.     

Events surrounding the early development of euglena chloroplasts: v. Control of paramylum degradation

The degradation of the storage carbohydrate, paramylum, is induced by light in wild-type Euglena gracilis Klebs var. bacillaris Pringsheim and in a mutant, W₃BUL, which lacks detectable plastid DNA. Treatment of wild type with cycloheximide in the dark produces 60% as much paramylum breakdown as light, whereas treatment with levulinic acid in the dark yields a slightly greater response than light. Both cycloheximide and levulinic acid produce a greater paramylum breakdown in the light than they do in the dark. Treatment of W₃BUL with levulinic acid in darkness produces a larger paramylum degradation than light, with values similar to wild type in the light. Treatment of W₃BUL with cycloheximide induces paramylum degradation in darkness, and as with wild type, light is slightly stimulatory in the presence of both cycloheximide or levulinic acid. Streptomycin brings about only a very small amount of paramylum breakdown in the dark and only slightly inhibits breakdown in the light. Thus paramylum breakdown induced by light does not require the synthesis of proteins on cytoplasmic or plastid ribosomes. A model which explains these results postulates the existence of a protein which inhibits paramylum breakdown. When the synthesis of this protein is prevented either by light, cycloheximide, or by levulinic acid acting as a regulatory analog of delta amino levulinic acid, paramylum breakdown takes place. Because levulinic acid is a better inducer than light in W₃BUL, W₃BUL may not be able to form as much delta amino levulinic acid in light as wild type. The small amount of induction by streptomycin is viewed as a secondary regulatory effect attributable to interference with plastid protein synthesis which affects regulatory signals from the plastid to the rest of the cell.     

Changes in Stoichiometry among PSI, PSII and Cyt b6-f Complexes in Response to Chromatic Light for Cell Growth Observed with the Red Alga Porphyra yezoensis

Stoichiometry among 3 thylakoid components, PSI and PSII andCyt b6-f complexes, was determined with the red alga Porphyrayezoensis with special reference to the regulation of PSI/PSIIstoichiometry in response to light regime. The ratio of PSIto PSII abundance was four times greater in thalli grown underorange light which excites mainly phycobilisome, thus PSII,than that under red light which excites preferentially Chl a,thus PSI. Cyt b₆-f abundance remained almost constant. The PSIand PSII content was regulated separately under the two growthlight conditions as was also observed with the red alga Porphyridiumcruentum by Cunningham et al. [(1990) Plant Physiol. 93: 888].This differs from the cyanophyte Synechocystis PCC 6714 whereadjustment occurs only in the PSI content [(1987) Plant CellPhysiol. 28: 1547]. However, results on the marine cyanophyteSynechococcus NIBB 1071 indicate that changes in the PSI/PSIIsoichiometry is similar to red algae. In this species, as inthe red algae, more than one PSII is associated with each phycobilisome.The light regime also induced changes in the phycobiliproteincomposition in Porphyra yezoensis. Under PSII light, phycoerythrinincreased, and phycocyanin decreased, while under PSI lightthe response was reversed. The change suggests an occurrenceof complementary chromatic adaptation. (Received April 8, 1994; Accepted June 1, 1994)     

Carbonic Anhydrase of a Unicellular Red Alga Porphyridium cruentum R-l. I. Purification and Properties of the Enzyme

Cells of Porphyridium cruentum R-l, a unicellular red alga,grown under ordinary air (0.04% CO₂) showed much higher activityof carbonic anhydrase (CA) than those grown under CCvenrichedair (2% CO₂). CA activity was not detected in a suspension ofintact cells, and was detectable only after the cells had beenhomogenized, indicating that this enzyme was localized onlywithin the algal cells. After partial purification of Porphyridium CA, its mol wt wasestimated as 59 kDa by SDS-PAGE and 55 kDa by gelfiltration.This suggests that the native enzyme is a monomer. Its activitywas not affected by benzensulfonamides, potent inhibitors ofCAs isolated from Chlamydomonas and other organisms. Chloride(or bromide) ions was essential for CA activity. CA activitymarkedly decreased when the cell extract had been incubatedat pH lower than 7 before assay. Upon readjusting the pH ofthe preincubation medium to 9 or higher, the enzyme activitywas restored, indicating that the inactivation is reversible. (Received April 17, 1987; Accepted July 21, 1987)     

Old-Culture Flowering of Lemna paucicostata 6746 in Aged Hutner's Medium

Lemna paucicostata 6746, a short-day plant, flowers in agedHutner's medium even under continuous light, when the endogenousnitrogen level decreases to below 1.6 µmg fr wt. At thesenitrogen levels, daylength-independent flowering of the plantcan be induced even in fresh Hutner's medium. Thus, old-cultureflowering in Hutner's medium is due to nitrogen deficiency inthe plants. ¹Present address: Biological Institute, Faculty of Science,Shizuoka University, Shizuoka 422, Japan. (Received February 12, 1987; Accepted August 28, 1987)     

[14C]-Glucose Metabolism during Shoot Bud Development in Cultured Cotyledon Explants of Pinus radiata

Excised cotyledons of Pinus radiata D. Don cultured under shoot-forming(plus benzyladenine) and non shoot-forming (minus benzyladenine)conditions for 10 and 21 days were fed U-[¹⁴C]-glucose for 3h in the light followed by a 3 h chase period. The labellingof individual metabolites as well as ¹⁴C incorporation intoprotein was assessed. It was found that the general metabolicpatterns were qualitatively the same in shoot-forming and nonshoot-forming conditions, however, metabolism leading to respirationas well as to the synthesis of some amino acids and proteinsynthesis was enhanced in the shoot-forming cultures. (Received February 16, 1987; Accepted July 8, 1987)     

Environmental Factors Controlling Capitulum Opening and Closing of Dandelion, Taraxacum albidum

The capitula of Taraxacum albidum kept in darkness opened whenthe temperature rose. The higher the temperature before thechange and greater the temperature rise, the larger the openingresponse was. The opening was promoted by light. The capitulakept in darkness at 20?1?C opened after exposure to light withoutthe temperature rise. The capitula closed 8–10 h afterthe beginning of the opening under constant light and temperatureconditions. (Received November 8, 1986; Accepted March 13, 1987)     

Chlorophyll-Protein Complexes and other Thylakoid Components at the Low Intensity Threshold in Euglena Chloroplast Development

The plastids of dark-grown resting cells of Euglena gracilisKlebs var. bacillaris Cori undergo only limited developmentwhen illuminated at the developmental threshold for light intensity7 foot-candles (ft-c) (27 µW/cm²). In the present work,we have found that these low intensity cells have substantialamounts of electron transport components such as ferredoxin-NADPreductase and Cyt c-552 but only trace amounts of the majorantenna components such as the light-harvesting Chl-proteincomplex (LHCP), the LHCP oligomer, CP la, Chi b and the 26.5kDa apo-LHCP; CP I and CPa are at levels comparable to the electrontransport components. Exposure of the low intensity cells tonormal light intensity causes large increases in major antennacomponents and small increases in electron transport components.The kinetics of accumulation of the antenna components Chi band apo-LHCP during greening of dark-grown resting cells atnormal intensities are the same as for Chi a. The low intensitywild-type cells strongly resemble mutants of Euglena low inChi b grown at normal intensities in lacking major antenna components. (Received April 7, 1987; Accepted June 19, 1987)     

Modulation of Sucrose Content by Fructose 2,6-bisphosphate during Photosynthesis in Rice Leaves Growing at Different Light Intensities

Reddy, A. R. and Das, V. S. R. 1987. Modulation of sucrose contentby fructose 2,6-bisphosphate during photosynthesis in rice leavesgrowing at different light intensities.—J. exp. Bot. 38:828–833. The relationship between the rate of CO₂ fixation and sucroseconcentration in the leaves of rice (Oryza sativa L.) grownat different light intensities was investigated. Maximum sucrosecontent coincided with maximum rates of CO₂ fixation, achievedat a photon flux density of 1600 µmol m^–2 s^–1.The levels of sucrose and fructose 2,6-bisphosphate were alsocompared in the leaves under different light intensities. Fructose2,6-Msphosphate accumulated during growth at low light. Theactivity of fructose-6-phosphate 2-kinase was high in the leavesgrown at low light while that of fructose-2,6-bisphosphatasewas low. The activities of phosphoglucose isomerase and phospho-glucomutasewere slightly increased by growth at low light The activitiesof UDP glucose pyrophosphorylase were adversely affected invitro with increased concentrations of fructose 2,6-bisphosphatewhile those of sucrose phosphate synthase were moderately affected.Phosphoglucose isomerase and phosphoglucomutase were activatedby fructose 2,6-bisphosphate (8-0 mmol m^–3) by 12-15%.The results suggested that low light intensities during growthresult in an accumulation of fructose 2,6-bisphosphate whichmodulates the key enzymes of sucrose biosynthesis thus regulatingcarbon flow under conditions of limited photosynthesis. Key words: Oryza sativa, photosynthesis, sucrose synthesis, fructose 2,6-bisphosphate, light     

Environmental relevance of heavy metal-substituted chlorophylls using the example of water plants

Following experiments which studied the substitution of thecentral ion of isolated chlorophylls by heavy metal ions invitro, in vivo experiments with submersed water plants werecarried out. It was discovered that the substitution of thecentral atom of chlorophyll, magnesium, by heavy metals (mercury,copper, cadmium, nickel, zinc, lead) in vivo is an importantdamage mechanism in stressed plants. This substitution preventsphotosynthetic light-harvesting in the affected chlorophyllmolecules, resulting in a breakdown of photosynthesis. The reactionvaries with light intensity. In low light irradiance all thecentral atoms of the chlorophylls are accessible to heavy metals,with heavy metal chlorophylls being formed, some of which aremuch more stable towards irradiance than Mg-chlorophyll. Consequently,plants remain green even when they are dead. In high light,however, almost all chlorophyll decays, showing that under suchconditions most of the chlorophylls are inaccessible to heavymetal ions. Key words: Heavy metal chlorophylls, submersed water plants, antenna pigments, copper, zinc     

Photoregulation of Phytochrome Synthesis in Germinating Embryos of Avena sativa L.

Hilton, J. R. and Thomas, B. 1987. Photoregulation of phytochromesynthesis in germinating embryos of Avena sativa L.—J.exp. Bot. 38: 1704–1712. The effect of light on the accumulation of phytochrome in germinatingAvena embryos was determined. A quantitative ELISA using monoclonalantibody AFRC MAC 56 was used to measure specifically type 1(or dark) phytochrome. A pulse of red light given after 14 himbibition but prior to the onset of type 1 phytochrome synthesis,strongly inhibited subsequent type 1 phytochrome accumulation.This effect of red light at 14 h was reversible by far-red lightindicating the involvement of phytochrome. Red light also inhibitedphytochrome synthesis after 18 h and 24 h imbibition but after24 h, far-red light did not reverse the effect. The effect ofred light treatment at 18 h was reversed by giving a pulse offar-red light at any time up to 30 h. Seed germination was notinfluenced by light under the conditions of these experiments.It is proposed that type 2 (or light) phytochrome may be responsiblefor photoregulation of type 1 phytochrome synthesis in germinatingAvena embryos. Key words: Photoregulation, phytochrome, seed.     

Rapid Phytochrome-Controlled Protein Phosphorylation and Dephosphorylation in Avena sativa L.

The time course for in vivo changes in the protein phosphorylationpattern was measured after red and red/far-red light. Avenacoleoptile tips were incubated in ³²P-labeled phosphate andirradiated. The supernatant fractions of homogenates were subjectedto SDS-poly-acrylamide gel electrophoresis and then autoradiographed.Within seconds, the radioactive label of two proteins decreasedand the radioactive label of one protein increased. These datasuggest that the phosphorylation states for these proteins maybe under phytochrome control. (Received July 20, 1987; Accepted July 20, 1988)     

Photoinhibition of Glucose Uptake in Chlorella

In colorless mutant cells of Chlorella vulgaris (M125), endogenousrespiration in the dark was not affected by 30-min preilluminationwith white light (9,000 mW?m^–2), while exogenous respirationof glucose or fructose was inhibited significantly by the sametreatment in air, but not under N₂. This light effect on exogenousrespiration was accompanied by an inhibition of hexose uptake. When autotrophically grown wild-type cells of Chlorella vulgaris(211-11h) were incubated in glucose medium with DCMU, lightalso greatly inhibited glucose uptake and growth. Blue lightwas very effective, while red light had only a slight effect.This photoinhibitory effect was also observed in algal cellsthat had been grown in a glucose-containing medium in the dark. Using SDS-gel electrophoresis, a new protein peak with a molecularweight of 35–40 kDa was detected in plasma membrane-richcell wall fractions when Chlorella vulgaris (211-11h) cellswere transferred to a glucose-containing medium. This peak disappearedafter the algal cells were returned to the glucose-free medium.These findings suggest that this protein includes the hexose-carrierprotein. Blue light significantly inhibited the formation ofthis protein during incubation in a glucose-containing medium. ¹ Present address: Laboratory of Chemistry, Faculty of PharmaceuticalSciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan. (Received July 31, 1986; Accepted March 12, 1987)     

Blue Light-Induced In Vivo Absorbance Changes and In Vitro Activation of Nitrate Reductase in a Nitrate-Starved Chlorella Mutant

Illuminating a colorless mutant of Chlorella vulgaris 11h (M125)with blue light caused a reversible photoreduction of b-typecytochrome, i.e., absorbance increases at 423, 525 and 557 nm.This light-induced reduction of cytochrome b was most pronouncedin nitrate-starved cells, which showed some blue light responsesin carbon metabolism, including enhancement of respiration byblue light as reported previously. Prolonged illumination withblue light caused a decrease in the rate of the reduction. The photoactivation of nitrate reductase in the mutant cellswas studied in both cell-free crude extract and purified enzyme.The absorption spectrum of purified enzyme showed three peaksat 423, 525 and 557 nm after the addition of a reductant, indicatingthat the spectrum is that of cytochrome b associated with nitratereductase. Nitrate reductase activity was easily enhanced byblue light illumination after 1 min; red light had no effecton it. The blue light activation of nitrate reductase was notsignificant in growing cells, which showed its high activity. The relationship between the blue light-induced reduction ofcytochrome b and carbon metabolism is discussed. (Received September 30, 1987; Accepted February 9, 1988)     

Free Cellular Riboflavin is Involved in Phase Shifting by Light of the Circadian Clock in Neurospora crassa

Phase shifting by light of the circadian conidiation rhythmof the Neurospora crassa strain band, of the riboflavin-deficientdouble mutant band rib2 and of the temperature-sensitive doublemutant band ribl was measured. Fluence response curves of theband strain exhibited two distinct steps, whereas those of bandribl and band rib2 revealed only one step. Maximum phase advancesobserved were 5.5 h in band and 10.4 h in the band rib strains.Sensitivity of band rib2 to light was proportional to the riboflavinconcentration in the growth medium over a 100 fold range. Extracellularflavin in the medium did not sensitize the strains. Riboflavinapplied after exposure to light showed no effect. Light sensitivitycorrelated with the level of cellular riboflavin. Four analogsof riboflavin, none of which can be phosphorylated, increasedthe sensitivity of Neurospora to light. Even at high riboflavinconcentrations in the medium, the sensitivity of the band rib2strain to light was not saturated. In addition, four riboflavinderivatives with bulky substituents at positions 3, 8 or 10of the isoalloxazine nucleus sensitized both strains. From ourdata, we conclude, that a) a cellular flavin controls the sensitivityof Neurospora crassa to light; b) that this flavin compoundis riboflavin; and c) that the active riboflavin is not proteinbound. ⁴Present address: Teikoku Women's University, Department ofHome Economics, 6-173 Touda-cho, Moriguchi-shi, Osaka, 570 Japan. (Received November 27, 1987; Accepted March 15, 1989)     

The Accumulation of Protochlorophyllide in Cells of Synechocystis PCC 6714 with a Low PSI/PSII Stoichiometry

Changes in intracellular levels of Chl a precursors were examinedin relation to changes in the PSI/PSII stoichiometry in thecyanophyte Synechocystis PCC 6714. Protochlorophyllide (Pchlide)accumulated markedly in cells with a low PSI/PSII stoichiometrygrown under light that is absorbed by Chl a (PSI light) whereasno accumulation occurred in cells with a high PSI/PSII stoichiometrygrown under light absorbed by phycobilisomes (PSII light). Levelsof Pchlide in cells grown under PSI light decreased rapidlyupon a shift to PSII light. The rapid decrease in Pchlide accompanieda transient increase in chlorophyllide a, indicating that reductionof Pchlide was enhanced by shift to PSII light. The action spectrumindicated that the Pchlide decrease upon the shift to PSII lightdepended on excitation of Pchlide, suggesting that the accumulationof Pchllide was due to limited excitation of Pchlide, so thatPchlide photoreduction, under PSI light. However, comparisonof levels of Pchlide and the photosystem complexes in wild-typePlectonema boryanum with those in a mutant that lacked the darkPchlide reductase (YFC 1004) indicated that dark reduction compensatedfor the limited photoreduction under PSI light. Similar compensationby dark reduction was confirmed with Synechocystis PCC 6714.In cultures of Synechocystis under conditions where Pchlidecould not be photoreduced, accumulation of Pchlide and low PSI/PSIIstoichiometry occurred only when cells were illuminated withlight that preferentially excited PSI. The results indicatethat the low PSI/PSII stoichiometry in cells grown under PSIlight is not a result of inefficient synthesis of Chl a witha reduced rate of Pchlide photoreduction. They suggest furtherthat accumulation of Pchlide under PSI light results from retardationof the Chl a synthesis due to suppression of PSI synthesis. ¹Present address: Tsurukawa 5-15-11, Machida, Tokyo, 195 Japan.     

CHANGES IN RESTING POTENTIAL AND ION ABSORPTION INDUCED BY LIGHT IN A SINGLE PLANT CELL

1. Effect of light on ion absorption and resting potential of theinternodal cell of Nitella flexilis was investigated under variousconditions.
2. On illumination, the resting potential increasedby about 30mVin 10^–4 M KCl and by about 60 mV in 10^–4M NaClsolution. A similar photoelectric response was also observedin 10^–3 M KCl, 10^–2 M CaCl₂ and 5 x 10^–2 MCaCl₂ solutions, but not at all in 10^–2 M KCl solution.
3. Absorption of ions by the cell took place in parallel withthelight-induced change in resting potential.
4. Red and bluelights were very effective in increasing the restingpotential,while green light was almost ineffective. These differenteffectsof color lights were in good agreement with their effectsinincreasing the osmotic value of the cell.
5. The photoelectricresponse was not affected by phenylurethane,which, on the otherhand, strongly inhibited the light-inducedion absorption.
6. Theuptake of ions by the cell from the external medium intothevacuole is assumed to proceed in two different steps: thefirstis the process involving the ion movements across theoutermostplasmalemma, and the second is that involved in thetransportof ions through the cytoplasmic layer and tonoplast.The formerprocess is considered to be influenced by the increasein restingpotential probably caused by the light absorbed bychlorophyll.The process was, however, suggested to be independentof photosynthesis.On the other hand, the latter process issupposed to be relatedto photosynthesis. A discussion was madealong this line.

(Received July 26, 1962; )     

Carbonic Anhydrase of a Unicellular Red Alga Porphyridium cruentum R-l. II. Distribution and Role in Photosynthesis

Antibody was raised against Porphyridium carbonic anhydrase(CA) which was electrophoretically recovered from the gel afterSDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) of thepartially purified enzyme. The antiserum reacted with CA ofPorphyridium, but not with that of Chlamydomonas reinhardtii.Even though the antiserum did not react with CA from P. cruentumR-l in Ouchterlony's double immunodiffusion, it blocked theenzyme activity in the presence of 1% Nonidet P-40 and 1% TritonX-100. After Western blotting and enzyme-linked immunostaining(ELIS), only one band which reacted with the antiserum was detectedin the extract of low-CO₂ cells (grown under ordinary air) ofP cruentum, while no significant band was detected in that ofhigh-CO₂ cells (grown under air enriched with 1–5% CO₂).Immunogold electron microscopy of low-CO₂ cells of P. cruentumR-l using this antibody revealed that most of the CA was localizedin the chloroplast, with some in the cytoplasm. No specificbinding of gold particles was observed in the high-CO₂ cells. ¹Present address: National Institute for Basic Biology, Myodaiji,Okazaki 444, Japan (Received May 18, 1987; Accepted September 7, 1987)     

Mercury-Induced Inhibition of Photosystem II Activity and Changes in the Emission of Fluorescence from Phycobilisomes in Intact Cells of the Cyanobacterium, Spirulina platensis

Addition of low concentrations of mercury chloride (HgCl₂ tointact cells of the cyanobacterium, Spirulina platensis causedan enhancement in the intensity of fluorescence emitted fromphycocyanin at room temperature and induced blue shifts in theemission peak suggestive of changes in energy transfer withinthe phycobilisomes. HgCl₂ also suppressed the whole-chain electrontransport activity (H₂O methylviologen) at much lower concentrationsthan that required to inhibit Hill activity supported by para-benzoquinone.The extent of inhibition of Hill activity was much higher underhigh-intensity light than that under low-intensity light. Ourresults indicate that mercury ions at low concentrations affectthe transfer of energy within phycobilisomes and at high concentrationsthey inhibit electron transport in this cyanobacterium. (Received February 21, 1989; Accepted October 2, 1989)