Glycollate Formation during the Photorespiration of Acetate by Chlorella

WhenChlorella pyrenoidosa photoassimilates ³H-¹⁴C-acetate theglycollic acid formed shows a high ³H/¹⁴C ratio, the only othercompounds showing similar ratios being glycerate and serine.The ³H/¹⁴C ratio of glycollate was unaffected by the TCA cycleinhibitors MFA, diethylmalonate and arsenite showing that ³Hin glycollate does not result from the oxidation of acetatevia the TCA cycle, the resulting NADP³H₂ or NAD³H₂ being usedfor the reduction of the glycollate precursor. Although DCMUdecreased the ³H/¹⁴C ratio, complete inhibition of glycollatelabelling was not observed with 10^–6 M DCMU, at whichconcentration complete inhibition of the Hill reaction is achieved.Although the ³H/¹⁴C ratio was unaltered, total dpm of both ¹⁴Cand ³H in glycollate were increased by INH. The ³H/¹⁴C ratiosof glycerate and serine were decreased by INH, as were the totaldpm of ³H and ¹⁴C incorporated into these compounds. Thus, INHinhibits the further metabolism of glycollate to glycerate andserine. The effect of INH on incorporation of ¹⁴C-I-acetateinto various cell fractions was investigated. The incorporationof ¹⁴C into polysaccharide and lipid was decreased, while theincorporation of ¹⁴C into the water-soluble fraction of cellsand therelease of ¹⁴CO₂ were little affected. Although glycollicacid was an early product of acetate photoassimilation in Chlorellapyrenoidosa, glycollate excretion does not take place undera wide range of environmental conditions shown to favour glycollateexcretion by other algae. However, small amounts of labelledglycollate were detected in the supernatant from the cells duringthe photoassimilation of ³H-¹⁴C-acetate, but this glycollatedid not show the high ³H/¹⁴C ratio of glycollate present withinthe cell. The failure of Chlorella pyrenoidosa to excrete appreciableamounts of glycollate when photoassimilating acetate or carbondioxide was considered to result from the presence of glycollateoxidase (EC 1.1.3.1 [EC] ) which allowed the further metabolism ofglycollate. Besides glycollate oxidase, glyoxylate reductasewas also demonstrated in Chlorella pyrenoidosa so that glycollatecould function in hydrogen transfer during the photoassimilationof acetate.     

Effects of Glycollate on the Growth of a Planktonic Chlorella

The duration of the lag phase in cultures of a planktonic strainof Chlorella pyrenoidosa growing from small inocula under conditionsof light limitation was shortened by the addition of 1 mg 1^–1of glycollate to the medium. Of eight related compounds tested,none, with the possible exception of lactate, had a similareffect. Addition of 1 mg 1^–1 of glycollate also considerablyincreased the relative growth-rate of the alga at low (500 lux)but not at higher light intensities. This effect was not, however,specific to glycollate. Concentrations of glycollate (20 mg1^–1) higher than those normally produced in cultures byexcretion had an inhibitory effect on growth.     

Factors Affecting Growth and Aggregate Dissociation in Batch Suspension Cultures of Datura innoxia (Miller)

Growth kinetics of Datura innoxia batch suspension cultureswen monitored by a Klett-turbidimetric technique. While cultured. wt varied linearly with Klett units, f. wt and packed cellvolume did not. Turbidimetrically determined doubling timeswere highly reproducible. The method proved to be useful inthe determination of acutely lethal conantrations of a seriesof anti-metabolites. In certain circumstances, aggregate dissociation in batch suspensioncultures of D. innoxia was found to be coupled to growth rate.Suspensions maintained with 10^–5 M 2,4-D exhibited a relativelyslow growth rate with a high degree of aggregate dissociation:10^–4 M 2,4-D promoted a maximum growth rate, but dramaticallysuppressed aggregate dissociation. At 10^–5 M 2,4-D, themitotic index of smaller-aggregate fractions was greater thanthe mitotic index of the large-aggregate fraction. At 10^–5M 2,4-D the converse was observed. Supraoptimal 2,4-D concentrationsthus enhanced both aggregate dissociation and the growth ofsmaller aggregates. When present in concentrations promoting optimal growth. malicand succinic acids caused a decrease in aggregate dissociation.Casein hydrolysate dramatically enhanced growth, but did notaffect aggregate dissociation to the same degree as 2,4-D orthe Krebs cycle organic acids. Suggestions are made concerningmedium composition to be used in future mutant selection schemesusing D. innoxia. Datura innoxia (Miller), suspension culture, growth kinetics, mitotic index, 2,4-dichorophenoxy acetic acid     

Effect of Auxin and Gibberellin on Conidial Germination in Neurospora crassa II. "Conidial Density Effect" and Auxin

Conidial germination in liquid shake culture of Neurospora crassawas affected by the number of conidia in the medium (conidialdensity effect) with the optimum density being around 2?10⁶conidia/ml. The conidial density effect at 2?10⁴ and 2?10⁵ conidia/mlwas eliminated by the addition of auxin, IAA or 2,4-D with theoptimum concentrations of IAA and 2,4-D being around 10^–6M. IAA and 2,4-D had no effect on the effect at 2?10⁷ conidia/ml.An active substance(s) for conidial germination which was relatedto the conidial density effect was detected in the cell-freefiltrate of the germination medium, and was found to be non-dialyzableand thermolabile. At the early stage of germination, the concentrationof active substance(s) in the medium increased in proportionto the conidial density and reached a supraoptimum amount forgermination at 2?10⁷ conidia/ml. IAA (10^–6M) enhancedthe concentration. Endogenous auxin concentrations in filtratesof the germination media containing 2?10⁵, 2?10⁶ and 2?10⁷ conidia/mlwere 5.8?10^–12, 4.6?10^–11 and 1.7?10^–10M IAAequivalent, respectively. The conclusion reached was that auxinmay control conidial germination with mediation by the activesubstance(s). (Received June 8, 1982; Accepted September 27, 1982)     

Morphological and physiological characterization of IAA-less-sensitive and IAA-sensitive phases in rooting of Azukia cuttings

In disbudded epicotyl cuttings taken from light grown 5-dayold Azukia angularis Phaseolus angularis) seedlings, all adventitiousrootlets appeared on the second day of incubation. No root primordiawere observed within the first 24 hr and no increase in thenumber of roots occurred after 48 hr. Puromycin (5.5?10^–5M), p-fluorophenylalanine (1?10^–3M),2-thiouracil (2.3?10^–4M) and 2,6-diaminopurine (2?10^–5M)inhibited rooting when applied to cuttings on the second day,but showed no inhibition when applied on the first day. Unlike these inhibitors, pyrithiamine (7.2?10^–5M) inhibitedrooting when it was applied to cuttings on the first day. A rooting promoting effect was observed with actinomycin D (2.4?10^–6M),2,4-dinitrophenol (3?10^–5M) and p-fluorophenylalanine(1?10^–4M) applied to the cuttings on the first day, whereasindoleacetic acid (1.7?10^–4M) showed its promoting effectmost effectively on the second day. ¹Contribution No. 17 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. (Received June 4, 1969; )     

Auxin-Binding Protein in Etiolated Mung Bean Seedlings: Purification and Properties of Auxin-Bindmg Protein-II

An auxin-binding protein (ABP-II) was purified from the extractof etiolated mung bean seedlings by affinity chromatographyon 2,4-D-linked Sepharose 4B and by gel filtration on Sepharose4B and Sephacryl S-200. The molecular weight was estimated tobe about 190,000 by gel filtration on Sephacryl S-200. ABP-IIgave a single band corresponding to a molecular weight of about48,000 on SDS-polyacrylamide gel electrophoresis. The dissociationconstants of ABP-II for 2,4-D determined by amrnonium sulfateprecipitation and equilibrium dialysis were 9.5?10^–6 Mand 1.1?10^–5 M, respectively. ¹⁴C-2,4-D-binding to ABP-IIwas reversible and inhibited by addition of IAA, naphthalene-1-aceticacid, 2,4,5-trichlorophenoxyacetic acid or p-chlorophenoxyisobutylicacid to the assay mixture. (Received September 5, 1984; Accepted November 5, 1984)     

Studies on the Growth in Culture of Plant Cells: XX. UTILIZATION OF 2,4-DICHLOROPHENOXYACETIC ACID BY STEADY-STATE CELL CULTURES OF ACER PSEUDOPLATANUS L

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore produced an immediate reduction in ratesof cell division and eventually in rates of biomass accumulation.The sequential responses of a chemostat and of turbidostat culturessubjected to gradual withdrawal of 2,4-P were: (i) a transientincrease in biomass accumulation, (ii) increased accumulationof p-coumaric acid, flavonoids, and lignin, (iii) increasedcell aggregation, (iv) reduced rates of cell division, and (v)death. During stepwise reduction of 2,4-D supplied to turbidostatcultures, rates of 2,4-D uptake were reduced when the spentmedium concentration fell to 3?5–1?0 ? 10^–7 M. Underthese conditions the 2,4-D concentration in soluble and insolublecell fractions declined. The growth responses were correlatedwith the spent-medium 2,4-D concentration but not with its concentrationin the intracellular fractions.     

Studies on the Growth in Culture of Plant Cells: XXI FINE STRUCTURAL FEATURES OF ACER PSEUDOPLATANULS L. CELLS RESPONDING TO 2,4-DICHLOROPHENOXYACETIC ACID WITHLDRAWAL IN TURBIDOSTAT CULTURE

A culture of Acer pseudoplatanus L. grown in the presence ofan equilibrium level of 2,4-dichlorophenoxyacetic acid (2,4-D)of 1?5 ? 10^–7 M (state IV culture) showed, in comparisonwith one of a similar specific growth rate but in which theequilibrium level of 2,4-D was 2?3 ? 10^–6 M (state Iculture), an enhanced degree of cell aggregation, enhanced meancell volume, and the presence of cells giving a generalizedlignin reaction with extracellular lignin-positive material.The state IV culture showed a proportion (10–15 per cent)of cells having ultrastructural features not observed in thestate I culture. Some of the cells, located at the surface ofthe cellular aggregates, were small, rounded, highly cytoplasmic,and rich in rough endoplasmic reticulum. Further within theaggregates there occurred some cells showing abnormal or incompletecytokinesis and having irregularly thickened walls. Locatedcentrally in the aggregates were cells showing massive accumulationsof electron-dense material and with cell walls showing bandsof thickening alternating with thinner wall regions traversedby plasmodesmata. The latter cells are interpreted as cellsshowing intense polyphenol metabolism and imperfect xylogenicdifferentiation.     

Induction of DNA synthesis and callus formation from tuber tissue of Jerusalem artichoke by 2,4-dichlorophenoxyacetic acid

Cellus induction was observed from Jerusalem artichoke tubertissue on a synthetic medium containing 2,4-D at 10^–6,10^–5 (optimum conc.) and 10^–4 M. The first DNA synthesis(thymidine incorporation) was observed only at 2,4-D concentrationsof 10^–5 to 10^–4M. In 10^–5 M 2,4-D treatedtissue, DNA synthesis increased after a 20 hr lag and reacheda maximum at 36 hr, after which it decreased. Actinomycin Dand 8-aza-guanine; inhibitors of RNA synthesis, inhibited DNAsynthesis completely. 2,4-D caused the characteristic changesin RNA and protein syntheses. In comparison with the control,RNA and protein syntheses were first repressed then inducedbefore the peak of DNA synthesis. Treatment with cycloheximide(10^–4M) for one hour before inoculation inhibited proteinsynthesis completely for 12 hr; consequently DNA synthesis wasalso delayed. The results suggest that RNA and protein synthesesneeded for callus induction are regulated by 2,4-D in the firstDNA synthesis. (Received July 19, 1973; )     

Purification and Properties of an Auxin-Binding Protein from the Shoot Apex of Peach Tree

A soluble auxin-binding protein was purified from the shootapices of peach trees by chromatography on columns of CM-Toyopearl,Sephacryl S-200, 2,4-D-linked-Sepharose 4B and ConA-Sepharose.The molecular mass of the purified protein was estimated tobe about 100 kDa. After electrophoresis on a denaturing gel,the protein gave a single band with a molecular mass of 20 kDa.From Scatchard analyses, the dissociation constant for 2,4-Dwas calculated to be 4.1 10^–5 M and the specific bindingof 2,4-D at saturating concentration was 42 nmol (mg protein)^–1.The binding of [¹⁴C]-2,4-D to the protein was reversible andwas inhibited by IAA, 1-naphthylacetic acid and p-chlorophenoxyisobutyricacid. (Received June 25, 1992; Accepted October 20, 1992)     

The Kinetics of Extracellular Glycollate Production by Chlorella pyrenoidosa

Extracellular glycollate is liberated by Chlorella pyrenoidosaduring growth in medium bubbled with air or 3 per cent carbondioxide in air. With air the rate of release of glycollate percell decreases, with 3 per cent carbon dioxide it increases,with increase in cell number. Glycollate is released duringshort-term experiments when C. pyrenoidasa, grown under lowlight and high carbon dioxide, is transferred suddenly to highlight and low carbon dioxide. No other combination of thesefactors produces a comparable release of glycollate. The quantityof glycollate released in short-term experiments increases exponentiallywith the relative growth-rate of the culture from which thecells are derived. A crucial condition for maximum glycollaterelease is that growth of the culture prior to the experimentshould not be limited by carbon-dixoide concentration. The effectof pH is related to its effect on growth-rate; i.e. C. pyrenoidosahas a lower relative growth-rate at pH 8.3 and produces correspondinglyless glycollate than faster growing cultures at pH 6.4. Duringshort-term experiments under high light and low carbon dioxidethe rate of glycollate release drops after 50–100 minutessuggesting exhaustion of the glycollate precursor.     

Effects of Auxin and Gibberellin on Elongation of Young Hyphae in Neurospora crassa

IAA, 2,4-D and GA₃ promoted the elongation of young hyphae inNeurospora crassa at the optimum concentrations of 10^–6,10^–6 and 10^–4 M, respectively. The effects of IAAand GA₃ were additive. (Received June 17, 1983; Accepted December 22, 1983)     

Ethylene and Ethane Production in 2,4-D Treated and Salt Treated Tobacco Tissue Cultures

Gas chromatography was used to measure ethylene (ethene) andethane production by tobacco (Nicotiana tabacum cv. Wisconsinno. 38) callus tissues grown on media containing inorganic saltsaccording to Murashige and Skoog (1962), sucrose, myo-inositol,thiamine-HCl kinetic according to Linsmaier and Skoog (1965),and either 2,4-dichiorophenoxyacetic acid (2,4-D) in the range0–100 mgl^–1 or 2 mgl^–1 indoi-3-ylacetic acidplus NaCl in the range 0–200 Meq l^–1. Ethylene productionrates were high (> 500 nl h^–1 g^1– fresh weight)initially in all treatments. Subsequently, ethylene productiondeclined in rapidly growing cultures but remained high in moderatelyand severely 2,4-D (> 0·5 mgl^–1) stressed andin severely NaCl (150 Meql^–1) stressed cultures. Highinitial rates of ethane production (> 200 nl h^–1 g^–1fresh weight) were obtained under conditions of severe stresscaused by 2,4-D or NaCl but not in control or moderately inhibitedcultures. With further incubation ethane production declinedin the severely stressed cultures. It is concluded that ethyleneproduction can be used as an index of moderate 2,4-D stressand severe NaCl stress by virtue of the high persisting ratesof ethylene production in stressed cultures. Ethane productioncan be used as an early index of severe stress caused by either2,4-D or NaCl in vitro. Nicotiana tabacum L., tobacco, ethylene, ethenen, ethane, 2,4-dichlorophenoxyacetic acid, auxin, stress, callus tissue     

Vessel element formation in cultured carrot-root phloem slices

The effects of light, auxin and cytokinin on vessel elementformation in phloem slices of carrot root were examined. When slices of carrot cultivars, ‘Nakamura-senko-futo’and ‘Yamada-hyakunichisenk6- naga’, preculturedin the dark on modified Murashige and Skoog's medium for twodays were cultured on a medium containing 5x10^–6 M 2,4-Din the dark, no vessel element formation occurred. When preculturedslices were cultured in the light with 5x10^–6M 2,4-D,vessel element formation was remarkable. But when 5x10^–7Mkinetin, benzyladenine or zeatin was added, vessel elementswere readily formed even in the dark. When slices were cultured in the light, a cytokinin-like substance(s)that causes vessel element formation was produced in the slices,then was released to the medium. The substance(s) was fairlystable to heat. In slices of carrot cultivars, kuroda-gosun, ‘Kintoki’and ‘Kokubu-senk6-6naga’, a different result forvessel element formation was obtained. When slices of thesecultivars were cultured on a medium containing 5x10^–6M2,4-D in the dark, vessel element formation was remarkable.It seemed, therefore, that these cultivars contain enough ofa cytokinin-like substance(s) to form vessel elements. In fact,vessel element forming activity was found in the alcohol extractof carrot root phloem from these cultivars. (Received June 8, 1971; )     

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. Gibberellin-coumarin and kinetin-coumarin interactions on stem elongation

In azuki bean (Azukia angularis = Vignia angularis) epicotylsections, 5 ? 10^–4 M coumarin inhibited the incorporationof radioactivity from [U^–14C]glucose into the cellulosefraction by 35% in the absence of indole-3-acetic acid (IAA)and by 40% in the presence of 1 ? 10^–4 M IAA. There wasno inhibitory effect on the incorporation of radioactivity intothe other fractions. Coumarin at 5 ? 10^–4 M reversed thepromoting effect of 1 ? 10^–5 M gibberellin A₃ (GA) andthe inhibitory effect of 1 ? 10^–5 M kinetin on IAA-inducedelongation of sections with no significant effects on IAA-inducedelongation. Neither GA nor kinetin had any appreciable effectson cellulose synthesis. No inhibition of cellulose syntheiswas observed with 1 ? 10^–3 M colchichine, which has beenreported to have effects similar to those of coumarin on GA-or kinetin-affected stem elongation. Coumarin at 5 ? 10^–4M was ineffectual in breaking up wall microtubules, while adisrupting effect on wall microtubules was clearly demonstratedwith 3 ? 10^–4M colchicine. From these results, the possible involvement of cellulose synthesisin cell expansion controlled by GA or kinetin was suggested. (Received August 3, 1973; )     

The Oxidation of 14C-labelled Glucose by Chlorella vulgaris: 2. The Fate of Radioactive Carbon

Most of the ¹⁴C added as glucose to carbohydrate-starved cellsof Chlorella Vulgaris can be recovered as alcohol-soluble compoundsor as polysaccharide. Only 5–I6 per cent., depending onthe position of ¹⁴C in the glucose supplied, is released ascarbon dioxide. Similar results were obtained with Chlorellapyrenoidosa and Ankistrodesmus. The labelled alcohol-solublecompounds in Chlorella vulgaris include amino-acids, particularlyglutamic acid, aspartic acid, and alanine, and, when glucose-I-¹⁴Cis metabolized, the amount of ¹⁴C recovered in these amino-acidsis about the same as that recovered as carbon dioxide. Degradationof the glucose incorporated into polysaccharide shown that theC₁ and C₆ atoms of glucose rapidly interchange when in the cells.The bearing of these results on attempts to estimate the relativeimportance of different pathways of glucose breakdown is discussed.     

Somatic Embryogenesis from Clonal Leaf Tissues of Cassava

Leaf lobes were isolated from palmate leaves of clonal cassava(Manihot esculenta Crantz) material growing in vitro or in glasshouseconditions and subjected to a two-stage culture procedure involvingincubation on Murashige and Skoog (MS2) basal medium supplementedwith 2–12 mg l^–1 2,4-D for 20 d (Stage I) beforetransfer to MS2 basal medium supplemented with 0.01 mg l^–12,4-D and 0.1 mg l^–1 6-benzylamino purine (BAP) (StageII medium). Embryogenetic tissues, foliose structures and somatic embryosdeveloped from leaf lobes at all Stage I 2,4-D concentrations,except on those explants isolated from shoot-tip cultures incubatedon MS2 basal medium supplemented with 0.1 mg l^–1 NAA and1.0 mg l^–1 BAP. Leaf lobes isolated directly from glasshouse plants showed optimalembryogenetic competence when subjected to a Stage I cultureperiod of 17 d, although foliose structure initiation was optimalwith shorter Stage I durations. Leaf lobes of 2–4 mm lengthand those isolated from phyllotaxic leaf numbers 4 and 5 showedthe greatest embryogenetic competence. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, morphogenetic competence     

The Role of Glycollic Acid in the Photoassimilation of Acetate by Chlorella pyrenoidosa

The kinetics of ³H-acetate assimilation by Chlorella pyrenoidosain the light were examined. The primary products of assimilationwere glycollate and succinate. After 10 sec glycollate contained45 per cent and succinate 25 per cent of the tritium incorporatedby the cells. The percentage of the total tritium in glycollateand succinate fell with time while that in citrate increased.Initially the specific activities (µc of ³H per µmoleof acid) of succinate and glycollate were greater than citrate.When ³H-¹⁴C-2-acetate was added to the cells, total dpm for³H and ¹⁴C in glycollate rapidly reached a steady state andgave a ³H/¹⁴C ratio of 10, compared with a ³H/¹⁴C ratio of 4in the acetate. This ³H/¹⁴C ratio in glycollate is found because³H is derived from ³H-¹⁴C-2-acetate and because the ¹⁴C is dilutedwith cold carbon from elsewhere. The addition of ¹⁴CO₂ at thesame time as ³H-¹⁴C acetate decreased the ³H/¹⁴C ratio in glycollatebut incorporation of ¹⁴C from ¹⁴CO₂ into glycollate was slowerthan incorporation from ¹⁴C-2-acetate. Although ¹⁴C from acetaterapidly appeared in glycollate, ¹⁴C-labelled glyoxylate wasnot detected. The ³H/¹⁴C ratio observed in glycollate rulesout formation of glycollate from acetate via glycoaldehyde.The available evidence did not support glycollate formationvia the Calvin cycle. ¹⁴C from ¹⁴C-Z-acetate appeared in glycollatebefore it did in phosphoglyceric acid. Total dpm for ³H, ¹⁴C,and ³H/^l4C ratio in Calvin cycle intermediates were not in equilibriumwith glycollic acid.     

Ion Fluxes Across the Transfusion Zone of Secreting Limonium Salt Glands Determined from Secretion Rates, Transfusion Zone Areas and Plasmodesmatal Frequencies

Faraday, C. D., Quinton, P. M. and Thomson, W. W. 1986. Ionfluxes across the transfusion zone of secreting Limonium saltglands determined from secretion rates, transfusion zone areasand plasmodesmatal frequencies.—J. exp. Bot. 37: 482–494. The epidermal salt-secreting glands of Limonium (Plumbaginaceae)are enclosed in a cuticular envelope. Ions and metabolites enterthe glands from the mesophyll through gaps in the cuticularenvelope, the transfusion zones. Net influxes of ions acrossthe transfusion zone were calculated from measurements of secretionrates and transfusion zone areas. When leaves of L. pereziiF. T. Hubb. were treated with 300 mol m^–3 NaCl, transfusionzone influxes of Na⁺ K⁺, Ca⁺⁺ and Cl^– as high as 7?0?10^–5,1.7?10^–5, 5?8?10^–7 and 8.5?10^–5 mol m^–2s^–1 respectively, were calculated. Assuming a transmembranepathway, these fluxes would be some of the highest reportedfor ions in plant cells. Key words: Salt glands, ion fluxes, ultrastructure     

Differential Responses of Buckwheat Leaf Cells to Growth Substances Stimulating Cell Division

Isolated buckwheat cotyledons form calli, roots or buds whencultured in an appropriate medium. A medium containing high2,4-D (5 mg 1^–1) and low KN (01 mg I^–1), which inducescallus formation, was found to stimulate cell division in thelayer between palisade and spongy parenchyma tissue after 72h. Low 2,4-D and low KN (01 mg I^–1 each), which stimulatesroot formation in buckwheat cotyledons, induces divisions primarilyin spongy parenchyma cells. In a high benzylaminopurine (10^–5M) and a low IAA (10^–6 M) medium, which favours bud induction,cell divisions were localized to the palisade layer. The differentialresponsiveness of leaf cells to various hormone treatments isdiscussed.