CHANGES IN RESPONSE TO PURINE AND PYRIMIDINE DERIVATIVES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURING

1. The growth of the carrot root callus which had been subculturedfor a long period (CCL) was promoted by the addition of 5l0^–8and 5l0^–7 M kinetin, whereas in the callus subculturedfor a short period (CCS) no growth promotion was observed atany concentrations of kinetin tested.
2. CCL showed an increasedgrowth in response to the applicationof kinetin, guanine, adenine,hypoxanthine, uracil, thymine,and cytosine in the presenceof fractions A and C of carrotroot extract, whereas no suchresponse was observed in CCS.CCL required fraction C to respondto uracil and probably purineand pyrimidine derivatives ingeneral.
3. The growth of CCL was promoted by kinetin, guanine,adenine,or hypoxanthine in the medium containing inositol andaminoacids mixture. In this case the growth-promoting actionof guanine,adenine, or hypoxanthine was nullified by kinetin.

(Received December 24, 1964; )     

AUXIN-INDUCED GROWTH OP TUBER TISSUE OP JERUSALEM ARTICHOKE: III. EFFECT OF ACTINOMYCIN D ON AUXIN-INDUCED EXPANSION GROWTH AND RNA SYNTHESIS

1. The following results were obtained using tissue slices excisedfrom cold-stored Jerusalem artichoke tubers.
2. Actinomycin Dat the concentration of 20 µg/ml given duringthe agingperiod did not affect the subsequent expansion growthcausedby auxin or auxin plus kinetin.
3. Actinomycin D given in thegrowth period, on the other hand,strongly inhibited the expansiongrowth of tissue slices agedin the absence of the antibiotic.
4. In the growth period, auxin or auxin plus kinetin promotedtheincorporation of uracil-2-¹⁴C into RNA fraction.
5. ActinomycinD inhibited the incorporation of ³²P orthophosphateinto ribosomalRNA during the aging period.
6. In the growth period, the incorporationof ³²P into RNA wasenhanced by auxin and was inhibited by actinomycinD, more remarkablyin ribosomal RNA than in lighter RNA.

¹A part of this paper was presented at the Conference on PlantGrowth Regulators held by the New York Academy of Sciences onMay 16, 1966.     

Correlation, between {beta}-galactosidase and auxin-induced elongation growth in etiolated pea stems

ß-Galactosidase, -galactosidase, ß-glucosidaseand ß-xylosidase were studied in relation to auxin-inducedelongation of etiolated pea stems. Frozen-thawed sections wereincubated in substrate solutions and individual enzyme activitywas determined. Results were as follows:

1. Only ß-galactosidase activity was remarkably enhancedby 2,4-D in the concentration range which induced remarkableelongation.
2. Auxin-induced increase in ß-galactosidaseactivityreached maximum after 1 hr treatment of the sectionwith 2,4-D(10^–5 M), and was nearly constant afterwards.
3. Auxin-enhanced ß-galactosidase activity was observedeven if auxin-induced apparent elongation was osmotically suppressedby mannitol.
4. Correlation analysis indicated that ß-galactosidaseactivity had a high positive correlation with auxin-inducedelongation and increase in lateral face area of the section,while none of the other enzyme activities did.
5. Measurementof in vivo distribution of four glycosidase activitiesin intactthird internode of the epicotyl made clear that onlyß-galactosidaseshowed maximum activity in the zonewhere endogenous elongationwas maximum.

(Received February 5, 1976; )     

CHEMICAL CONTROL OF MORPHOGENESIS IN MOSS PROTONEMA

1. The protonema of the moss, Funaria hygrometrica, grows continuouslyin calcium-free liquid media.
2. The growth was promoted by additionof oxalate, although themorphogenesis resulting in formationof gametophytic buds onthe protonema was suppressed by theaddition.
3. Calcium oxalate promoted the growth of protonema,while at ahigh concentration (10^–2 M) it caused the formationofclumped protonema (falsebuds).
4. Addition of plant growthhormones, such as IAA, NAA, 2,4-D andgibberellin retarded thegrowth of protonema, while 2,4-D ata low concentration stimulatedthe growth of protonema.
5. Kinetin greatly stimulated the formationof gametophytic budsin the protonema, but these buds were foundto be morphologicallyand physiologically abnormal.

(Received January 29, 1965; )     

STUDIES ON PROTEIN SYNTHESIS IN TOMATO COTYLEDONS AND LEAVES. II. INTERMEDIATE STAGES OF PROTEIN SYNTHESIS

1. The activities of RNase, ATPase and proteolytic enzymes inextractsof tomato leaves have been investigated with regardto detrimentaleffects on protein synthesis. The use of coppersulphate asan inhibitor of RNase is described.
2. The effectof microbial contamination on amino acid uptake hasbeen studiedin relationship with cell-free systems, and thehigh activitynoted underlines the necessity for aseptic procedures.
3. Methodsfor the preparation of cell-free systems capable ofamino acidactivation and incorporation into protein in an asepticenvironmentare discussed.

¹Present address: Dept. of Horticultural Science, Universityof Wisconsin, Madison, Wisconsin, U.S.A.     

STUDIES ON PROTEIN SYNTHESIS IN TOMATO COTYLEDONS AND LEAVES. I. PROTEIN SYNTHESIS AND TURNOVER IN INTACT COTYLEDONS AND LEAVES

1. Details of aseptic culture of virus-free tomato seedlings usedin comparative in vivo and in vitro studies on protein synthesisare described.
2. Developmental changes in the levels of DNA,RNA, protein andchlorophyll content of seedling cotyledonsand leaves were recorded,and are related to protein synthesis.
3. Incorporation of isotopically labeled carbon into proteinwasfollowed both by photosynthetic uptake of ¹⁴CO₂ and by theuptakeof ¹⁴C-amino acids through the roots.
4. A marked stimulationby light of ¹⁴C uptake was observed, andthe higher rate of¹⁴C incorporation from ¹⁴CO₂ than from ¹⁴C-aminoacids intothe protein fraction is discussed in relation tothe pathwaysof protein synthesis in tomato leaves, and alsowith regardto protein turnover.

¹Present address: Dept. of Horticultural Science, Universityof Wisconsin, U.S.A.     

Biological activity of phyllosinol, a phytotoxic compound isolated from a culture filtrate of Phyllosticta sp.

1. Phyllosinol is a phytotoxic metabolite of Phyllosticta sp. Thissubstance at 100 µg/ml produced dark grey necrotic lesionson the leaf of red clover. Sensitivities of various plant speciesto phyllosinol differed both quantitatively and qualitatively.
2. Phyllosinol reduced root growth in rice seedlings by 60% at10^–4 M, whereas stimulation of root elongation occurredat a concentration range from 10^–9 to 10^–5 M.
3. Phyllosinolat 2.5x10^–4M promoted adventitious root formationin epicotylsof Azukia cuttings by about 100%. Promotion waspartly reducedby simultaneous application of cysteine.
4. IAA-induced elongationof isolated Avena coleoptile sectionswas inhibited by phyllosinolat a concentration range from 10^–5to 10^–3M.
5. Sulfhydrylcompounds, i.e. cysteine and glutathione relievedinhibitioncaused by phyllosinol in IAA-induced elongation ofAvena coleoptilesections.
6. GA₃-induced elongation of wheat leaf sections wasslightly inhibitedby phyllosinol at 10^–4M.
7. Phyllosinolalso has antibiotic activity. Among the organismstested, Phycomycetesand Gram-negative bacteria appeared mostsusceptible to phyllosinol.

(Received April 21, 1970; )     

The Water Permeability of Unplasmolysed Tissues2

1. The permeability of unplasmolysed cells of beetroot, v. ‘CrimsonGlobe’, was determined from the rate of water loss ofbeet slices on placing in sucrose solutions having O.P. greaterthan the suction pressure of the beet. The absolute values obtainedwere about 0?7µ³ water per µ² cell-surface per hourper atmosphere osmotic pressure difference, i.e. 0?7 µ/hr./atm.
2. The permeability of similar beet cells plasmolysed withintheircell walls was found to be about 13µ/hr./atm.
3. Thepermeability of beet cells which had been plasmolysed andallowedto recover was shown to be approximately the same asthat ofunplasmolysed cells.
4. The hypothesis is advanced that the increasein water permeabilityon plas-molysis is due to those partsof the plasma-membranewhich had formerly been pressed againstthe micelles of thecell wall becoming free and able to takepart in water transfer.
5. The energy requirement for the maintenanceof an excess hydrostaticpressure of five atmospheres withina cell by its vital activitywas shown to be about one-tenthof the total respiratory energyreleased in freshly cut beetslices.

     

STUDIES ON A CAROTENE-PROTEIN COMPLEX ISOLATED FROM GREEN LEAVES

1. Two new methods of preparing a carotene-protein complex fromchloroplasts were described, in addition to the one previouslyreported by the present authors.
2. Light-scattering measurementswere performed and from the dissymmetryratio of the scattering,the particle size of the complex wascalculated. The mean radiuscorresponding to the weightaverageparticle weight was foundto be 56 mµ. This result wasdiscussed with referenceto the previously reported values obtainedfrom sedimentationmeasurements and electron micrographic determinations.
3. Thedepolarization of scattered light was measured. Large valuesobtained for the degree of depolarization of the incident lightrevealed a high degree of anisotropy in the particle of thecarotene-protein complex.

¹Present address: Johnson Foundation, University of Pennsylvania,Philadelphia, Pa., U.S.A. (Received July 8, 1960; )     

Studies on the destruction of indole-3-acetic acid by a species of Arthrobacter I. On the induction of the enzyme responsible for the destruction

1. The induction of an IAA-destroying enzyme in Arthrobacter sp.that can utilize IAA as its sole source of carbon and nitrogenwas investigated.
2. 1. The enzyme was most effectively inducedby 10–³ to2x10–³ M IAA, at pH 6.5.
3. 2. All testedIAA analogs were unable to induce the enzyme.Analogs otherthan indole-3-lactic acid were rather inhibitoryon the inductionwith IAA.
4. 3. The induction period was shortened with the ageof culturein both polypeptone and acetate media.
5. 4. Pretreatmentof the bacterium with IAA caused a shorteningof the inductionperiod.
6. 5. The induction was inhibited by various antibiotics,aminoacid analogs and nucleobase analogs.
7. 6. The inductionwas less remarkable in actively proliferatingcells than itwas in slowly proliferating ones.

(Received July 1, 1967; )     

Effects of 3-Indolylacetic Acid and 3-Indolylacetonitrile on the Growth of Excised Tomato Roots

1. The inhibition by IAA (3-indolylacetic acid) and by IAN (3-indolylacetonitrile)of the growth of excised tomato roots cultured for 7 days at27 C. in a modified White's medium is described. 510^–9g./ml, IAA or 510^–6 g./ml, IAN cause approx, 50 per cent,inhibition of the linear growth of the main axis. With IAA decreasein number of laterals closely parallels the decrease in lineargrowth of the main axis; with IAN reduction in linear growthof the main axis occurs at concentrations above 10^–8 whereasnumber of laterals does not decrease until the concentrationexceeds 10^–6.
2. Study of the course of cell elongationin the exodermal cellsshowed that in the standard medium andin media containing 510^–9IAA or 510^–6 IAN theprocess takes about 7 hours; thefinal cell lengths in IAA andIAN media are lower than in standardmedium owing to a slowerrate of elongation. The decrease inlinear growth of the mainaxis in presence of IAA could be accountedfor by the decreasein cell length; this was not the case withIAN. The implicationsof this are considered.
3. Determinations of the distance (mm.)between, and of the numberof exodermal cells separating, theadjacent laterals in oneorthostichy showed that IAN enhancesthe frequency of lateralswhereas this is either unaffectedor decreased by IAA. The enhancementof lateral frequency inIAN arises from shortening of the cellsof the main axis anddecrease in the number of cells separatingadjacent laterals.
4. The results are considered to support the view that IAN haseffects on root growth different from those of IAA. Study ofthe degree of inhibition of main axis growth and of alterationsin lateral frequency resulting from treatment with mixturesof IAA and IAN provided data which could also be most easilyexplained on this hypothesis.

     

DISTRIBUTION AND TURNOVER OF PHOSPHATE COMPOUNDS IN GROWING CHLORELLA CELLS

1. Using the Chlorella cells which had been uniformly labeled with³²P, the distribution of phosphorus in various fractions ofcell material was investigated. Uniformly ³²P-labeled Chlorellawas further grown in a P-free medium or in a standard "cold"medium, and the change of distribution of ³²P (as well as theuptake of exogenous P) in various cell fractions was followed.
2. Analysis of the ³²P-labeled algal cells showed that the highestin P-content was the fraction of RNA followed by those of polyphosphates,lipid, nucleotidic labile phosphate compounds, DNA and protein(in decreasing order). ATP and ADP were found to be only minorfractions of the total labile phosphates.
3. On incubating the³P-labeled alga in a P-free medium, the P.contentsin the fractionsof DNA, protein, lipid and ATP increased, thosein polyphosphatesand ADP decreased, and that in RNA remainedalmost unchanged.When the ³²P-labeled alga was further grownin the normal "cold"medium, DNA and protein increased withthe expenditure of endogenous³²P, but with practically no incorporationof external P. Inthe meantime the P in polyphosphates decreasedconsiderably,and the RNA fraction incorporated a large amountof externalP but only a little of endogenous³²P.
4. It was inferred that,under the experimental conditions of thepresent study, thephosphorus used in the syntheses of DNA andprotein was primarilytaken from polyphosphates, while thatused in the synthesesof RNA, phospholipid and polyphosphateswas, for the most part,taken from the extracellular P-source.

¹A part of this paper was read at the Vth International Congressof Biochemistry, Moscow, August 10–16, 1961. (Received June 4, 1961; )     

The mineral nutrition of cultured chlorophyllous cells of tobacco I. Effects of {pi}salts, {pi}sucrose, Ca, Cl and B in the medium on the yield, friability, chlorophyll contents and mineral absorption of cells

1. The growth rate of cultured free-cells and their contents ofchlorophyll, Mg, K and N reached peaks on media containing M_n+= 60 and 80 meq per liter, or in which _salts=2.7 and 3.6 atm.These concentration levels in the media were considerably higherthan those (_Salts=1.5–2.0 atm) which produced good growthof intact plants. This discrepancy in media concentrations maybe due to differences in the culture conditions and in the morphologyof the materials.
2. Very low Ca²⁺/(Mⁿ⁺–Ca²⁺) equivalentratios of 0.1 to 0.01in a medium were sufficient for good growthof free cells, whilegenerally about unity of the ratio is requiredin a medium forgood growth of intact plants. The differencein the medium compositionmay also be due to morphological differencesin the materials.
3. Addition of Cl^– to the medium furtherpromoted the growthrate and friability of cell clumps.
4. Alow concentration of 0.01 mM BO₃^3– in the medium alsopromoted the friability of cell clumps.

(Received August 15, 1972; )     

ETUDE DES COMPLEXES DE PIGMENTS CHLOROPHYLLIENS D'EUGLENA GRACILIS AU COURS DE LA CROISSANCE ET A L'ETAT ADULTE

Une tude des cellules d'Euglnes et de leurs extraits prparsavec les ultra-sons, chauffs ou non chauffs (quelques heures 35), montre que:

1. le chauffage ne dtruit pas la chlorophylle de faon notable;
2. en prsence d'alcool, un agrgat de chlorophylle ayant unebanded'absorption 740–750 mµ apparait dans lesextraitsd'Euglnes ges de plus de 11 jours;
3. les jeunesEuglnes et les Euglnes ges et chauffes ne peuventformerl'agrgat absorbant aux grandes longueurs d'ondes;
4. deux outrois complexes chlorophylliens diffrents sont impliqusdansla formation des agrgats qui absorbent 740 mµ.

¹ Travail rendu possible par l'attribution d'une bourse d'tudede l'universit de Montral l'un des auteurs (DE K.) et l'octroid'une subvention de recherches (A-1196) du Conseil Nationaldes Recherches du Canada.     

Studies in Stomatal Behaviour: IV. THE WATER-RELATIONS OF THE EPIDERMIS

1. It is shown that a dry external atmosphere exerts the followingeffects on stomatal movement:

1. A striking accelaration ofclosure in darkness.
2. A slight acceleration of opening in light.
3. If the water-supply to the leaf is impaired, an inabilitytomaintain full opening in the light.

Conversely, a saturatedexternal atmosphere induces sluggishness of movement and a tendencyto incomplete closure in darkness.

1. These results are consideredto support La Rue's contentionthat the epidermal water-supplyis drawn solely by lateral movementfrom the main veins, andnot from the underlying mesophyll.The stomatal phenomena themselvesdo not appear capable of anysimple explanation based on currentknowledge of guard-cellphysiology.
2. The biological significanceof these results is discussed, withparticular reference tothe problem of xeromorphic structures,for which a new interpretationis suggested.

     

PROMOTION OF ADVENTITIOUS ROOT FORMATION BY HELIANGINE AND ITS REMOVAL BY CYSTEINE

1. Heliangine at 10^–4M promoted the adventitious root formationin hypocotyls of cuttings taken from light-grown (1,900 lux)seedlings of Phaseolus mungo. The promotion was almost completelyreduced by simultaneously supplied 310^–4M cysteine or1.510^–4M cystine, but not suppressed by 310^–4Mof reduced glutathione, alanine or serine.
2. A 4 hr pretreatmentwith 310^–4M cysteine made Phaseoluscuttings less sensitiveto heliangine, but cysteine suppliedafter the treatment withheliangine brought about no effecton the action of heliangine.
3. Cysteine also removed the inhibiting effect of heliangineonthe indoleacetic acid-induced elongation of etiolated Avenacoleoptile sections.
4. In an aqueous solution heliangine formedan addition productwith cysteine, indicating that cysteinecan inactivate helianginewithout any biological processes.
5. On Phaseolus adventitious rooting, no effect was observedofp-chloromercuribenzoic acid, N-ethylmaleimide, 1,4-naphthoquinone,coumarin or penicillin. Reactivity toward sulfhydryl groupsalone does not qualify a substance to be a promotor of rootformation.
6. Maleic hydrazide at 10^–4M promoted root formation,butits effect was not removed by cysteine.

¹ Contribution No. 13 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo.     

INHIBITORY EFFECT OF 5-BROMOURACIL AND 5-FLUOROURACIL ON PHOTOPERIODICALLY INDUCED GERMINATION OF ERAGROSTIS SEED

1. The inhibitory effects of 5-bromouracil and 5-fluorouracil onthe photoperiodically induced germination of Eragrostis ferrugineaseed were investigated. 5-BU was more effective than 5-FU ininhibiting germination, but less effective in suppressing seedlingelongation.
2. The inhibition of germination by 5-BU was specificallyex ertedon the inductive dark process, which i9 essential tothe photoperiodicgermination induction. A high concentration(10^–1:M) of5-FU also elicited a complete inhibition ofgermination. 5-FU,however, did not affect the photoperiodicinduction processes,but inhibited the processes subsequentto them. The inhibitionof germination by 5-BU or 5-FU was alleviatedby neither uracil,thymidine nor orotjc acid.
3. The 3eedlinggrowth was effectively inhibited by 5-FU, and theinhibitionwas reversed by uracil, uridine and orotic acid,but not bythymine and thymidine. The effect of 5-FU may, therefore,possiblybe by virtue of its depressing action upon ribonucleicacidsynthesis.

(Received April 18, 1963; )     

THE DISTRIBUTION OF FORMYLTETRAHYDROFOLATE SYNTHETASE IN PLANTS, AND THE PURIFICATION AND PROPERTIES OF THE ENZYME FROM PEA SEEDLINGS

1. Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was foundto be widely distributed in higher plants and the high enzymeactivity was observed in green leaves of Brassica and Alliumspecies, spinach, and in pea seedlings. In pea seedlings, theenzyme activity changed during the course of germination, andmost of the enzyme activity was located in a soluble fractionof the cytoplasm.
2. The enzyme was labile and lost the activityrapidly, even whenstored at 5 in the presence of 0.1 M mercaptoethanol.It was,however, found that ammonium sulfate was very effectivein stabilizingthe enzyme activity.
3. The enzyme has been purifiedapproximately 500-fold from extractsof pea seedlings by treatmentswith ammonium sulfate, protaminesulfate, hydroxylapatite, calciumphosphate gel, and DEAE-cellulosecolumn chromatography.
4. Thepurified enzyme was specific for formate, ATP and FAH₄,andthe Michaelis constants for these reactants were 2.1 10^–2M, 5.1 10^–4 M, and 5.6 10^–3 M, respectively.
5. The optimum pH was found to be 8.0, and the optimal temperaturewas observed at 37. Both NH₄^$ and a divalent cation (Mg^SS orMn^SS) were required for the optimal activity.

¹ Studies on the Enzymatic Synthesis and Metabolism of FolateCoenzymes in Plants. II. (For the previous paper see reference(8)) A part of this paper was presented at the Meeting of theKansai Division of the Agricultural Chemical Society of Japan,Kyoto, January 29, 1966.     

ORGANIC ACID SYNTHESIS IN RESPONSE TO EXCESS CATION ABSORPTION IN VACUOLATE AND NON-VACUOLATE SECTIONS OF CORN AND BARLEY ROOTS

1. ¹⁴CO₂ fixation into organic acids in tips and proximal sectionsof both corn and barley roots was studied as a function of thenature and concentration of the salt in the external solution.
2. In comparison with the level of ¹⁴CO₂ fixation by vacuolateproximal sections in KCI, incorporation was markedly enhancedin K₂SO₄ and diminished in CaCk. By contrast, non-vacuolateroot tips were indifferent to the type of external salt withrespect to UCO₂ incorporation into organic acids.
3. The effectof salt type on organic acid formation from ¹⁴CO₂was most pronouncedat relatively high concentrations.
4. The conclusion was reachedthat organic acid synthesis in responsetoexcess cation uptakeis the result of cation movement into thevacuole, and thattransport into the vacuole is mediated bythe low-affinity componentof the dual mechanisms involved inion absorption.

¹Present address: Government Forst Experiment Station, Meguro,Tokyo.     

INFLUENCE OF LIGHT ON ACETATE UTILIZATION IN GREEN EUGLENA

1. The kinetics of adaptation to exogenous acetate, measured asthe ability to stimulate respiration, has been studied in Euglenapreviously grown with autotrophic nutrition.
2. The continuedpresence of light significantly inhibits the fulldevelopmentof respiratory adaptation to acetate.
3. Carbon fixed in photosynthesisis routed almost exclusivelyinto protein when acetate is present.Acetate incorporated withconcomitant photosynthesis largelyenters lipid and polysaccharide,with only a small fractionincorporated into protein.

(Received December 25, 1964; )