An Examination of a Method of Auxin Assay using the Growth of Isolated Sections of Avena Coleoptiles in Test Solutions

1. Methods of auxin assay using the Avena coleoptile are discussed.
2. A review is given of experimental procedure and evaluationofresults in the straight-growth method using isolated sectionsof coleoptiles in test solutions.
3. Possible sources of variationin the straight-growth methodare investigated and discussed.
4. A revised experimental procedure for the straight-growth methodis described.

The author wishes to thank Professor E. Ashby for his adviceand encouragement during the course of the experiments. Thanks are also due to Mr. D. Payne, a technical assistant inthe Botany Department, for his assistance in some of the assays.     

Studies on Plant Growth Hormones: I. BIOLOGICAL ACTIVITIES OF 3-INDOLYLACETALDEHYDE AND 3-INDOLYLACETONITRILE

1. Two neutral plant hormones, one isolated recently from plants(3-indolylacetonitrile) and the other (3-indolylacetaldehyde)reported to be present in plants, are avaible as pure syntheticcompounds for investigation of their biological activities.This paper is mainly concerned with their effects on cellelongationin the Avena coleoptile
2. 3-Indolylacetaldehyde is considerablyless active than 3-indolylaceticacid in the Avena straight-growthtest; for example, a 1.0 mg./l.solution of the aldehyde showsan activity equivalent to thatof a 0.1 mg./l. solution of theacid
3. An acidic substance is produced in solutions of the aldehydeduring the period of assay. In some experiments it accountsfor all of the activity shown by the aldehyde solutions, onthe assumption that it is 3-indoylacetic acid, and in otherexperiments it shows a greater activity than that of the aldehydesolutions from which it was obtained. Therefore, it is concludedthat 3-indolylacetaldehyde, itself is either inactive or inhibitory.Acid production in aldehyde solutions in vitro is much lower,a fact which suggests that there is enzymatic oxidation of aldehydeto acid in the presence of coleoptiles.
4. The activities of3-indolylacetaldehyde in the pea test andin root-inhibitionand of 3-indolylacetone in the straight-growthtest are brieflyreported.
5. 3-Indolylacetonitrile is considerably more activethan 3-indolylaceaticacid in the Avena straight-growth test;for erample, a 0.1 mg./l.solution of the nitrile shows an activityequivalent to a 1.0mg./l. solution of the acid. The inhibitoryeffect at concentrationsabove 1.0–10.0 mg./l. is lesswith the nitrile than withthe acid.
6. There is negligible productionof acid in solutions of the nitrileboth in vitro and in thepresence of Avena coleoptiles at temperaturesranging from –18?to 25? C. for varying lengths of time.The possibility of enzymaticconvesion of nitrile to acid insidethe cells of the coleoptileis discussed
7. The activities of 3-indolylacetamide and of 2:4-dichlorophenoxyaceticacid and the corresponding nitrile are considered in this connexion
8. The nitrile is destroyed by treatment with alkali but notbyacid. In the light of these results, it is desirable to re-examineprevious work on identification of auxins in plants by theiracid and alkali sensitivity. Evidence for the existence of thenitrile in a number of other plants is presented.

     

Organic Acid Metabolism of Sedum praealtum

1. The organic acids present are citric, isocitric, and l-malic,with a small residue of unidentified acids.
2. The diurnal variationin acidity is due chiefly to changes,in malic acid, with aparallel fluctuation shown by citric acid.Under these conditionsisocitric acid shows little change.
3. The importance of carbondioxide during acidification is confirmed,and it is shown thatat room temperatures or higher the CO₂produced in respirationis sufficient to produce maximum acidification.At lower temperaturesthe supply of CO₂ limits acid production.
4. In the absence ofoxygen no acidification occurs, but even smallquantities (approx.1 per cent.) are sufficient to cause someacid production.
5. Completebalance-sheets are presented for acids, carbohydrates,CO₂ andoxygen for leaves maintained in the dark at high andlow temperatures.As acids are produced there is a correspondingloss of carbohydrate(chiefly starch). A scheme of reactionsis suggested to explainthe experimental results.

     

Observations on the Gaseous Exchanges which take place between Menyanthes trifoliata L. and its Environment: I. THE COMPOSITION OF THE INTERNAL GAS OF THE PLANT

1. As part of a general investigation into the exchange of gasesbetween the bogbean, Menyanthes trifoliata, and its environment,determinations have been made of the composition of the internalatmosphere of the plant at five different levels, viz. leafyshoot, three successive portions of stem, and roots.
2. Procedureis described for the analysis of small gas samplesusing a modificationof the Bonnier and Mangin apparatus.
3. In darkness with thelower part of the plant surrounded by anatmosphere of nitrogen,it has not been possible to show thepresence of a regular downwardsgradient of oxygen, such aswould be expected on a basis ofnormal gaseous diffusion.
4. Under these Air Top/Nitrogen Bottomconditions the roots areshown to contain between 12?5 and 17?5percent, of oxygen intheir intercellular gas system. The portionof stem immediatelyadjacent to the roots showed an oxygen levelof from 14?0 to18?0 per cent.
5. In plants kept under NitrogenTop/Air Bottom conditions in thedark for 48 hours, the concentrationof oxygen in the rootsfell to a level of 3?1 per cent, or belowin three plants andto 6?o per cent, in a fourth. The oxygenconcentration in theadjacent lowermost part of the stem wasas high as 18?0 percent., depending on the degree of aerationof the solution bathingthe stem. It is suggested that thisdistribution of oxygen canbe related to the relative impermeabilityof roots and of stemendodermis to gaseous diffusion from without,and it is thoughtthat the major part of the oxygen supply tothe roots is transportedto them through the stelar air passages.
6. Some evidence is presented to show that oxygen passes fromthelower parts of the plant into the medium surrounding it.

     

The Metabolism of Glucose and Acetate in Aspergillus niger

1. The metabolism of a citric-acid-forming strain of A. nigerwhengrown on a glucose-acetate medium has been investigated.
2. Acetate greatly accelerated the rate of utilization of glucose.
3. Citric acid production from glucose was much increased bythepresence of acetate.
4. The formation of oxalate from glucose-acetatecultures was muchless than from acetate alone.
5. In some cultureslarge amounts of glucose and acetate were consumedbut no acidicproducts were formed.

     

Studies on Plant Growth Hormones: II. FURTHER BIOLOGICAL PROPERTIES OF 3-INDOLYLACETONITRILE

1. 3-Indolylacetonitrile is more active than 3-indolylacetic acidin the Avena straight-growth test, but less active in the Avenacurvature test at comparable concentrations. Reasons for thisare discussed, and results of previous work on plant extractsusing the curvature test as a means of assay are considered.
2. Transport of both the acid and the nitrile is polar, fromapexto base of the coleoptile. The nitrile can reach the growingcells as easily, and possibly more easily, than the acid. Thesignificance of these findings for a theory on the mechanismof action of the nitrile is discussed.
3. The nitrile is inactivein the pea curvature test and straight-growthof pea stem sectionsexcept at high concentrations. It is alsoinactive or only slightlyactive in lateral bud inhibition,root initiation, and petioleabscission at the concentrationstested.
4. It is less activethan the acid in root inhibition in cress,but approximatelyas active in Avena. It is approximately asactive as the acidin parthenocarpic fruit development, andinitiation of cambialactivity.
5. The significance of these results is discussed.

     

Studies in the Nitrogen Metabolism of Apple Fruits: THE CLIMACTERIC RISE IN RESPIRATION IN RELATION TO CHANGES IN THE EQUILIBRIUM BETWEEN PROTEIN SYNTHESIS AND BREAKDOWN

1. A close parallelism in the drift of the rate of respirationand the protein-N/ non-protein-N ratio is shown to occur bothin apple fruits attached to the tree and when detached fromthe tree at various stages of development and stored for severalmonths at 12 C.
2. In detached fruits the fall in respirationwhich occurs immediately(during the first 48 hours) after pickingis only accompaniedby a concomitant fall in net protein invery young fruits inwhich active cell division is taking place.Subsequently, infruit of all ages when a climacteric rise inrespiration occursit is accompanied by a net increase in protein.
3. It is argued that the climacteric rise in respiration is aresultof increase in the level of protein which will be expectedtoreduce the ATP/ADP ratio.
4. Over the climacteric period,although rate of respiration andnet protein content both rise,R rises more rapidly than proteinand, subsequently, falls ata faster rate than P. It is suggestedthat this may be due tothe ‘new’ protein containinga higher proportionof enzyme(s) directly involved in respirationand leading, forexample, to a reduction in the ATP/ADP ratio.

     

The Time Factor in Studies of Growth Inhibition in Excised Organ Sections

1. Apprehension over the adequncy of current techniques stimulateda detailed study of the time factor in the arsenate inhibitionof growth and respiration in excised stem and root sectionsof Pisum sativum.
2. Growth inhibition by arsenate sets in veryslowly, its rateof onset being related to the molar concentration(C) of arsenateate by the relation where T₅₀ is the time taken in hours to reduce the growthrateto 50 per cent of the control and K is a constant. An explanationof the physiological basis of this relationship is attempted.
3. Estimates were made of the final steady growth rate (relativeto control) in various arsenate concentrations. The inhibitionscalculated from this rate are held to approximate to the truearsenate effect and are shown to be very different from thosecalculated from ‘total growth’ measures.
4. Respirationof growing stem sections is not inhibited by thelow arsenateconcentrations that inhibit growth. Some inhibitionis indicatedat high concentrations (3 ? 10^–4M. and over)but onlyafter 15-20 hours of exposure.
5. Two per cent sucrose has noeffect on the arsenate inhibiitionof stem growth. Sucrose,however, markedly stimulates respirationin stem sections, butthis stimulation is prevented by arsenate.
6. The misinterpretationswhich may arise as a result of ignoringthe time factor in inhibitionstudies in excised organ sectionsare discussed and the desirabilityof constructing completegrowth curves in all such studies isstressed.

     

Factors concerned in the Rooting Responses of Isolated Leaves

The rooting responses of isolated leaves of the dwarf Frenchbean and the ivy were studied by applying solutions of growthsubstances to the petiole: ß-indolyl-butyric acid(I.B.A.) for the former and -naphthyl-acetic acid (N.A.A.) forthe latter. The following factors were investigated:

1. The variation in response of successive leaves from the apextowards the base of the plant.
2. The optimal concentrationsof the growth substances and therelation between duration ofapplication and concentration.
3. The relation of age of theleaf and optimal concentration(ivy).
4. The nutritive factorsconcerned in the response were studied,(a) by varying periodsof contact between lamina and petiole,(b) by varying durationof light, (c) by varying the amountof light, and (d) by feedingwith sucrose and asparagine.

The most responsive leaf in the bean was the 2nd from the apex,in the ivy the 9th leaf. Very young leaves were killed. Withpetioles immersed for 24 hours the optimal concentrations wereI.B.A. at 2·5 parts per million (p.p.m.) for the bean,and N.A.A. 100 p.p.m. for the ivy. It was shown that the activesubstance entered mainly through the cut end in the transpirationstream; entry through the cuticle was much slower. Starvation of the cuttings whether by low light intensity, darkness,or separation of the lamina from the petiole reduced root formation.Feeding with sucrose and asparagine increased the response. Analysis of the leaves was carried out at regular intervalsfor total sugar, total and soluble nitrogen. In the bean solublenitrogen increased slightly in the petiole during the first5 days after treatment, after which insoluble nitrogen continuedto accumulate until the 7 day, when the roots first emerged.After this the total nitrogen decreased. Sugar content of thepetiole at first increased but rapidly fell before the rootsemerged and then again increased. In the ivy petiole the increasein soluble and total nitrogen was very slow, the former beinggreatest on the 15th day and the latter on the 20th day aftertreatment. The total sugar increased to the 10th day, but thendecreased until the emergence of roots, after which it againincreased. Treated leaves of the bean and ivy of different agesand the leaves starved for varying periods were also analysedfor total sugars and total nitrogen. Treated bean leaves receivingvarying periods of daily illumination were also analysed toelucidate their rooting response. Histological examination showed that in the bean the roots arosein the rays between the vascular bundles and in the ivy externalto the vascular bundles opposite the phloem. The growth of the rooted leaves was followed for more than amonth in the bean and over one year in the ivy. Some growthwas found in the immature bean leaves which survived treatment,but the final size was much below that of the control leavesattached to the plant. In the ivy very little further growthoccurred although the leaves produced a large root system andafter 2 years are still alive: the original differences in sizeof the successive leaves at the time of removal from the plantstill remain.     

FLAVOPROTEIN IN CHLORELLA ELLIPSOIDEA CATALYZING TPNH-LINKED REDUCTION OF PLASTOCYANIN

1. An enzyme possessing a capacity of catalyzing reduction of thecopper protein, plastocyanin, with reduced pyridine nucleotides(TPNH-plastocyanin reductase) was isolated from Chlorella ellipsoidea.The procedures of purification and the properties of the purifiedenzyme are described.
2. From the results of chromatographicand enzymic tests, the prostheticgroup of the enzyme was identifiedas FAD. No evidence was obtainedto indicate the participationof metal ions in the reaction.
3. The enzyme utilizes both TPNHand DPNH as electron donors, butthe reaction is about 12 timesfaster with TPNH than with DPNH.
4. The enzyme, with TPNH aselectron donor, catalyzes the reductionof Chlorella plastocyanin,cytochrome c, 2,6-dichlorophenolindophenol and oxygen in adecreasing order of reaction rate.The reaction with oxygenas electron acceptor was found to bemuch more strongly acceleratedby the addition of higher concentrationsof flavins as comparedwith the reaction with other acceptors.FAD and FMN added tothe reaction mixture are not appreciablyreduced.
5. The propertiesof the enzyme are compared with those of alliedchloroplastenzymes reported by various investigators and thepossible roleof the new enzyme in photosynthesis is discussed.

(Received January 18, 1961; )     

Further Studies on the Acid Metabolism of Aspergillus niger: THE FORMATION AND UTILIZATION OF OXALIC ACID

1. Some recent works on the formation of oxalic acid by variousfungi are critically considered.
2. The present work deals withthe role of oxalic acid in the metabolismof Aspergillus niger.
3. When glucose solutions were supplied to preformed mats ofthefungus oxalic acid accumulated, attaining an equilibriumlevelwhich was not exceeded despite the presence of a considerableconcentration of glucose.
4. When the glucose supplies were depletedthe oxalic acid concentrationfell steeply to a low level.
5. Theconcentration of oxalic acid was dependent on the glucoseconcentration.In three separate series of experiments it wasshown that theoxalic acid concentration diminished with increasingglucoseconcentration.
6. Similar results were obtained when the cultureswere rearedfrom spores on culture solutions with the normalamounts ofnutrient salts but different glucose concentrations.
7. In all cases the CO₂ output increased with the glucose concentration.
8. When cultures were supplied with glucose+oxalic acid, theconcentrationof the latter fell steeply to the equilibriumlevel attainedon glucose only. In a culture receiving glucose+oxalicacid,with the oxalic acid concentration somewhat below thenormalequilibrium concentration, the formation of oxalic acidfromthe glucose ceased as soon as the equilibrium level hadbeenattained.
9. When 1 per cent. oxalic acid only was suppliedto the fungusthe concentration gradually diminished to a lowlevel. When3 per cent. oxalic acid was supplied the rate ofacid utilizationsoon fell to low value.
10. In several experimentsit was shown that the rate of CO₂ outputwas higher from culturessupplied with glucose+excess oxalicacid than from culturessupplied with glucose only.
11. The rate of oxalic acid carbonloss was always below that ofthe CO₂ carbon output both incultures supplied with oxalicacid only and in cultures receivingglucose+oxalic acid.
12. The cultures were incapable of utilizingneutral sodium oxalateand the presence of this substance hadno effecft on the ofCO₂ output.
13. The results indicate thatthe utilization of oxalic acid isassociated with the liberationof at least an equivalent amountof CO₂.
14. It is suggested thatthe utilization of oxalic acid is promotedby the presence ofglucose, thus accounting for the lower oxalicacid concentrationsand higher rates of CO₂ output of cultureswith higher glucoseconcentrations.

     

PHOTOOXIDATION REACTIONS BY CHLOROPLASTS SOLUBILIZED WITH SURFACE-ACTIVE AGENTS

1. Solubilization of chioroplasts with a mixture of 1 per centDuponol C and 1 per cent Span 80 (3: 1) caused a destructionof activity in the HILL reaction, but the treatment broughtabout an increase by about 60 per cent in the rate of ascorbatephotooxidation in the presence of DPIP. Heating the broken chloroplastscaused a marked decrease in the photooxidation activity. Byadding surface- active agents to the boiled preparation, theactivity was restored up to almost 80 per cent of the originallevel.
2. With colloidal suspensions of isolated chiorophylls,ascorbatewas only slightly photooxidized in the presence ofDPIP. Byaddi tion of the surface-active agents, the activitywas greatlyenhanced.
3. Dependency of the photooxidation bywhole and solubilized chloroplastsand isolated chlorophylla on the presence of DPIP was examined.DPIP can serve as anintermediate electron carrier in solubilizedchloroplasts aswell as in whole chloroplasts.
4. Effect of o-phenanthrolineon ascorbate photooxidation by thesethree preparations wastested. With solubilized chloroplastsand isolated chlorophylls,the addition of the inhibitor hadno influence on their ascorbatephotooxidation either in thepresence or absence of DPIP.
5. Treatmentof whole chloroplasts with the surface-active agentsinducedan activity of photooxidation of cytochrome c. The electron-flowpattern for the photooxidation of ascorbate by whole and solubilizedchloroplasts was briefly discussed.

¹ Contribution No. 130 from the Department of Biology, Facultyof Science, Kyushu University. Aided in part by Grant-in-Aidfor Fundamental Scientific Research from the Ministry of Education. (Received August 23, 1962; )     

Microbiological Synthesis of Fat: PRELIMINARY SURVEY OF THE FAT-PRODUCING MOULDS

1. A survey of potential fat-producing moulds has shown that atleast 40 strains from 10 species are of interest.
2. These havebeen grown on five different media and A. nidulans,P. spinu-losum,P. javanicum, P. piscarium, P. flavo-cinereum,P. oxalicum,A. flavus, A. flavipes have shown the most promise.
3. The fatcontent on felt weight was maximal at 39?7 per cent,with A.flavipes and 34?6 per cent, with F. lini, and on usedsugarwas maximal at 9?3 per cent, with A. flavipes and 6?7per cent,with A. nidulans.
4. The potential value of micro-organisms asfat producers is discussedwith reference to the moulds.

     

COMPONENTS OF THE ELECTRON-TRANSFERRING SYSTEM IN ACETOBACTER SUBOXYDANS AND RECONSTRUCTION OF THE LACTATE OXIDATION SYSTEM

1. Cytochromes a₁⁵⁹⁰, b⁵⁶⁰, c₁⁵⁵⁴ and c₁⁵⁵² were isolated andpurifiedfrom a strain of Acetobacter suboxydans. The proceduresusedwere described in detail.
2. The main cytochrome band at550-560 mµ in intact cellssplitted at liquid air temperatureinto two bands, 551 mµ(strong) and 559 mµ (weak).
3. Optical and physiological properties of the four cytochromeswere investigated. Lactic dehydrogenase activity was found tobe associated with cytochrome c₁⁵⁵⁴. The two c₁-type cytochromes,especially cytochrome c₁⁵⁵⁴, persisted in their reduced formafter the purification through many steps.
4. By some combinationsof isolated components reconstruction ofthe oxygen uptake systemcould be realized.
5. The oxygen-consuming activity of purifiedoxidase preparationswas accelerated by a-tocopherol but notby Emasoll 4130 andTween 80.
6. Some discussions were made onthe nature of terminal oxidase,the role of cytochrome c₁⁵⁵²in the electron-transport system,and persistence of reducedstate of c₁-type cytochromes.
7. A possible scheme of the electron-transferringsystem of Acetobactersuboxydans was presented.

(Received May 16, 1960; )     

Biosynthesis of alicyclic acids from 14C-labeled glucose and erythrose by isolated cells from Vigna angularis leaves

Mesophyll cells isolated enzymatically from Vigna angularisleaves were fed ¹⁴Cglucose or ¹⁴C-erythrose and the time-courseof ¹⁴C incorporation into shikimic and quinic acids was examined.When ¹⁴C-glucose was fed to the cells, the highest radioactivityin quinic acid was observed after 10 hr of incubation, whilethat in shikimic acid was after 14 hr. In the experiment with¹⁴C-erythrose, the radioactivity in shikimic acid rose strikinglyup to the 3rd hour, but ¹⁴C in quinic acid increased graduallyduring the incubation. The incorporation of ¹⁴C into shikimicacid was enhanced when unlabeled shikimic or quinic acid wassupplied to the cells simultaneously with either ¹⁴C-glucoseor ¹⁴G-erythrose, whereas that into quinic acid was not significantlyincreased by these alicyclic acids. The difference in incorporationrate of ¹⁴C into quinic acid from that into shikimic acid wasmore conspicuous in the isolated mesophyll cells than in theepicotyls of V. angularis seedlings. ¹ Present address: Department of Biology, Faculty of Science,Kumamoto University, Kumamoto 860, Japan. (Received September 22, 1978; )     

Gametogenesis and Embryogeny of Tamarix ericoides Rottl

1. The haploid chromosome number in Tamarix ericoides Rottl., reportedfor the first time, is twelve.
2. The archesporial cell is single,rarely double.
3. There is no tetrad formation, division of themegaspore mother-cell;instead, a four nucleate embryo-sac results.
4. The nuclei are originally arranged in a cruciform type, butlater form a I+3 arrangement.
5. As a result of the third division,the embryo-sac is again four-nucleate,with two haploid nucleiat the micropylar end and two triploidnuclei at the chalazalend.
6. The cells of the embryo-sac are formed at the end ofthe fourthdivision, the embryo-sac thus representing the sixteennucleatecondition.
7. The development of the embryo-sac of Tamarixericoides conformsto the Fritillaria-type.
8. The endospermis free nuclear, and the embryo is characteristicin developinga massive suspensor and a large pad of cellulartissue fromthe proximal cell.
9. Double embryo-sacs and the occurrence ofpolyembryony are reportedfor the first time in the family.

     

STUDIES ON THE MECHANISM OF GROWTH INHIBITING EFFECT OF LIGHT

1. A substance which inhibits indoleacetic acid (IAA)-and naphthaleneaceticacid (NAA)-induced elongation of Avena coleoptile section andIAA-induced Avena coleoptile curvature was found in an ethersoluble neutral fraction of water extract of sunflower leavesand in agar blocks containing the diffusate from young sunflowerleaves.
2. This substance also inhibits the growth of isolatedsunflowerepicotyl.
3. The R_f value (0.9) of the substance ona paper chromatogramdeveloped with ammoniacal iso-propanolindicates that it isidentical with the inhibitor reported byAUDUS et al. (1956),but not with inhibitor-ß.
4. Theinhibitor can be transported from leaf to stem, and thetransportseems to be accelerated by illuminating the leaf.
5. The auxindiffused from sunflower leaf into agar block may beidenticalwith IAA.
6. A substance, which has the same properties as theinhibitorfrom sunflower leaf, was obtained in crystalline formfrom theleaf of Jerusalem artichoke.
7. The mechanism of growthinhibition caused by this crystallinesubstance seems to involveinactivation of a sulfhydryl group.
8. The reason why the stemgrowth of sunflower seedlings is reducedby strong light isdiscussed: the amount of the inhibitor transportedfrom leafto stem is increased under strong light, and in thestem, growthinhibition is caused by a direct effect of thisinhibitor ongrowth and by its inhibiting effect on the transportof IAAfrom leaf to stem.

¹ Present address: Botanical Garden, Faculty of Science, Universityof Tokyo, Tokyo (Received February 15, 1961; )     

The Effect of Prolonged Chilling on Water Movement and Radial Growth in Trees

1. The woody shoots of young saplings of Fraxinus.excelsior andAcer Pseudo-platanus in pots were subjected to continuous coolingto about 2° C. during the growth season, with the resultthat radial growth was almost completely inhibited throughoutthe woody stem.
2. The chilling did not adversely affect extensiongrowth exceptthat it was later in commencement and proceededmore slowly.
3. If the temperature around the stem is loweredfrom 2° C.to 0° C., water conduction is cut down tosuch an extentas to cause wilting of the leafy shoots; turgidityis recoveredwhen the temperature is again raised to 2°C.
4. This wilting effect is discussed particularly in relationtothe part played by living cells in the upward movement ofwaterin the wood.

     

The 'Diffuse' Centromere or Polycentric Chromosomes in Spirogyra

1. Among ten species of Spirogyra types are distinguished withlarge-, medium-, and small-sized chromosomes.
2. Evidence ofcompound structure is presented with regard to thelarge-andmedium-sized chromosomes.
3. Parallel separation of chromatids,first described by Geitlerfor S. crassa, is found in all specieshaving large chromosomesand to some degree in those with medium-sizedchromosomes.
4. It is suggested that the parallel separationof chromatids iscaused by their polycentric nature.
5. Polyploidyin the genus is only doubtfully comparable with thatin thehigher plants.
6. It is suggested that in Spirogyra the localizedcentromere mayhave evolved in a polycentric chromosome.
7. Comparisonsare drawn between Spirogyra and other organismsstated to have‘diffuse’ centromeres and it is suggestedthat theirchromosomes also are polycentric.

     

Stereospecificity of hydrogen transfer by pyridine nucleotide-dependent enoyl reductases in fatty acid synthesis: Studies with enzymes obtained from developing castor bean seeds and Chlorella vulgaris

The mechanism of hydrogen incorporation into fatty acids wasinvestigated with fatty acid synthetase systems from developingcastor bean seeds and Chlorella vulgaris. Fatty acids synthesizedin the presence of D₂O or stereospecifically deuterium-labeledNADPH or NADH were isolated and analyzed by mass spectrometryto examine the localization of deuterium atoms in the molecule.The stereospecificities of ß-ketoacyl-acyl carrierprotein (ACP) reductase and enoyl-ACP reductase for reducedpyridine nucleotide were determined with acetoacetyl-ACP andcrotonyl-ACP as substrates. The products were also analyzedby gas chromatography-mass spectrometry. The following resultswere obtained:

1. ß-Ketoacyl-ACP reductases from both castor bean seedsand C. vulgaris used the B-side hydrogen of NADPH.
2. Enoyl-ACPreductase from C. vulgaris required NADH for the activity.
3. Enoyl-ACPreductase from castor bean seeds used the A-side hydrogenofNADPH, whereas that from C. vulgaris used the B-side hydrogenof NADH.
4. When stearate was synthesized with the crude fattyacid synthetasefraction from castor bean seeds, hydrogen atomsfrom water werefound on the even-numbered methylene carbonatoms (two hydrogenatoms per carbon atom) and some were foundon the odd-numberedmethylene carbon atoms. Hydrogen atoms fromthe B-side of NADPHwere found on the odd-numbered methylenecarbon atoms (one hydrogenatom per carbon atom). Hydrogen atomsfrom the A-side of NADPHwere also found on the odd-numberedmethylene carbon atoms,but the number of incorporated hydrogenatoms was less thanexpected.

(Received October 17, 1979; )