CHANGES IN THE RESPONSE OF CARROT ROOT CALLUSES TO THE ROOT EXTRACT OCCURRING DURING THEIR SUCCESSIVE CULTURES

1. Alcohol extract of carrot root promoted the growth of the carrotroot callus which had been succesively cultured for more than18 months (CCL) on the medium containing WHITE'S inorganic salts,sucrose, yeast extract and 2, 4-D, but only a weak promotionwas observed for the growth of the carrot root callus whichhad been cultured for less than 14 months (CCS).
2. The activesubstances were fractionated by Amberlite IR-120and AmberliteIRA-400 into four fractions; C, D, E, and F. Eachfraction seemedto act synergistically to produce the effectof the whole carrotroot extract on the growth of CCL.
3. Fraction F of the carrotroot extract, which was adsorbed byAmberlite IRA-400 but notby Amberlite IR-120, promoted thegrowth of CCL in the presenceof other fractions, but had noeffect on the growth of CCS.So the different responses to thealcohol extract of the carrotroot calluses having differentlengths of successive cultureperiod seemed to depend mainlyon the ability of respondingto fraction F.
4. Using four strains of carrot root callusesof different origin,it was ascertained that different responsesof carrot root callusesto fraction F depended on the lengthof their culture and noton their strain-specific characters.
5. The substances active for the growth of CCL in the carrotrootextract passed through a dialysis membrane. These substanceswere little affected by autoclaving and remained in the aqueouslayer when shaken with several organic solvents: n-butanol,ethyl acetate, chloroform, benzene, ethyl ether and carbon tetrachloride.
6. Alcohol extract of carrot root also promoted the growth ofcarrotroot explant, tobacco stem callus and sunflower crowngall tissue.

(Received December 24, 1964; )     

Absorption and Assimilation of Inorganic Nitrogen from Different Sources by Storage Root-Tissue

1. The rate of ammonium and nitrate absorption and assimilationby storage tissues of sweet potato tubers, radish, and carrotroots cultured in aerated dilute solutions (5 mg. eq./1.) ofNH₄Cl, KNO₃ or NH₄NO₂ at 25° C. and the effect of the sourceof nitrogen on the nitrogenous composition of the tissues werestudied.
2. Both ions were absorbed at more or less equal ratesunder theprevailing experimental conditions by cells of thedifferentplant materials used.
3. The rate of nitrate assimilationwas lower than absorption,leading to the accumulation of nitrate-Nin the cells of alltissues examined. Ammonium-N, on the otherhand, was assimilatedalmost as soon as it entered the cellsof sweet potato tubersand carrot roots, and there was onlya small increase in thelevel of arnmonium-N; but in radishroot cells ammonium assimilationlagged very much behind absorption,resulting in a great increasein the level of arnmonium-N.
4. Sweetpotato tubers and carrot tissues were shown to be adaptedtothe utilization of more ammonium than nitrate nitrogen, butradish root tissues are adapted to the utilization of more nitratenitrogen.
5. Ammonium or nitrate-N caused similar increases inthe complexorganic-N level (protein-+rest-N) in radish root,but the rateof synthesis was higher with ammonium in sweetpotato tubersand with nitrate in carrot roots.

Furthermore, with both sources changes in the content of amideand amino-N were negligible in radish tissues of both series.In contrast to radish, carrot tissues showed marked increasesin the two fractions. The increase of these fractions in sweetpotato tuber tissues was much lower. The marked contrast in nitrogenous composition in the threedifferent plant materials fed with ammonium and nitrate saltsseparately indicated that the actual course of protein metabolismis most probably dependent on the source of nitrogen utilized.     

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; )     

CHANGES IN ORGAN FORMING CAPACITY OF CARROT ROOT CALLUSES DURING SUBCULTURES

1. The formation of buds and roots in seven strains of carrotrootcallus successively cultured for various periods on a mediumcontaining WHITE'S inorganic salts, sucrose, 2,4-D and yeastextract was investigated. 2,4-D completely suppressed organformation during stock subculturing. It was confirmed that theorgan forming capacity of the callus in a 2, 4-D-free test mediumdiminishes and finally completely disappears with prolongedperiods of previous subculturing of the callus.
2. IAA promotedthe root formation. Yeast extract, casein hydrolysateand aminoacids mixture promoted the bud formation of callusesat earlieststeps of subculturing (Phase I).
3. At next steps of subculturing(Phase II), IAA-dependent rootforming capacity of calluseswas lost although the bud formingcapacity induced by yeastextract, casein hydrolysate and aminoacids mixture was retained.
4. At further advanced steps of subculturing (Phase III), yeastextract induced only root formation, while casein hydrolysatestill could induce buds. IAA and amino acids mixture did notaffect the organ formation.
5. No organ formation was observedin calluses subcultured over38 months under any conditionsattempted (Phase IV).
6. Single cells or small cell clumps obtainedfrom the callus subculturedfor 2 months formed only roots onthe medium containing IAAand formed buds and roots on the mediumcontaining yeast extract.
7. These differences in organ formingcapacity and in responsestowards various factors are interpretedto reflect the changesin physiological states of the callusduring successive cultureson the stock culture medium.

(Received December 24, 1964; )     

EFFECT OF LIGHT ON THE CHLOROPHYLL FORMATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. Previous studies have shown that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), apparently chlorophyll-less cells with profoundly degeneratedplastids—referred to as "glucose-bleached cells—areproduced either in the light or in darkness. When the glucose-bleachedcells are incubated in a medium enriched with the nitrogen sourcebut without added glucose, an active formation of chlorophylloccurs after a certain lag period under illumination, whilein darkness a very small amount of chlorophyll is formed atabout the same time as in the light. The stimulating effectof light on the chlorophyll formation is not appreciably affectedwhen the photosynthetic CO₂-fixation of greening algal cellsis blocked by the addition of CMU. In the present study, itwas further found that the light-enhanced chlorophyll formationproceeds, although at a somewhat lower rate, under aerationof CO₂-free air. All the experiments in this work were doneunder these non-photosynthetic conditions to exclude any influenceof photosynthates.
2. The effect of light (from daylight fluorescentlamps) on thechlorophyll formation in the glucose-bleachedalgal cells wassaturating at about 1,000 lux. Blue light wasfound to be mosteffective; yellow, green and red light followingin the orderof decreasing effectiveness.
3. When the bleachedalgal cells were illuminated for a short periodin the lag phaseof chlorophyll formation and subsequently incubatedin darkness,there occurred an appreciable enhancement of chlorophyllformationin the dark. When the short illumination was appliedat differenttimes of the lag phase, the enhancement was inducedto almostthe same extent. But the longer the duration of theilluminationduring the lag phase, the greater was the enhancementof chlorophyllformation in the subsequent dark incubation.In such experimentsblue light was most effective and red lightleast, as it wasthe case in the experiments of continuous illumination.An intervenientillumination of the bleached cells at lowertemperatures orunder the atmosphere of N2 produced little orno enhancementof the chlorophyll formation in the subsequentdark incubation.
4. Based on these results, it was concluded that the light enhancementof chlorophyll formation in the glucose-bleached algal cellsis mediated by a non-chlorophyllous photoreceptor(s), absorbingmaximally blue and yellow light, and that a light-induced changeof the photoreceptor is immediately followed by a certain dark(temperaturedependent and aerobic) process(es) which is connected,directly or indirectly, to the chlorophyll synthesis.

(Received August 10, 1967; )     

MODE OF NUCLEAR DIVISION IN SYNCHRONOUS CULTURES OF CHLORELLA: COMPARISON OF VARIOUS METHODS OF SYNCHRONIZATION

1. Chlorella ellipsoidea was grown synchronously using variouspossible techniques and the mode of nuclear division in eachcase was followed by staining the nuclei according to FEULGEN.
2. A satisfactory synchrony in respect to nuclear and cellulardivision was obtained by starting the culture from a homogeneouspopulation of young and small cells and by discontinuing theillumination at the stage which was called the L₃-stage. Thestarting young cells were invariably mononuclear and the L₃-cellswere either dinuclear or tetranuclear. When the L₃ were incubatedin the dark, they ripened further, and after passing througha tetranuclear stage (referred to as the L₄) divided into fourmononuclear daughter cells which have been called the D_n-cells.The most clear-cut and repetitive synchronous culture was obtainedwhen the culture (in the light) was started from the D_n-cellsand the illumination was discontinued at the L₃-stage untilthe fully ripened cells divided into four each of D_n-cells.
3. An apparently "synchronous" culture was also obtained by themethod of programmed light-and-dark regimen, in which a randomculture is subjected to a regular alternation of light and darkperiods of adequate durations. In this case, however, the cellsat different stages of culture showed irregular nuclear patterns,and the average "division number" of mother cells was not constant,being subject to change between 4.0 and 4.9.

(Received May 25, 1961; )     

Changes in respiratory metabolism in tissue cultures of carrot root

Changes in respiratory metabolism accompanied by callus formationin cultured explants of carrot root were followed and the followingresults were obtained. 1) When the explant was cultured on amedium containing kinetin and 2,4-D, active cell division occurredand resulted in callus formation by the 9th–12th days.2) Fresh weight remarkably increased after a lag-time of about5 days. Changes in protein content on fresh weight basis weresimilar to changes in fresh weight. 3) Respiration rate increasedduring the first few days, when growth could not be distinctlymeasured. Accompanying the rise in respiration, the C₆/C₁ ratioalso increased. As callus developed, the respiratory rate andC₆/C₁ ratio gradually decreased and RQ, became higher than unity.4) Alcohol dehydrogenase activity increased between the 4thand 9th days after culture. 5) When sub-cultured callus tissuewas fed with G-U-¹⁴C, some radioactivity was detected in thealcohol of the tissues. 6) These results suggest that duringthe first 4–6 days after culture the activity of the EMBDEN-MEYERHOF-PARNAS-TCApathway was remarkably increased and, as callus developed, therelative participation of the pentose phosphate pathway graduallyincreased and simultaneously alcohol fermentation occurred. (Received December 13, 1968; )     

The Effects of Gibberellins on the Growth of Excised Tomato Roots

1. At appropriate concentrations both gibberellic acid (GA) and1-naphthalene-acetic acid (NAA) enhance the main axis growthof excised tomato roots grown in culture media containing sucroseat concentrations below 1 per cent. Lateral root extension growthis enhanced by GA at all sucrose concentrations tested; onlyat the lower sucrose concentrations is this effect observedwith NAA. Both GA and NAA increase the number of emergent lateralroots and this effect is most marked in media of low sucrosecontent. Both GA and NAA at higher concentrations inhibit rootgrowth but NAA exhibits its full range of growth effects overa much narrower concentration range than GA.
2. GA, like NAA,speeds up the loss of meristematic activity whichoccurs whenindividual meristems are repeatedly subculturedin media containing1 per cent, or higher concentrations ofsucrose.
3. The promotionof main axis growth by both GA and NAA involvesenhanced cellelongation and cell division. At a moderatelyinhibitory concentrationGA reduces both cell elongation andcell division; this is notthe case with NAA.
4. Gibberellins A₁, A₂, and A₄ resemble GA(gibberellin A₃) intheir growth effects. Allogibberic acidlike G A promotes lateralroot extension growth but causes markedinhibition of root growthat a much lower concentration thanGA.

     

EFFECT OF ULTRAVIOLET LIGHT UPON VARIOUS PHYSIOLOGICAL ACTIVITIES OF CHLORELLA CELLS AT DIFFERENT STAGES IN THEIR LIFE CYCLE

1. Using Chlorella ellipsoidea as material, investigations weremade of the effects of ultraviolet irradiation upon variousactivities of cells at different developmental stages in theirlife cycle. Cell activities investigated were photosynthesis,respira tion, over-all growth, modes of synchronous growth andcell division as well as the formation of nucleic acids. Theu. v.- light applied was 30 µµW/cm²in intensityand 2537 Å in wavelength.
2. The most u. v.-sensitive wasthe over-all growth activity, andin this respect the irradiationapplied at the L₂-stage wasmore inhibitive than that givenat the D-stage. The next mostvulnerable was the photosyntheticactivity, the sensitivitybeing the same in the D- and L-cells.The most resistant towardu.v. was the endogenous respirationof D-cells followed by theirrespiration using exogenous glucoseas substrate. The L₂-cellsappeared to be unable to use exogenousglucose as substrateof respiration, but their endogenous respirationwas considerablystronger than that of D-cells, and its u. v.-sensitivitywasthe same as that of glucose respiration of D-cells.
3. WhenD-cells were u. v. irradiated immediately before the startofsynchronous culture, growth and cell division as well astheformation of DNA and RNA were retarded in proportion totheu. v.-dose applied. The division number (n) was normal (around4) at lower u.v.-doses (1-2 minute irradiation), but was reducedto a half (about 2) at a higher dose.
4. When, during the synchronousculture, 1-minute u.v.- irradiationwas applied at various stagesof the ripening phase, the divisionwas retarded, but the cells,after attaining an abnormally largesize, divided into about8. If the irradiation was given atthe L₄-stage, the divisionnumber was practically unmodified(n=4.5), although the divisionwas somewhat retarded comparedwith that of the control culture.When a 1-minute irradiationwas given at the L₂-stage, thereoccurred an apparent stimulationof DNA- and RNA-formation,a phenomenon which corresponds tothe production of a largernumber of daughter cells than itwas the case in control cultures.
5. Thus the cells which were moderately u.v.-irradiated at differentstages of synchronous culture were able to complete their lifecycle, but later a certain portion of irradiated cells becameunable to grow normally.

¹Present address: Department of Biochemistry, Dartmouth MedicalSchool, Hanover, New Hampshire, U.S.A. (Received March 6, 1961; )     

Physiological activity of a viridicatin derivative, 3-(4-phenylcarbostyriloxy) acetic acid

A carboxymethylene derivative (V-OCH₂COOH) of viridicatin (V-OH)promoted the root growth of rice and sesame seedlings. V-OCH₂COOHhad no known hormonal activities, per se, but did have an inhibitoryeffect on IAA and 2,4-D-induced growth of Avena coleoptile sectionsand of carrot root callus. However, inhibition by VOCH2COOHof 2,4-D-induced growth in carrot root callus was to some extentreversed by increasing the concentration of 2,4-D. V-OCH₂C0OHseemed to competitively inhibit IAA-induced elongation of Avenacoleoptile sections. (Received September 14, 1970; )     

INFLUENCE OF INORGANIC NITROGENOUS COMPOUNDS ON TOBACCO SEED GERMINATION INDUCED BY GIBBERELLIN A3, KINETIN AND AMMONIUM SALTS OF ORGANIC ACIDS

1. Investigations were made on the influence of inorganic nitrogenouscompounds upon the the germination of tobacco seeds (Nicotianatabacum L. var. virginica (AGDH.) COM. "Bright Yellow") inducedby GA₃, kinetin and ammonium salts of organic acids. Potassiumnitrate, ammonium chloride, ammonium sulfate and ammonium nitrategreatly increase the germination of the seeds induced by theabove reagents, while these inorganic salts, given alone, arealmost ineffective in causing germination.
2. Kinetin was shownto induce germination of tobacco seeds inthe dark. The discrepancywith the results of previous investigationsin this respectwas discussed.
3. It was inferred that nitrogenous metabolismis involved in theprocess of dark-germination of tobacco seedsas induced by theabove-stated stimulating factors and promotedby inorganic nitrogenoussubstances.

(Received July 17, 1961; )     

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURES

1. The longer the period of stock culture, the more remarkableis the growth inhibition by 8-azaguanine in callus.
2. Chloramphenicol,5-methyltryptophane and mitomycin C exert greaterinhibitionon growth in CCL than in CCS.
3. Bud formation is inhibited bysome concentrations of chloramphenicolwithout accompanyinginhibition of the growth.
4. Cell size and the contents of RNA,DNA, protein and lipid percell of CCL are greater than thoseof CCS, respectively. Thecontents per cell of RNA and lipidin "mitochondrial fraction"are higher in CCL than in CCS.
5. Incorporationof guanine-8-¹⁴C into RNA of CCS occurs rapidlyin the first12 hr and slows down thereafter, but that in CCL-RNAincreasessteadily for 16 hr. This difference in rate of theincorporationafter 12 hr between CCS and CCL is principallydue to the differencein rate of the incorporation into RNAof nuclear, mitochondrialand soluble fractions.

1. The rate of RNA breakdown in CCL wasnot so great as the rateof synthesis.
2. 8-azaguanine (10^–3and 10^–4M) inhibits incorporationof guanine-8.¹⁴C intoRNA of both CCS and CCL during 14 hr,but thereafter (up to25 hr) it inhibits the incorporation intoCCL-RNA alone leavingthat into CCS-RNA unaffected.

1. In CCL 510^–5M 8.azaguaninedoes not affect total radioactivityincorporated into bulk RNA,but inhibits incorporation intoRNA of "mitochondrial fraction".

(Received December 23, 1964; )     

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.

     

EFFECT OF CHROMATIC LIGHTS ON PHYCOBILIN FORMATION IN A BLUE-GREEN ALGA, TOLYPOTHRIX TENUIS

1. The formation of phycobilin pigments in a blue-green alga Tolypothrixtenuis was investigated with special reference to the effectsof preillumination with colored lights.
2. It was discoveredthat the algal cells are capable of formingphycobilin pigmentsin the dark, if they have been previouslyilluminated for severalhours in the presence of CO₂.
3. The color of light applied inthe later period of preillumination(chromatic illumination)was found to affect the ratio of phycoerythrinto phycocyaninformed in the subsequent dark period. A greenlight acceleratesthe dark-formation of phycoerythrin, a redlight that of phycocyanin,and the two lights counteractingwith each other in their effects.
4. These directive effects of the "chromatic illumination" canbe accomplished within a very short period, for instance, in3 minutes if it is preceded by sufficient "preillumination"with an incandescent or day light fluorescent light. The reactionsoccurring during the period of chromatic illumination does notrequire the presence of CO₂ and the aerobic condition.
5. Thealga can be grown heterotrophically when supplied with casaminoacids and glucose. Under such a condition the alga forms phycocyanintogether with chlorophyll and carotenoids, but not phycoerythrin.
6. On the basis of the results obtained, a tentative scheme forthe biosynthesis of phycobilin pigments in the alga was proposed,assuming the light-induced formation of unknown precursors whichare converted into phycocyanin and phycoerythrin in the subsequentdark period.

(Received July 4, 1960; )     

PARTIAL PHOTO-REPRESSION OF THE GLYOXYLATE BY-PASS IN EUGLENA

1. Addition of exogenous acetate or ethanol to autotrophic culturesof Euglena gracilis strain Z induces formation of the glyoxylateby-pass.
2. Visible light decreases the activity of malate synthasein greenEuglena by about 50%. No such effect was found in apermanentlybleached mutant.
3. Aconitase activity parallelsthat of malate synthase, but isocitricdehydrogenase activityis constant under all conditions examined.
4. Oxygen consumptionis proportional to the activities of malatesynthase and aconitase,but not to that of isocitric dehydrogenase.
5. The results ofsimilar studies with other growth substrates(pyruvate, malate,succinate) suggest that some of the oxygenconsumed by C₂-grownEuglena may not be associated with energyproduction.

(Received March 25, 1966; )     

Measurement of Stomatal Aperture by Digital Image Processing

We developed a new digital image processing technique for exactlymeasuring the degree of stomatal opening, that is, the ratioof the width to the maximum length of a stomatal pore, and thepore area. We applied this technique to evaluate responses toSO₂ of neighboring stomata in a small region of an intact attachedleaf, with the following results:

1. The pore region could be exactly extracted even when the originaldigital image was of poor quality. The standard errors in theevaluation of the pore area, the pore length, and the pore widthwere 10 µm², 0.3 µm and 0.3 µm, respectively.
2. A wide variety of responses to SO₂ was found among neighboringstomata in a small region of an attached leaf. The differenceswere especially remarkable before water-soaking and wilting.When these began, k₁ showed a local maximum value, and thenall the stomata began to close. These results suggest that stomatalresponses of intact growing plants to air pollutants shouldbe examined from the standpoint of not only the average movementof many stomata but also the movement of each stoma and thatthe interrelationship among movements of neighboring stomatashould be investigated to clarify the mechanism of stomatalresponses to air pollutants.

(Received June 4, 1984; Accepted August 23, 1984)     

Culture of Clostridium pasteurianum in Defined Medium and Growth as a Function of Sulfate Concentration

Clostridium pasteurianum strain W-5 was selected as an anaerobe which may be grown from large inocula in defined media with sulfate as its primary sulfur source. Since it is important to keep inocula small in minimizing transfer of sulfur sources, culture conditions were optimized. The medium devised decreased lag period and generation time when compared with other media, but growth could not be induced consistently with 6 x 10(6) cells per ml or less. Addition of trace elements, chelating agents, reducing agents, metabolites, and spent medium from various stages of growth did not stimulate growth from small inocula. Generation time was 85 min on inoculation with 10(7) or more cells per ml taken from young stocks, but the lag period decreased somewhat with larger inocula. On the other hand, generation time and lag period increased with age of the inoculum. The total yield of cells increased when buffer capacity was increased. Growth of C. pasteurianum W-5 was dependent upon sulfate at relatively low sulfate concentrations, and the organism is thus suitable for study of sulfur metabolism. No evidence of a maintenance requirement for sulfate was detected.     

INTERACTION OF GIBBERELLIN A3, AND INORGANIC PHOSPHATE IN TOBACCO SEED GERMINATION

1. Investigation was made on the influence of inorganic phosphateupon the germination of positively photoblastic tobacco seed(Nicotiana tabacum L. var. uirginica (AGDH.) COM. "Bright Yellow")induced by GA₃, GA₃M, kinetin, red light, and ammonium saltsof various organic acids.
2. Inorganic phosphate increases theGAs-induced germination, andinhibits the germination causedby ammonium citrate, while itdoes not influence the germinationbrought about by GA₃M, kinetin,and red light.
3. The optimumpH for the GA₃-induced germination lies in the acidicpH range,indicating that the undissociated form of GA₃ is operative.The stimulatory effect of phosphate is, however, not ascribedmerely to the pH control in the mediurr. Phosphate exerts somespecific influence for which the presence of the free carboxylgroup of GAs is required.
4. The observed contrasting effectsof phosphate on the GA₃-inducedgermination (i.e., acceleration),on the one hand, and on theammonium citrate-induced germination(i.e., inhibition), onthe other, were explained by assumingthat the phosphate effectsultimately consist in acceleratingthe uptake of the carboxylicacid into the seeds.
5. GA₃M alsohas an activity of inducing the germination of tobaccoseedwithout light.

¹Present address: Department of Vegetable Crops, Universityof California, Davis, California, U.S.A. (Received March 12, 1962; )     

Physiological Studies in Plant Nutrition: XVI. The Mineral Nutrition of Bracken

1. The nutritional requirements of the bracken sporophyte wereexamined in a factorial combination of 3 potassium levels x2phosphorus levels x3 solution types, in which the cations weremainly Na, Ca, or NH₄.
2. The effects of nitrogen and phosphorusdeficiency and of shadingunder conditions of high and low potassiumsupply were alsoexamined.
3. Leaf area, total dry weight, andnet assimilation rates aremuch depressed by lack of eitherK or P.
4. Water contents of leaves and rhizomes are generallyincreasedby lack of K when Na is present in the culture mediumbut notwhen Ca is in excess.Phosphorus causes diminished succulence.
5. Starch content increases as phosphorus supply is lowered.Withincreasing doses of K, starch content falls in the highcalciumsolution, while increasing in the other two solutiontypes.
6. Reduction of light intensity is shown to have a beneficialeffectunder conditions of K deficiency.
7. Analysis revealscomplex interactions between the various factors,and possibletoxic effect of Na, NH₄, and excess P. An estimateof the relativeimportance of net assimilation rate, leaf numberand area perleaf in determining total plant size has been calculatedforthe different nutrient treatments. Comparisons are madewithprevious results for barley and flax.

     

STUDIES ON THE PATHWAY OF SULFIDE PRODUCTION IN A COPPER-ADAPTED YEAST

Metabolism of some sulfur-containing substances was studiedin a copper-resistant strain of yeast (R), its parent strain(P) and respiratory-deficient(RD) mutants from them. The resultsobtained are as follows:

1. Using sulfate, sulfite and thiosulfate as sulfur sources, Rproducedmore H₂S than P, and both of these had the activityhigher than their RD mutants. All of them produced a large amountof H₂S from cysteine, but only little from methionine, cysteinesulfinic acid and S-sulfocysteine.
2. From sulfite and thiosulfate,P and R produced more H₂S inaerobicthan in anaerobic condition.With sulfate and cysteine, however,H₂S production did not differunder those conditions.
3. In both P and R, the sulfate-to-sulfiteand sulfite-to-sulfidereactions were remarkably lowered byiron and zinc deficiencies.But the cysteine-to-sulfide reactionwas not affected by themetal-deficiencies.
4. H₂S productionfrom sulfate was remarkably depressed by highconcentrationsof pantothenate.
5. Rates of reaction steps on a plausible pathway from sulfatetosulfide and to organic sulfur compounds areestimated forthe strainsused. R is characterized by its largecapacity ofthe reaction step from sulfate to sulfite, and excessivesulfitethus formed is liberatedas sulfide not by the way ofcysteine.

¹Present address: Research Reactor Institute, Kyoto University,Kumatori-cho, Sennan-gun, Osaka