A study on senescence in tobacco leaf disks I. Inhibition by benzylaminopurine of decrease in protein level

The drop in protein level as an index of senescence in tobaccoleaf disks was examined in the dark in the presence of BA and/orinhibitors of protein synthesis. Cycloheximide at 10^–6–10^–5M and 10^–5 g/ml actinomycin-D accelerated the senescenceand interfered with the anti-senescence action of 10^–6M BA. Chloramphenicol (10^–8– 10^–4 M) and puromycin(10^–8–10^–4M) did not modify the senescenceor the action of BA. Cycloheximide was more effective at earlierstages of the dark culture of the disks. The rate of ¹⁴C-leucineincorporation into the protein fraction was increased by BAand decreased with senescence, and this drop was removed byBA. The incorporation was also suppressed by cycloheximide equallyin the presence and absence of BA. The drop in labeled proteinof the disks during the chasing period was retarded by not onlyBA but cycloheximide also. The conclusion reached, based onthese and relevant findings, was that BA affects the anti-senescenceaction by promoting synthesis and at the same time inhibitingdegradation of protein. (Received December 2, 1974; )     

Effects of 6-Benzylaminopurine and Potassium on {delta}-Aminolevulinate Dehydratase Activity of Excised Cucumber Cotyledons

Changes in -aminolevulinate dehydratase (ALAD) activity werefollowed in 7-day excised cotyledons maintained on water ortreated with 6-benzylaminopurine (BA 1 µg.ml^–1),KCl (10 mM) or a combination of BA + KCl. Cotyledons were incubatedfor 3 days in the dark or in the light, or preincubated for18 h in the dark before illumination. Cycloheximide treatments(10µg.ml^–1) were performed to differenciate betweenALAD biosynthesis or activation. The excision entailed an increase in ALAD activity. Then a decreaseappeared after a 24-h incubation on water in the dark. It wasmore precocious in the light, but was delayed when the cotyledonswere illuminated after a dark preincubation. BA had little effect on ALAD activity in the dark. In the light,BA completely prevented the decrease in ALAD activity observedin the control, and enhanced this activity when a dark preincubationpreceded illumination. KCl had a slight stimulating effect inthe dark, but was uneffective in the light. The combinationBA + KCl was always devoid of stimulating effect. Stimulatingeffects of BA or KCl seemed to be due to an increase in ALADbiosynthesis. No treatment markedly affected ALAD exportationfrom cytoplasm to plastids. These results are discussed in relation with cotyledon greening. (Received November 25, 1986; Accepted February 5, 1987)     

Light-induced changes in membrane potential in Spirogyra

Spirogyra cells exhibited changes in membrane potential whenthey were exposed to light. Cells made chloroplast-free didnot show any light-induced potential change (LPC) upon illuminationwith white light and also monochromatic red (680 nm) and farred (720 nm) light. LPC was observed when the cell containedonly a small fragment of chloroplast, whether the cell had anucleus or not. The magnitude of LPC depended on the amountof chloroplast in the cell. DCMU at 10^–5 M, CCCP at 10^–5 M and DNP at 10^–4M at pH 5.5 suppressed LPC, while CCCP at 1–5 ? 10^–6M, NH₄Cl at 5 ? 10^–2 M and DNP at 10^–4 M at pH 7.0stimulated LPC. PMS at 10^–4 M stimulated LPC and couldinduce LPC which was completely inhibited by DCMU. These factssuggest that LPC is related to noncyclic and cyclic electronflows. The influences of light and dark conditions and various metabolicinhibitors (DCMU, DNP, CCCP, NH₄Cl) on ATP level have been investigated.No significant difference in the ATP level was observed betweencells in the light and dark. DNP at 10^–4 M (pH 5.5) andCCCP at 5 ? 10^–6 M decreased the ATP level significantly,while DCMU and NH₄Cl only slightly. Good correlation was notfound between the total ATP level and LPC in Spirogyra. LPC occurred even when the external medium contained only asingle salt such as KCl, NaCl or CaSO₄. LPC was also recorded in chloroplasts in situ and in vitro.The mode of LPC of chloroplasts was quite different from thatof the cell. On illumination, the chloroplast potential changedvery rapidly and transiently in the positive direction thenrecovered spontaneously to almost the original potential level. Possible causes of LPC are discussed in relation to the electrogenicion pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo, Tokyo 113, Japan. (Received November 9, 1977; )     

Removal by chemicals of photoperiodic light requirements of Lemna gibba G3

Both the initial and the terminal 1 hr portions of the subjectiveday fraction, namely the L1- arid L2-phases, of a 24 hr daymust be illuminated in order for the day to be perceived asa long day in the min-LD determination by the long-day plant,Lemna gibba G3 (9). The light requirement of the L1-phase wassatisfied by a 10 min red light pulse given at the beginningof the phase. The red light effect was erased by a subsequent10 min far-red light, indicating phytochrome-mediated processesoccurring in the L1-phase. The light requirement of the L2-phasewas satisfied by blue or far-red light given during the terminal10 min period of the phase; there was no indication of phytochromeinvolvement. The light action on the L1-phase was replaced by10^–5 M of cyclic AMP or 10^–7 M of DL-isoproterenol.The isoproterenol action was antagonized by 10^–7 M ofDL-propranolol. Cyclic AMP (10^–5 M) combined with salicylicacid (10^–6 M), which can remove the light requirementof the L2-phase (10), rendered a completely dark day physiologicallyequivalent to a long day. Acetylcholine (10^–5 M) exertednyctomimetic action on the L1-phase of the second light day.The action of acetylcholine was antagonized by cyclic AMP (10^–5M). The L2-phase required no light in the presence of 10^–7M of DL-propranolol, and this propranolol action was not affectedby isoproterenol. These findings suggest changes in membranepermeability caused by the light given during the L1- and L2-phases. (Received July 7, 1976; )     

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; )     

Effects of Light and Inhibitors of Photosynthesis and Respiration on the Multiplication of Tobacco Mosaic Virus in Tobacco Protoplasts

The multiplication rate of tobacco mosaic virus (TMV) in tobaccoprotoplasts in light was several times than in the dark. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea(DCMU) at 10^–5M completely antagonized this illuminationeffect. KCN at 10^–4 M and antimycin A at 10^–5 M,which prevented the protoplasts from surviving in the dark,did not block TMV multiplication in light. Inhibitor experimentsshowed that photosynthesis and respiration were indirectly associatedwith the TMV multiplication. Either of them was found to benecessary for TMV multiplication but neither was indispensable.They play complementary roles in the supply of energy and materialsrequired for virus production. (Received August 2, 1984; Accepted July 9, 1985)     

Control of Plasma Membrane Cl- Fluxes in Chara corallina by External Cl- and Light

The efflux of Cl^– at the plasma membrane of Chara wasstudied in relation to two treatments known to affect the flux:that of removal of external Cl^– and of light. It is shownthat although removal of external Cl^– results in a rapidreduction in Cl^– efflux (consistent with a direct effectof external Cl^– on the transport system) the magnitudeof this reduction in the dark is greater than the measured darkinflux. Therefore, in the dark at least, it is proposed that1:1 exchange diffusion cannot account for the trans-stimulationof efflux by external Cl^–. Light induces an inhibition of efflux and a concomitant stimulationof Cl^– influx at 20 °C, but at 10 °C the responsesto light of the two fluxes can be separated temporally. It istherefore suggested that the fluxes are not reciprocally dependenton the same factor which mediates in the light response. Furtherconsiderations show that it is unlikely that the cytoplasmicCl^– concentration mediates in the light response of eitherflux, but that changes in cytoplasmic pH may do so.     

The Effect of Selected Growth-Promoting and Growth-Inhibiting Substances on the Extension of Segments of Etiolated Tomato Seedling Hypocotyls

The effects of a number of growth-promoting and growth-inhibitingsubstances, including two fungal toxins, were studied on theextension of segments of etiolated tomato seedling hypocotyls.The bioassay was sensitive to small quantities of NaF, coumarinand ₂, ₄-DNP and inhibition was observed at all concentrations.₂, ₄-DNP or Iodoacetate stimulated growth at concentrationsbetween 1? 10^–4 and 5 ? 10^–6M. or 1 ? 10^–6and 1 ? 10^–7M. respectively. Inhibitor experiments inbuffered nutrient solution were approximately 10 per cent. moresensitive than those in deionized water. By means of paper partition chromatography small quantitiesof two fungal toxins, fusaric and alternaric acid were chromatographedand bioassayed. The effect of fussric acid (5, n-butyI picolinicacid) on hypocoty1 growth was detected at concentrations aslow as 1 ? 10^–5M. Experiments with recongnized growth-promoting substances showedthat Kinetin inhibited growth at concentrations up to 1 ?10^–8M.in both light and dark. IAA inhibited growth up to 1 ? 10^–6M.At 1 ? 10^–7 and 1 ? 10^–8 only small increases occurredwith IAA and the effect of light was negligible. Gibberellicacid (GA₂)stimulated growth at concentrations from 10^–3to 10^–7M. and significant increases up to 17 per cent.were recorded in the light. Since the light induced inhibitionwas only partly restored, the existence of some other naturallight sensitive growth substance is suggested. The value ofthe bioassay as a method for estimating natural growth-inhibitingand growth-promoting substances is discussed.     

EFFECTS OF CHLORAMPHENICOL AND KINETIN ON UPTAKE AND INCORPORATION OF AMINO ACIDS BY TOBACCO LEAF DISKS

The effects of chloramphenicol and kinetin on uptake and incorporationof ³⁵S-methionine and some ₁₄C-amino acids have been investigatedin leaf-disks of Nicotiana rustica in light and dark. Chloramphenicolin a concentration of 1 mg per ml inhibits the uptake of aminoacids from 30 to 60 per cent compared with the water control.The incorporation of amino acids into bulk protein is stronglyinhibited in light (40 to 70 per cent), but only to a smalldegree in dark (10 to 20 per cent), as revealed also by ¹⁴CO₂-photosynthesisof the disks and following treatment with chloramphenicol indark. The stimulating effect of kinetin on uptake and incorporationof amino acids is dependent upon its concentration (10^–5to 10^–6 M ; but 10^–4 M solution inhibits stronglyboth uptake and incorporation). The stimulation seems to influencemore incorporation than uptake processes. Possible interactionsof chloramphenicol and kinetin in the protein metabolism oftobacco leaves have been discussed. (Received April 27, 1964; )     

Bl?tenbildung bei Lemna paucicostata 6746 durch kombinierte Anwendung von Abscisins?ure und GCG

To a certain extent the flowering of Lemna paucicostata 6746is evoked in continuous light by application of abscisic acid(ABA) and CCC. Moreover, the action of the combined substancesappears in two separate concentration ranges. In the range ofABA 2?10^–9 M/CCC 10^–7–10^–6 M floweringis initiated without inhibition of vegetative growth and proceedsonly in the presence of high intensity light and sucrose. Acombination of ABA 2?10^–5 M/CCC 10^–3M simultaneouslycauses a strong inhibition of frond multiplication. Here theeffect can be observed also under low intensity light conditionsand without sucrose in the medium. A range with flower inhibitingactivity lies between the two flower promoting concentrationranges. (Received November 16, 1972; )     

Bisulphite-induced Inactivation of Growth and Chlorophyll Formation in Chlorella pyrenoidosa

Treatment of S-sufficient or S-deficient Chlorella pyrenoidosacells with NaHSO₃, during an 8-h period in the light, significantlydecreased their chlorophyll and dry matter contents when thecells were incubated in the presence or absence of SO₄²⁺. Incontrols lacking HSO₃^–, when the starting pH was 7.5,dry matter and chlorophyll contents increased slightly, whereasno significant changes in either occurred at a starting pH of3.0 when the cells and medium contained SO₄^–. In the dark,at both pH 3.0 and 7.5, dry matter and chlorophyll contentsdecreased slightly. Bisulphite treatment in the dark causedlittle decrease of either dry matter or chlorophyll when cellsand medium contained SO₄^2–. However, in its absence, drymatter decreased markedly, but there was little change of chlorophyllcontent in the dark. The interactions between HSO₃^– asa source of S and as an inhibitor of growth and chlorophyllformation are discussed in the context of the changes inducedby light and alternative sources of S. Overall, the harmfuleffects of HSO₃^– outweigh any role it has as a sourceof S, since its effects are ameliorated by SO₄^2–.     

Bisulfite compounds as metabolic inhibitors: nonspecific effects on membranes

Bisulfite compounds are shown to be nonspecific inhibitors ofphotosynthetic processes and of ion transport in green tissues.CO₂ fixation and light-dependent transient changes in externalpH are inhibited about 50% by 5x10^–4 M glyoxal-Na-bisulfite.Chloride uptake in the light and in the dark is inhibited tothe same extent at this concentration. At 5x10^–3 M theinhibitor reduces ATP levels in the light and in the dark, andeffects on glycolate oxidase and PEP carboxylase are observed.The extent of inhibition is dependent on time of treatment withglyoxal-Na-bisulfite and freshly prepared NaHSO₃ is equallyas effective as the addition compound. Possible explanations of the bisulfite effects and the relationshipsto SO₂ effects on photosynthesis are discussed. (Received September 1, 1971; )     

Induction of Flowering in a Duckweed, Wolffia microscopica, under Non-inductive Long Days, by 8-Hydroxyquinoline

Wolffia microscopica, a duckweed, flowers in response to a singlephotoinductive SD cycle of 16 h dark and 8 h light. Floweringin W. microscopica could be induced, under non-inductive longdays, by 8-hydroxyquinoline (8-HQ). Flowering was initiatedwith 10^–6 M 8-HQ and maximum flowering (ca. 75%) was obtainedat 5 x 10^–6M level. Flowering was accentuated furtherwhen plants, supplied with 8-HQ, were subjected to SD cycles. (Received September 13, 1985; Accepted December 4, 1985)     

Effects of Preillumination on Dark Nitrogen Fixation and Respiration by Anabaena Cylindrica

In whole filaments of Anabaena cylindrica dark nitrogen-fixingactivity (measured as acetylene reduction) and respiration increasedwith the light intensity of a fixed period of preillumination,saturating at ca. 10,000 lux. With saturating light during preillumination,the amount and duration of dark nitrogen-fixing activity increasedwith length of preillumination, but respiration declined rapidlyin the dark. At dark respiration rates below 250 nmol O₂ uptake mg protein^–1?h^–1(State 1) no significant nitrogen-fixing activity is observed.From 250 to 550 nmol O₂ uptake?mg protein^–1?h^–1(State 2), nitrogen-fixing activity depends on O₂ uptake whileabove 550 nmol O₂ uptake?mg protein^–1?h^–1 (State3), nitrogen-fixing activity no longer increases with furtherincrease in O₂ uptake rate. (Received June 18, 1983; Accepted November 10, 1983)     

The Effect of L-Phenylalanine on Phenylalanine Ammonia-lyase and Cell Division in Artichoke Callus Culture

5 x 10^–5 M L-phenylalanine overcame the inhibitory effectof white light on cell division in artichoke callus culturesand increased extractable phenylalanine ammonia-lyase (PAL)activity compared to cultures grown in the presence of 5 x 10^–4M phenylalanine The lower concentration of the amino acid alsoenhanced rates of uptake and incorporation of ¹⁴C labelled phenylalaninethroughout G₁ and S. Differences between the two concentrationswere greatest during S with a 4-fold increase in uptake anda 3-fold increase in incorporation It is suggested thereforethat the capacity of 5 x10^–5 M phenylalanine to offsetthe light effect is due to an indirect stimulatory effect onamino acid and protein metabolism Increased levels of extractablePAL activity would then be reflected by this general stimulationof protein synthesis. Helianthus tuberosus L, Jerusalem artichoke, callus culture, cell division, phenylalanine ammonia-lyase     

Studies on CN--insensitive respiration in plant roots III. Induction of CN--insensitive respiration with low concentrations of respiratory inhibitors

Induction of CN^–-insensitive respiration with low concentrationsof respiratory inhibitors was studied. If roots were treatedwith 10^–3 M CN^– for 96 hr, the plants died, whilethose treated with 10^–4 M CN^– showed healthy growth. O₂ uptake in untreated rice and wheat roots showed a negativeresponse to 10^–2 M CN^– to a considerable extent.On the other hand, pretreatment with 10^–4 M CN^–for more than 6 hr did not greatly affect respiratory rate,but made respiration insensitive to 10^–2 M CN^–.A similar induction of CN^–-insensitivity was also broughtabout with 10^–4 and 10^–3 M H₂S and 10^–4 MNaN₃. (Received July 6, 1971; )     

O-Benzylhydroxylamine: An Inhibitor of Phenylpropanoid Metabolism in Plants

O-Benzylhydroxylamine (OBHA) is a potent inhibitor of phenylalanineammonialyase (PAL, EC 4.3.1.5 [EC] ) and phenylpropanoid metabolismas evidenced by its effects on three plant species [soybean(Glycine max (L.) Merr.), buckwheat (Fagopyrum esculentum Moench.),and mung bean (Vigna radiata L.)]. When supplied to roots, OBHA(10^–5 M) did not significantly inhibit light- or dark-growthof soybean seedlings, but reduced (25%) soluble hydroxyphenoliccompound accumulation in light-grown axes. Higher concentrations(510^–5 M) of OBHA caused reductions (25%) in axis freshweight of light-grown seedlings (72 h), but did not lower axisweight of dark-grown seedlings. Anthocyanin accumulation inhypocotyls of intact mung bean seedlings was reduced by 25%after 3 days light growth after treatment with OBHA (10^–5M) via root feeding. Anthocyanin content of excised, etiolatedbuckwheat hypocotyls floated on solutions of OBHA (10^–5M) and incubated in the light for 24 h was reduced by 40%. L-Phenylalanineand t-cinnamic acid, intermediates of phenylpropanoid metabolism,were able to partially reverse this inhibition in buckwheat.Extractable PAL activity (specific activity basis) in soybeanaxes was substantially reduced (20% in dark, 40% in light) asearly as 24 h after root feeding with OBHA (10^–5 M). Reductionof PAL activity (specific activity or per axis basis) by OBHAcompared to control levels, continued throughout a time courseof 96 h. Kinetic studies on soybean PAL revealed a K_m of 1.1mM for L-phenylalanine and an apparent K_i of 3.5 µM forOBHA. (Received May 31, 1985; Accepted August 6, 1985)     

Light dependency of the mating process in Closterium acerosum

Sexual cell division and activation of gametangial cells forconjugation in Closterium acerosum were induced by light. L₂₀₀cells conjugated at maximum level under the following conditions;(i) a light intensity higher than 1,000 lux in a 16-hr lightand 8-hr dark regime and (ii) an illumination time longer than12 hr at 3,000 lux. L₂₀₀ cells also conjugated under continuousillumination at 3,000 lux. The action spectrum for the activation of gametangial cellshad peaks around 450, 611 and 665 nm. 3-(4'-Chlorophenyl)-l,l-dimethylurea (CMU) inhibited the accumulationof carbohydrates and sexual cell division at 10^–5 M andthe activation of gametangial cells for conjugation at 10^–4M. (Received August 15, 1977; )     

Effect of Cytoplasmic Ca2+ on the Membrane Potential and Membrane Resistance of Chara Plasmalemma

Effects of cytoplasmic Ca²⁺ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca²⁺ up to 2.5?10^–5M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca²⁺]_i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca²⁺]_i from1.4?10^–6 to 2.5?10^–5 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca²⁺]_i from zero to 1.38?10^–6M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca²⁺with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca²⁺ on the ATP-dependent H⁺ effluxwas measured. No marked difference in H⁺ effluxes was detectedbetween zero and 2.5?10^–5 M [Ca²⁺]_i; but, at 10^–4M the ATP-dependent H⁺ efflux was almost zero. Ca²⁺ efflux experimentswere done to investigate dependencies on [Ca²⁺]_i and [ATP]_i.The efflux was about 1 pmol cm^–2 s^–1 at all [Ca²⁺]_iconcentrations tested (1.38?10^–6, 2.5?10^–5, 10^–4M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]_i. Thepossibility of a Ca²⁺-extruding pump is discussed. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985)     

Effects of Cytokinin and Several Inorganic Cations on the Polyamine Content of Lettuce Cotyledons

Kinetin (4.7 x 10^–5 M) and 6-benzyladenine (2.22 x 10^–5M) were found to increase ca. 2-fold the putrescine contentin cotyledons of lettuce (Lactuca sativa L.) seedlings grownfor 3 days under fluorescent light. On the other hand, severalinorganic ions (K⁺, Na⁺, Ga⁺⁺, Mg⁺⁺) at a concentration of 3x 10^–2 M reduced the putrescine content. The combinationof kinetin with one of several inorganic ions at the same levelmarkedly increased the spermine content, but the putrescinecontent decreased; calcium and magnesium ions were less effective.The physiological significance of these findings is discussed. (Received July 4, 1982; Accepted November 6, 1982)