A study on senescence in tobacco leaf disks II. Chloroplast and cytoplasmic rRNAs

In tobacco leaf disks floated on water in the dark, chloroplastrRNA (23S and 16S rRNA) decreased rapidly, and this decreasewas inhibited by 10^–6M BA. The cytoplasmic rRNA (25S and18S rRNA) level changed little during a few days of dark incubationregardless of the presence or absence of BA. Cycloheximide at10^–5 M completely removed the effect of BA. ChloroplastrRNA and cytoplasmic rRNA respectively decreased and increasedduring a 4-day culture in the light, and BA had no influenceon these light effects. (Received December 2, 1974; )     

Effects of Cycloheximide and Light on Leaf Senescence in Maize and Hydrangea

The senescence of maize and hydrangea leaves after detachmentand darkening was studied in terms of the loss of chlorophylland protein. Chlorophyll contents of the detached leaves decreasedin the dark in both plants. Cycloheximide at 0.1 mM effectivelyinhibited the loss of chlorophyll in maize, but did not do soin hydrangea. Continuous irradiation with white light of 4.6Wm^–2 prevented the loss of chlorophyll in hydrangea leaves,while it caused bleaching of maize leaves. Reducing agents suchas ascorbic acid and glutathione did not prevent the bleachingby light. In maize leaves, the amount of protein decreased inthe dark more slowly than that of chlorophyll, and cycloheximideslightly prevented the protein decrease. Continuous light irradiationof 4.6 Wm^–2 delayed the loss of protein more effectivelythan cycloheximide did. (Received January 31, 1981; Accepted May 21, 1981)     

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

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

Remarkable retardation of the senecence of tobacco leaf disks by cordycepin, an inhibitor of RNA polyadenylation

The antibiotic cordycepin (3'-deoxyadenosine), a known specificinhibitor of nucleic acid synthesis and polyadenylation of RNA,remarkably retarded decrease in the chlorophyll and proteincontents of senescing tobacco leaf disks. The effectivenessof cordycepin, at its optimum concentration (ca. 4 x 10^–5M), was 53% as effective as a cytokinin, benzyladenine at 10^–6M. Adenosine and 2'-deoxyadenosine had no anti-senescence action. (Received May 29, 1975; )     

Factors Influencing Alanine Aminotransferase Activity in Leaves of Lolium temulentumL.: II. EFFECTS OF GROWTH REGULATORS AND PROTEIN BIOSYNTHESIS INHIBITORS

Effects of kinetin (K), gibberellin A₃ (GA₃), and 2-(chloroethyl)-trimethylammoniumchloride (CCC) on levels of alanine aminotransferase (GPT) andrates of protein synthesis were studied with both intact plantsand isolated leaf segments of Lolium temulentum L. In intactplants CCC stimulated and CA₃ reduced GPT activity, the effectsbsing much greater in 8.h than in 16-h photoporiods. CCC showedmaximum stimulatory effects at 10^–2 M and K at 5 x 10⁵M. No effect of GA₃ could be demonstrated with concentrationsup to 10^–4M. Both K and CCC retarded GPT decline in leafsections, the latter without associated effects upon pigmentbreakdown. Cycloheximide was highly effective in reducing proteinsynthesis in leaf sections. A close correlation between rateof protein synthesis and GPT activity was found over an inhibitorconcentration range from 10^–6 to 10^–4 M. The resultsare discussed in terms of possible methods of in vivo regulationof GPT activity.     

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)     

Effect of cycloheximide and cordycepin on auxin-induced elongation and {beta}-glucan degradation of noncellulosic polysaccharides of Avena coleoptile cell wall

The effect of cycloheximide (10^–5 M) and cordycepin (10^–4M) used as protein and RNA synthesis inhibitors, respectively,on auxin action in noncellulosic ß-glucan degradationof Avena coleoptile cell wall was investigated. Both depressedauxin-induced ßglucan degradation of the cell wallas well as auxin-induced elongation and cell wall loosening,suggesting that the process of ß-glucan degradationof the cell wall is closely associated with cell wall looseningand that auxin enhances the activity of an enzyme for ß-glucandegradation through de novo synthesis of RNA and protein butnot through activation of the enzyme in situ. Kinetic studywith the inhibitors showed that RNA metabolism involved in ß-glucandegradation was stimulated by auxin treatment of only 15 minwhile a longer lag phase (about 1 hr) existed for the synthesisof the enzyme. (Received December 16, 1978; )     

The Effect of Cycloheximide on Absorption of Ions by Bean (Phaseolus vulgaris L) Roots

10^–7 M cycloheximide inhibited bean (Phaseolus vulgaris)root elongation by about 20 per cent but it inhibited absorptionof rubidium, sodium, and phosphate ions to a much greater extent(34–71 per cent). Tips of intact plant roots grown inthe inhibitor showed more inhibition in ion uptake than adjacentproximal portions of the same roots and this is taken to indicatethat 10^–7 M cycloheximide does not exert its effect onion uptake by any uncoupling action. Sodium uptake from 0.5 or 10 mM NaCl solutions by root tipswas inhibited by 10^–7 M cycloheximide to twice the extentthat it was in the elongating region of the root. Assuming thatthe inhibitor affects the plasmalemma more than the tonoplast,Epstein's model of parallel operation of system 1 and system2 at the plasmalemma is supported.     

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; )     

Effect of Metabolic Inhibitors on the Formation of Cell Walls in a Green Alga, Micrasterias crux-melitensis

The effects of cytochalasin B, N-ethylmaleimide (NEM), colchicine,vinblastine and cycloheximide on the formation of birefringentcell wall layers were studied. Birefringent layers accumulatedoutside the plasma membrane of daughter semicells when cellswere cultured in a 0.16 M mannitol solution without any inhibitors.In cells treated with 2 x 10^–5 M cytochalasin B, 3 x 10^–5M NEM, 10^–4 M vinblastine or 10^–5 M cycloheximidefor 24 hr, birefringent layers were not observed outside theplasma membrane, but were present in cells treated with 10^–2M colchicine. The possibility is discussed that substances necessaryfor wall synthesis could be transported from the cytoplasm tothe outside of the plasma membrane by a system associated withmicrofilaments, microtubules and myosin-like structures. (Received June 26, 1981; Accepted September 24, 1981)     

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; )     

Studies on CN--insensitive respiration in plant roots IV. Effect of 10-4 M CN- pretreatment on mitochondrial properties and the effect of chloramphenicol and cycloheximide on the induction of CN--insensitive respiration

No significant difference in respiratory activities was foundfor mitochondria isolated from CN^–-sensitive and CN^–-insensitiveroots. The increase in CN^–-insensitive respiration inducedby 10^–4 M CN^– pretreatment was not influenced bychloramphenicol, but was partly reduced by cycloheximide. (Received October 16, 1972; )     

Ribulose-1,5-Diphosphate Carboxylase Protein during Flag Leaf Senescence

Ribulose-l,5-diphosphate (RuDP) carboxylase protein and activitywere determined in relation to net photosynthetic rate duringthe senescence of intact flag leaves of wheat on the plant.Initially the decrease in RuDP carboxylase activity was greaterthan the decline in net photosynthesis. The major decrease inRuDP carboxylase activity over this period resulted from a decreasein enzyme specific activity from 11 to 2 µmol CO₂ fixedh^–1 mg^–1 protein. Loss of RuDP carboxylase proteindid not occur until late in senescence by which time chlorophyllconcentration had decreased by more than 50%. Treatment of flagleaves at weekly intervals with either 1000 parts 10^–62-chloro-ethyltrimethylammonium chloride or 100 parts 10^–6gibberellic acid with 1 part 10^–6 kinetin did not significantlyaffect net photosynthetic rate, RuDP carboxylase protein oractivity during senescence.     

The Effect of Cycloheximide on Uptake and Transport of lons by Sunflower Roots

The effect of cycloheximide on uptake and transport of saltsby sunflower roots was investigated. Treatment with cycloheximideresulted in a reduction of uptake and transport of K⁺ and NO₃^–to the xylem. Cycloheximide stimulated O₂ uptake and appearedto act as an uncoupler of oxidative phosphorylation. The implicationsof these results regarding the use of cycloheximide as a meansof distinguishing between uptake and transport components ofion movement to the xylem are discussed.     

Correlation of cytokinins and abscisic acid with monocarpic senescence in soybeans

The involvement of cytokinins and abscisic acid (ABA) in themonocarpic senescence (foliar yellowing following fruit development)of soybeans was examined. Foliar sprays of cytokinin (10^–4M zeatin or 10^–5 M benzyladenine), begun when the plantsfirst set fruit and repeated every other day, significantlydelayed, but did not prevent, monocarpic senescence. Foliarsprays of 10^–4 M ABA, applied in the same manner, significantlyhastened senescence of fruiting soybeans but apparently hadno effect on depodded plants. Leaf and stem material from pre-senescentand senescent plants was extracted, chromatographed, and bioassayedfor cytokinin-lilce activity (Amaranthus betacyanin productionassay) and ABA-like activity (oat coleoptile straight growthassay for inhibitors). ABA-like activity increased, and cytokinin-likeactivity decreased in shoot tissue before the plants began tosenesce. Cytokinin-like activity in the fruit also declinedduring this period. These results implicate a decrease in cytokininsand an increase in ABA-like inhibitors in the control of monocarpicsenescence of soybeans, but neither alone is causal. ¹ Supported in part by Research Grant 416-15-79 from the USDACooperative State Research Service under PL 89–106. ² Present address: Biology Dept., College of St. Benedict'sSt. Joseph, Minn. 56374, U.S.A. (Received February 4, 1978; )     

Effect of auxin on Mg++-activated and -inhibited ATPases from mung bean hypocotyls

Mg⁺⁺-activated (A) and -inhibited (B) ATPases were isolatedfrom dark-grown mung bean hypocotyls and the effect of in vivoand in vitro IAA-treatment on these ATPases was observed. The activity of the A-ATPase, which was localized in the plasmamembrane-rich fractions, was enhanced by exogenously added l0^–10M IAA (ca. 35%). The stimulatory effect of in vitro IAA-treatmentwas particularly found in the A ATPase isolated from sectionstreated with 10^–5 M IAA for 60 min (ca. 280%). The activity of the B-ATPase, which was localized in the solublefraction of the cell homogenate, was markedly increased by invivo treatment with 10^–7 M IAA for 60 min (ca. 360%).The stimulatory effect of IAA-treatment was observed even ifprotein synthesis was inhibited by cycloheximide. These results suggest that the enhancement of the activitiesof the A- and B-ATPases caused by in vivo and in vitro IAA-treatmentsoccur as a result of the activation of already present enzymes,rather than of the synthesis of these enzymes. ¹Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received March 27, 1974; )     

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; )     

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; )     

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)     

Removal of the sugar inhibition of flowering in Lemna gibba G3 by catecholamines

DL-Epinephrine (10^–8–10^–7 M), DL-norepinephrine(10⁶ M) and DL-isoproterenol (10^–8–10^–6 M)alleviated floral inhibition due to 1% of sucrose, in Lemnagibba G3. The induction period extended by sucrose was curtailedby epinephrine, frond multiplication enhanced by the sugar beingleft unaltered. The pattern of action of catecholamines appearedto be very similar to that of cAMP. DL-Epinephrine, however,was ineffective in the presence of 10^–7 M DL-propranololwhich affected neither flower nor frond production by itself.Quabain and nicotinic acid also nullified the epinephrine actionon duckweed flowering. These and other relevant findings supportthe hypothesis that the cAMP-adenyl cyclase system participatesin the processes of flower induction in this long-day plant. (Received August 21, 1973; )