Effects of dikegulac, a new growth regulator, on RNA syntheses in Spirodela

The effects of dikegulac sodium on plastid RNA syntheses werestudied, as dikegulac induces the formation of yellow misshapenleaves. It depressed uridine incorporation into both plastidand cytoplasmic ribosomal RNAs of axenically cultured Spirodela(duckweed). With short labeling time (1 hr) dikegulac specificallysuppressed the synthesis of a plastid 1.2 ? 10⁶ MW RNA species,as well as nonspecifically depressing incorporation. With longerlabeling time (24 hr), the incorporation into mature plastidrRNAs was suppressed to a greater extent than that into cytoplasmicrRNAs. The inhibitions of uridine incorporation caused by dikegulacare probably indirect and a reflection of its effect on othergrowth parameters. (Received September 25, 1976; )     

Diurnal change of light-dependent uridine incorporation into RNA in a long-day duckweed, Lemna gibba G3

Light-dependent incorporation during subjective day and nightof radioactive uridine into RNA of a long-day duckweed, Lemmagibba G3, was examined. When the dark treatment was startedfrom the subjective night phase, the activity of uridine incorporationdropped approximately by half only after the very subjectivenight phase had passed or with the commencement of the subsequentsubjective day phase. However, when the dark treatment was startedfrom the subjective day phase, the incorporating activity promptlybegan to decrease and the inhibition increased with the lengthof the dark period until a final steady level (also at ca. 50%of the initial level) was reached after 24 hr of darkness. Thesetwo phases of different light sensitivities recurred daily undercontrol of the physiological clock and the rhythm was resetby a light-on signal. The lowered incorporating activity dueto the darkened day phase was recovered completely by a 12-hror even 15-min white light period perturbing the succeedingnight phase. That the incorporation of uridine in every RNAspecies, especially in chloroplast ribosomal RNA, was loweredby dark treatment of the day and night phases, was disclosedby MAK column chromatography and acrylamide gel electrophoresis. (Received August 21, 1974; )     

Uptake of uridine by a long-day duckweed, Lemna gibba G3

Uptake of uridine by a long-day duckweed, Lemna gibba G₃ wasexamined. Km and Vmax for uptake were in the range of 1 to 2x10^–5 M and of 5 to 10 x10^–8 moles/g fresh weight/2hr, respectively. Uptake rate depended on temperature, and theoptimum pH was 5.0. Uridine uptake was competitively inhibitedby some compounds structurally analogous to uridine. However,the activity of uridine kinase was not affected by these compounds,except for cytidine. Uridine uptake was inhibited by metabolicinhibitors, in which uridine taken up was left unconverted toother forms, especially in the presence of DNP. These resultssuggest that uridine was taken up into the duckweed celb bya specific transport system and immediately phosphorylated byuridine kinase. Phosphorylation of uridine was not associatedwith the uridine transport reaction. (Received November 15, 1976; )     

ROLE OF NUCLEIC ACID AND PROTEIN METABOLISM IN THE INITIATION OF GROWTH AT GERMINATION

When dry decotyledonized embryos of Raphanus are supplied withwater, a brief period of water absorption (phase A) is followedby a period of no fresh weight increase (phase B) which lastsfor 8 hr at 30°. In this period, embryos become ready toadvance into the period of fresh weight increase (phase C). When embryos were exposed to various concentrations of thiouracilor actinomycin D solution from 0 hr of water supply, increasesin fresh weight and in RNA content measured at 13 hr were inhibitedin parallel with each other. Chloramphenicol and puromycin inhibitedthe fresh weight increase without affecting the RNA increase.When embryos were exposed to thiouracil or puromycin for 2,4 and 6 hr, beginning at 0 hr of water supply, the start ofphase C delayed 2, 4 and 6 hr, respectively. When these drugswere given after phase B had progressed at least for 2 hr, thedelay of the start of phase C was shorter than the period ofthe drug treatment. If given at the end of phase B, thiouraciland actinomycin D inhibited the incorporation of ¹⁴C-uracilbut not the fresh weight increase, while chloramphenicol andpuromycin inhibited the latter without inhibiting the former. During phase B, protein content per dry weight of embryo didnot increase, but the rate of ¹⁴C-leucine incorporation increasedremarkably to reach the level in phase C. Incorporation of labeledleucine was inhibited if embryos were subjected to thiouracilor actinomycin D action during phase B, but not if the drugswere given when phase B had been completed. Puromycin and chloramphenicolinhibited the incorporation whenever they were given. The increase in respiratory activity during phase B was inhibitedrelatively little by the above mentioned four drugs. In conclusion syntheses of RNA and protein seem to be essentialfor the progress of phases B and C, protein synthesis havinga more direct effect. (Received September 17, 1965; )     

Further studies on the diurnal rhythm of uridine incorporation into RNA in the long-day duckweed, Lemna gibba G3

Using the long-day duckweed Lemna gibba G3, the changes in theactivities of RNA synthesis in isolated nuclei and chloroplastsand of the reaction prerequisite for the incorporation of exogenousuridine into RNA were examined. When the duckweed was exposedto either a light-dark cycle or to continuous light, the activityof RNA synthesis in the nuclear and chloroplast fractions changeddiurnally and reached its highest levels during the night phase.The changes coincided with uridine incorporation into RNA invivo. However, the amount of radioactive uridine taken up intothe acid-soluble fraction remained unchanged during the wholeday. The proportion of radioactivity incorporated into phosphorylateduridine compounds as well as UTP+UDP to the radioactivity inthis fraction remained constant. Thus, the diurnal rhythm ofuridine incorporation into RNA was related to the diurnal rhythmof RNA synthesis in isolated nuclei and chloroplasts. The loweractivity of uridine incorporation into RNA under continuousdarkness may be determined by the activity of RNA synthesisin nuclei and chloroplasts as well as the uptake rate of uridineinto the duckweed cells, not by the activity of its phosphorylation. (Received August 30, 1977; )     

EFFECTS OF GROWTH SUBSTANCES ON FROND AND FLOWER PRODUCTION IN LEMNA GIBBA G3

Frond and flower production in a long-day duckweed, Lemna gibbaG3, growing under different photoperiodic conditions in presenceand absence of varied concentrations of applied IAA, KIN andGA was investigated. The predominant actions of IAA, KIN and GA were revealed tobe respectively the depression of flowering, the promotion offrond multiplication and the enhancement of flowering. The rateof increase in FL % as an index of the concentration of floralstimulus in the tissues was curtailed greatly by IAA, to a lesserextent by KIN and little by GA. GA at 10^–5 M apparentlyreduced the critical day length (12 hr) and the induction period(48 hr) by 2 and 4 hr, respectively. No vegetative growth waspromoted by KIN in the light. On the basis of these and relevant findings the sites of actionof the growth substances examined in the flowering process ofthe duckweed were suggested. (Received May 30, 1965; )     

Diurnal change in temperature sensitivity gibba G3 induced by acetylcholine in continuous light

When Lemna gibba G3 was in contact with 10^–5 M Ach oreserine, floral response to chilling changed diurnally under[24(0)], as was the case for control cultures exposed to [16(8)].On the other hand, with a rise in temperature, the min-[24(0)]decreased discontinuously in 24-hr units in Ach cultures subjectedto [24(0)], as did the min-[16(8)] in control cultures. Thusin the presence of Ach or eserine, this long-day plant tookthe continuous light regime, i.e. [24(0)], for another typeof long-day regime consisting of a long light period and a shortdark period, e.g. [16(8)]. In Ach cultures, the lower limitof the min-[24(0)] (72 hr) was attained at 22.5?C and remainedunaltered at the higher temperatures examined. The min-[16(8)]for control cultures, however, known to reach its lower limit(48 hr) at 26?C. Sodium lauryl sulphate (10^–5 M) and ouabain(10^–6 M) also caused a similar diurnal change in temperaturesensitivity of 24(0)] cultures. We surmised that exogenous Ach or inserted dark period modifiesthe relative rates chemical and physical component reactionsinvolved in the floral evocation processes, resulting in therhythmical floral response to chilling. (Received August 1, 1974; )     

Cytokinin-Mediated Translational Control of Protein Synthesis in Cultured Cells of Glycine max

Polyribosome formation was stimulated by cytokinin treatmentof cultured cells of Glycine max cv. Funk Delicious. When suspensioncultures were given 0·5 µM zeatin after 24 h inculture in medium lacking a cytokinin, a nearly 2-fold increasein the polyribosome/monoribosome ratio occurred over the subsequent3 h. The effect of actinomycin D and of 5-fluorouridine on RNAsynthesis and on the polyribosome/monoribosome ratios of thesecells was examined. Actinomycin D at 5 and 20 µg/ml^–1inhibitedtotal RNA synthesis by 39 and 60%, respectively, as measuredby [³H]uridine incorporation into acid-precipitable material.The degree of inhibition of precursor incorporation into polyribosomalRNA was similar. At 0·1 mM, 5-fluorouridine inhibited[³H]uridine incorporation by 76%, and [³H]guanosine incorporationby 66% into polyribosomal RNA after 3 h of treatment. Fractionationof the polyribosomal RNA by oligo(dT)-cellulose chromatographydemonstrated that low concentrations of both actinomycin D (5µg ml^–1) and 5-fluorouridine (0·1 mM) inhibitedthe synthesis of ribosomal RNA to a greater extent than thepoly(A)-containing fraction of the messenger RNA. Synthesisof the poly(A)-containing RNA was inhibited by 24% with 5µgml^–1 actinomycin D and by 30% with 0·1 mM 5-fluorouridine.At the above concentrations, these two inhibitors reduced thepolyribosome/monoribosome ratio of the cytokinin-deprived cellsover a 3 h period, but they did not prevent cytokinin-inducedpolyribosome formation. These results provide further evidencethat cytokinin regulates polyribosome levels through an effecton protein synthesis at the translational level     

RNA Metabolism During Breakage of Seed Dormancy by Low Temperature Treatment of Fruits of Acer plataanoides(Norway Maple)

Stratification of Acer platanoides fruits at 4 °C led toan accumulation of RNA in the embryo axis and to breakage ofseed dormancy. The accumulated RNA was mainly rRNA. Storageof fruits at 17 °C led neither to an accumulation of RNAnor to breakage of dormancy. The proportion of embryo axis mRNA,as measured by poly(A) content, decreased during both fruitstorage and stratification, although levels of poly(A) wereconsistently lower in embryo axes from stored seeds. Isolatedembryos from both stored and stratified fruits were capableof incorporating [³H]uridine into embryo axis RNA. When assayedat 17–20 °C, however, this incorporation was significantlylower in embryos of stored fruits. The distribution of radioactivitybetween the different RNA species was similar in both storedand statified seeds. Acer platanoides, Norway Maple, dormancy, fruit, seed, ribonucleic acid, stratification, nucleic acid metabolism     

Effect of cell flotation on growth of Anabaena circinalis under diurnally stratified conditions

We tested the hypothesis that the growth rate of Anabaena circinalis,under diurnally stratified conditions, would increase as flotationvelocity increased owing to higher light availability. An insitu experiment compared the growth of diurnally stratifiedpopulations of A. circinalis with flotation velocities of 0.5and 1.0 m h^–1, with neutrally buoyant populations thatwere exposed to either mixed or persistently stratified conditions.The experiment was conducted in the turbid lower Murray Riverin South Australia (vertical attenuation coefficient = 4.52± 0.36 m^–1). To represent the mixing patterns,A. circinalis was contained in diffusion chambers that weremoved to different positions in the water column throughoutthe day. Diurnal populations with flotation velocities of 1.0and 0.5 m h^–1 grew at 0.23 ± 0.01 and 0.15 ±0.01 day^–1, respectively. Mixed populations grew at 0.19± 0.01 day^–1, whereas persistently stratified populationsgrew at 0.43 ± 0.01 day^–1. Results were used toextend a model that predicts growth of A. circinalis when exposedto the different mixing patterns. The model showed that bloomsare unlikely to be formed when the period of diurnal stratificationis <1 week, regardless of flotation velocity. When the diurnallystratified period is >1 week, flotation velocity is importantand a bloom may form depending on values assigned to the growthperiod and maximum mixed depth (Z_m).     

Effects of Light and Temperature on Flower-opening of Pharbitis nil

Flower buds of Pharbitis nil cut from plants growing in thefield opened rapidly when kept in darkness for 8 hr followedby continuous light at 20–25°C, but those kept indarkness for 4 hr opened promptly oniy when the temperatureduring the following light period was kept at 23°C or lower.Buds exposed to continuous light at 25°C did not open, butthose exposed to continuous light at 23°C opened slowly.At a lower temperature, the buds opened rapidly even in continuouslight. When the buds were placed in darkness at 25°C at13:30, 17:30 and 21:30 (artificial light from 17:30 to 21:30),they opened about 10 hr after the onset of darkness regardlessof the time of the onset of darkness, but when the buds werekept at 20°C in light from 13:30, 17:30 and 21:30, theyopened at 3:30–5:30 regardless of the time of transferto the lower temperature. The biological clock which controlsthe time of flower-opening is suggested to be easily reset bya light-off signal, but not by a shift from a normal to lowertemperature (20°C). At the lower temperature, the time offlower-opening probably is determined by the time of the latestpreceding light-off (or light-on) signal. ¹Dedicated to Professor Dr. Erwin Biinning on the occasion ofhis 75th birthday. (Received October 23, 1980; Accepted December 15, 1980)     

Maintenance of high Pfr-level in the dark period in relation to flowering in Lemna gibba G3

The relative Pfr-level in a long-day duckweed, Lemna gibba G3,was estimated by the null response method. The null % R value(% R in a R/FR-mixture that provides a null flowering response.This value was assumed to indicate the endogenous Pfr-levelof the duckweed.) remained high during the initial hours ofthe 15 hr nyctoperiod then decreased rapidly, if a 12 or 33hr photoperiod preceded the nyctoperiod. The null % R valuedropped immediately after the start of the 15 hr nyctoperiodsubsequent to a 1 or 24 hr photoperiod. Thus, the duration ofthe maintenance of a high Pfr-level changed rhythmically dependingon the length of the preceding photoperiod. Nyctoperiods ofup to 9 hr following a 12 hr photoperiod hardly affected flowering,but nyctoperiods given after a 24 hr photoperiod suppressedflowering in proportion to the length of the period. The Pfr-levelin the nyctoperiod, therefore, seems to be important for flowering,and phytochrome change, as a function of the length of photoperiod,may serve as a photoperiodic timer. Although floral responseto interruption with R or FR changed with the application period,the difference in response between R-treated and FR-treatedplants was relatively constant during a 15 hr nyctoperiod combinedwith a photoperiod of any length other than 1 hr. Apparently,the floral response to the R or FR pulse was regulated by ashift in the Pfr-level caused by the light pulse. (Received April 2, 1979; )     

Perithecial formation in Gelasinospora reticulispora III. Inhibitory effects of near-UV and blue light during the inductive dark period

Growing hyphae of Gelasinospora reticulispora required a continuousdark period prior to photoinduction of perithecia. The inductivedark period was interrupted by brief exposure of the hyphaeto white light so that the formation of perithecia no longertook place. Photosensitivity of the hyphae in terms of the light-breakeffect gradually changed during the inductive dark period. Sensitivityreached its maximum at the 18th hr of the dark period when anirradiation of 1?10⁵ ergs cm^–2 of near-UV light or 4?10⁴ergs cm^–2 of blue-light was sufficient for the light-break.Red and far-red light had no effect at all. The light-breakeffect was limited to the irradiated portion of the hyphae anddid not affect any unirradiated portions. Inhibitory effecton perithecial formation of continuous white light could betotally replaced for several days with intermittent irradiationof near-UV or blue light if given for 5 min every 4 hr. (Received December 18, 1973; )     

Effects of Temperature on Growth and Protein and RNA Syntheses of Excised Vigna mungo Embryonic Axis

Embryonic axes excised from dry Vigna mungo seeds cultivatedon wet filter paper grew well at 27°C, but did poorly below15°C. The increase in fresh weight at 27°C was completelyinhibited by both cycloheximide and a-amanitin. Excised axescould grow well at 15°C only if cultivated at 27°C forthe first 12 h. Also, excised axes cultivated at 15°C forseveral days grew well when transferred to 27°G. These resultssuggest that the ability of the axes to grow is retained duringcultivation at 15°C, even when no growth occurs, and thatonly the initial stage of cultivation requires high temperaturesfor growth. The incorporation of ³H-leucine into protein in the excisedaxes during the first 6 h of cultivation at 15°C was muchslower than that at 27°C. The incorporation rate at 15°Gduring the second 6 h was, however, considerably high. In contrast,the incorporation of ³H-uridine into the total RNA fractionat 15°C was much slower during the first and the second6 h as compared to that at 27°C, suggesting a differencein the response to temperature between protein and RNA synthesesin the axes. (Received February 9, 1983; Accepted August 4, 1983)     

Germination of Phaseolus vulgaris III. The role of nucleic acid and protein synthesis in the initiation of axis elongation

Excised embryonic axes of Phaseolus vulgaris L. (var. WhiteMarrowfat) begin cell elongation after approximately 4 hr ofincubation at 26°C. The incorporation of ³²P into nucleicacids and phenylalanine-l-¹⁴C into protein markedly increasesduring the 4th hr of incubation, prior to initiation of cellelongation. CH, which inhibits incorporation of phenylalanine-l-¹⁴C intoprotein by 93% during the 2nd hr after its addition, completelyprevents the initiation of axis elongation if added up to 2hr after the beginning of imbibition. Actinomycin D reducesthe fresh weight increase of the axes, and inhibits both ³²Pincorporation into nucleic acids and phenylalanine-l-¹⁴C incorporationinto protein. 5-FU inhibits ³²P incorporation into nucleic acidsbut not phenylalanine-l-¹⁴C incorporation into protein or thefresh weight increase of the axes. MAK column chromatography indicates that actinomycin D inhibitsthe synthesis of all types of nucleic acids to about the sameextent, while 5-FU almost completely inhibits the accumulationof ³²P in ribosomal RNA with lesser but significant inhibitoryeffects on accumulation of ³²P in tRNA. The results suggest an absolute requirement for protein synthesisprior to initiation of cell elongation and at least a partialrequirement for synthesis of nucleic acid species other thanribosomal RNA, tRNA and DNA. The kinetic data suggest that theaxes develop a greatly increased capacity for nucleic acid andprotein synthesis prior to initiation of axis elongation. ¹This research was supported by NSF grant GB 4145 and a grantfrom the U. S. Forest Service. (Received December 16, 1968; )     

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)     

Drought effects on cellular and spatial parameters of leaf growth in tall fescue

The effect of drought and recovery on cellular and spatial parametersof the growth process in tall fescue leaves was studied in twoexperiments. In both experiments plants grown on vermiculiteand maintained in a controlled environment were submitted toa 7 d drought period generated by withholding water. Droughtwas followed by a 3 d recovery period in experiment II. As leafelongation rate (LER) decreased during developing drought boththe growth zone length (initially 40 mm) and the maximum relativeelemental growth rate (initially 0.09 mm mm^–1 h^–1during the dark period of diurnal cycles) within the growthzone declined. But the growth zone still exhibited a lengthof approximately 15 mm when LER approached 0 under severe drought(–2.0 MPa predawn leaf water potential). The growth potentialof the basal 15-mm-long portion of the leaf was conserved duringthe period when drought effected the complete arrest of leafelongation. A (retrospective) analysis of the position-timerelationships of epidermal cells identified on leaf replicas(experiment II) indicated that the cell flux out of the growthzone responded very sensitively to drought. Before drought theflux was maximum at approximately 3.2 cells (cell file h)^–1during the dark period. Flux decreased to 0 when leaf elongationstopped. Flux also varied diurnally both under well-wateredand droughted conditions. In well-watered conditions it wasabout 30% less during the light than the dark period. Cell elongationwas also sensitive to drought. Under well-watered conditionsepidermal cell elongation stopped when cells attained a lengthof approximately 480 µm. During developing drought cellsstopped elongating at progressively shorter lengths. When LERhad decreased to almost nil, cells stopped elongating at a lengthof approximately 250 µn. When drought was relieved followinga 2 d complete arrest of leaf elongation then cells shorterthan 250 µm were able to resume expansion. Following rewateringcell flux out of the growth zone increased rapidly to and abovethe pre-drought level, but there was only a slow increase overtime in the length at which cell elongation stopped. About 2d elapsed until the leaf growth zone produced cells of similarlength as before drought (i.e. approximately 480 µm). Key words: Epidermal cell length, cell flux, (leaf) growth zone, leaf elongation rate, relative elemental growth rate, position-time relationships (path line, growth trajectory), drought, water deficit     

Influence of ethylene on nucleic acid synthesis in etiolated Pisum sativum

Ethylene applied to intact etiolated seedlings of Pisum sativumcv. Alaska inhibits incorporation of ³H-thymidine into DNA insubsequently excised plumular and subapical tissue segmentsbut has no influence on incorporation of ³H-uridine into RNA.The effect on DNA synthesis begins about 2 hr after ethyleneis applied, and intensifies progressively. A similar inhibitionof DNA synthesis occurs when ethylene is applied directly toplumular sections cut from control plants, but not with subapicalsegments under these conditions. Inhibition of DNA synthesisby ethylene is reversed by benzyl adenine in plumular sections.Brief exposure of dark grown seedlings to red light causes asubsequent increase in DNA synthesis in plumular tissue. Thechanges in DNA synthesis in tissues exposed to ethylene, benzyladenine and red light are correlated with the effects of thesetreatments on the mitotic index. (Received March 12, 1973; )     

Endogenous light-on rhythm in respiration of a long-day duckweed, Lemna gibba G3 II. On basic and rhythmic components of the rhythm

Effects of respiratory substrates (glucose, malate, citrateand pyruvate) and inhibitors (fluoride, iodoacetate, azide andDNP) on the O₂-uptake rhythm in a long-day duckweed,Lemna gibbaG3 in continuous light period were examined. Rates of O₂-uptake at the starting point (6 hr after the beginningof a continuous light period) and at the time of the first peakof the rhythm (18 hr after the beginning of a continuous lightperiod) were equally increased by exogenous substrates. Sensitivityof respiration to fluoride or iodoacetate was almost the sameat the 6th and 18th hr. The O₂-uptake (at the 6th, 18th, 30thand 42nd hr) was increased by DNP by the same amount. Azideat lower concentrations than 5X10^–4 M did not affect O₂-uptakeat the 6th hr, but inhibited uptake at the 18th hr. In the presenceof 5 X 10^–4 M of azide the rates of O₂-uptake at the 18th,30th or 42nd hr were down to the rate at the 6th hr, which wasinsensitive to azide. These results suggest that the O₂-uptakerhythm consists of two components, i.e. the basic respirationwhich is promoted by exogenous substrate, sensitive to DNP andinsensitive to azide; and rhythmic respiration, which is sensitiveto azide, but is not influenced by exogenous substrate and DNP. (Received February 19, 1971; )     

Alicyclic acid metabolism in plants 3. Fate of 14C-shikimate and 14C-quinate in mung bean plants

Young mung bean plants (Phaseolus mungo) were exposed to ¹⁴C-shikimateor ¹⁴C-quinate in the light. After 8 or 23.5 hr of incubationat 25°C, radioactivities in free and bound amino acids,organic acids, soluble and insoluble carbohydrates, ether-solublefraction and lignin were determined. Shikimic and quinic acidswere separated by the combined use of paper-chromatography andcolumn chromatography. Specific activity of formed quinate orshikimate was only slightly lower than that of fed shikimateor quinate. Specific activities of phenylalanine, tyrosine andbound tryptophan were high as compared with those of non-aromaticamino acids. Discussion is focused upon the interconversionbetween shikimate and quinate, and their roles in the biosynthesisof aromatic amino acids. (Received November 15, 1968; )