The synthesis and properties of polysomal RNA in potato tuber slices during the early stage of aging

The synthesis, molecular size, and coding properties of polysome-associatedpolyadenylated RNA[poly(A)(+)RNA]and non-polyadenylated RNA[poly(A)(–)RNA] were investigated in potato tuber discsduring the early stage of aging. Tissue discs were labeled for6 hr with ³H-uridine in the presence of 5-fluorouracil to suppressrRNA synthesis, and polysomal RNA was isolated from the discs.Poly(A)(+)RNA accounted for 70% of the radioactivity in polysomalRNA and had a molecular size ranging from 6S to 30S with a peakat about 15S, when measured by formamide-polyacrylamide gelelectrophoresis. The rest of the radioactivity was in poly(A)(–)RNAwhich had nearly the same range in molecular size, but had noconspicuous peaks on the gel. The polysomal RNA could programthe synthesis of a wide variety of polypeptides in a cell-freetranslation system of wheat germ. Seventy percent of the translationalcapacity of polysomal RNA was attributed to poly(A)(+)RNA. Theelectrophoretic behaviour of the majority of the products frompoly(A)(+)RNA was similar to that of products from poly(A)(–)RNA,but the former could program the synthesis of five polypeptidesin addition to those translated from the latter. There was atendency for poly(A)(–)RNA to be a more efficient messengerfor large polypeptides. ¹Present address: Department of Agricultural Chemistry, Facultyof Horticulture, Chiba University, Matsudo 271, Japan. (Received November 16, 1979; )     

Development of Ethylene Biosynthesis in Pear Fruits at --1 {degrees}C

Knee, M. 1987. Development of ethylene biosynthesis in pearfruits at — 1 °C.—J. exp. Bot. 38: 1724–1733. The regulation of ethylene synthesis in pear fruits was investigated.During storage for 60 d at — 1 °C the rate of ethylenesynthesis increased 100-fold but the concentration of 1-aminocyclopropane-l-carboxylicacid (ACC) increased only 2-fold and ACC synthase activity waslow. On transfer to 15 °C after storage at — 1 °Cethylene synthesis increased 10-fold within 10 h but ACC synthaseactivity only increased rapidly after 24 h; the decline in ACClevels during the first 16 h at 15 °C was insufficient tosustain ethylene synthesis. Ethylene synthesis was further investigatedusing discs cut from the mid cortex of pear fruits. Synthesiswas inhibited by aminoethoxyvinylglycine (AVG) and amino-oxyaceticacid at all stages of ripening. The rate of synthesis and ACCsynthase activity increased rapidly after slicing of pears heldat — 1 °C but more slowly in discs cut from pearsimmediately after harvest. Cycloheximide (CHI) inhibited theseincreases and reversed increases resulting from pre-incubationof discs. A combination of CHI and AVG abolished the capacityof discs to synthesize ACC and ethylene production was curtailed.Cordycepin and actinomycin-D were less effective as inhibitorsof the development of ethylene synthesis and ACC synthase activitythan as inhibitors of incorporation of 5-[³H] uridine into totalRNA or poly A rich RNA. The ability of discs to develop ethylenesynthesis and ACC synthase activity in the presence and absenceof cordycepin increased concurrently during storage of wholefruits at — 1 °C. This suggested that mRNA for ACCsynthase was formed at — 1 °C. Key words: 1-Aminocyclopropane-l-carboxylic acid, ethylene, fruit ripening, Pyrus communis L. (fruit ripening)     

Synthesis of RNA in tissue discs of sweet potato roots after cutting or mercuric chloride treatment

Time course analysis of RNA contents of tissue discs after cuttingdisclosed a remarkable increase in total RNA during the first12 hr after cutting and this elevated level remained unchangedfor 48 hr. The elevated RNA level at 24 hr of incubation wasnot changed by subsequent HgCl₂ treatment. The incorporationrate of the label from ³H-uridine into RNA rapidly increasedimmediately after cutting and reached a maximum at about 9 hrof incubation, then decreased sharply until 24 hr and continuedto decrease gradually thereafter. The incorporation rate at24 hr of incubation was not changed by subsequent HgCl₂ treatment.The results of polyacrylamide gel electrophoresis indicatedthat bulk RNA was synthesized most actively at 9 hr of incubationthen the rate of RNA synthesis decreased gradually. (Received August 26, 1977; )     

RNA synthesis required for DNA replication in Vicia seed embryos

The synthesis of DNA and RNA during germination of Vicia seedswas examined. Incorporation of ³H-thymidine into DNA reacheda maximum at about 32 hr after the beginning of imbibition,and RNA synthesis was shown to precede DNA replication. Sedimentationanalyses of ³H-uridine-labeled RNAs indicated that the embryossynthesize all types of rRNA, heterodisperse RNA and 4–5SRNA before and also during the phase of DNA replication. Actinomycin-treatments at lower concentrations (50 or 100 µg/ml)resulted in the specific inhibition of rRNA synthesis. Suchinhibition did not lead to a large reduction in ³H-thymidineincorporation during the replication phase. However, DNA synthesiswas drastically inhibited by a higher level (200 µg/ml)of actinomycin D. The results strongly suggest the involvementof synthesis of heterodisperse RNA in DNA replication. (Received May 28, 1976; )     

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; )     

Gene Activity during Germination of Spores of the Fern, Onoclea sensibilis: RNA and Protein Synthesis and the Role of Stored mRNA

Pattern of ³H-uridine incorporation into RNA of spores of Onocleasensibilis imbibed in complete darkness (non-germinating conditions)and induced to germinate in red light was followed by oligo-dTcellulose chromatography, gel electrophoresis coupled with fluorographyand autoradiography. In dark-imbibed spores, RNA synthesis wasinitiated about 24 h after sowing, with most of the label accumulatingin the high mol. wt. poly(A)^–RNA fraction. There was noincorporation of the label into poly(A) ⁺ RNA until 48 h aftersowing. In contrast, photo-induced spores began to synthesizeall fractions of RNA within 12 h after sowing and by 24 h, incorporationof ³H-uridine into RNA of irradiated spores was nearly 70-foldhigher than that into dark-imbibed spores. Protein synthesis,as monitored by ³H-arginine incorporation into the acid-insolublefraction and by autoradiography, was initiated in spores within1–2 h after sowing under both conditions. Autoradiographicexperiments also showed that the onset of protein synthesisin the cytoplasm of the germinating spore is independent ofthe transport of newly synthesized nuclear RNA. One-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis of³⁵S-methionine-labelled proteins revealed a good correspondencebetween proteins synthesized in a cell-free translation systemdirected by poly(A) ⁺RNA of dormant spores and those synthesizedin vivo by dark-imbibed and photo-induced spores. These resultsindicate that stored mRNAs of O. sensibilis spores are functionallycompetent and provide templates for the synthesis of proteinsduring dark-imbibition and germination. Key words: Onoclea sensibilis, fern spore germination, gene expression, protein synthesis, sensitive fern, stored mRNA     

Mechanism for the induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in HgCl2-treated sweet potaso root tissue

No 3-hydroxy-3-methylglutaryl coenzyme A reductase activitywas detected in microsomal fractions prepared from healthy andwounded sweet potato root tissues. However, there was considerableenzyme activity in the tissue discs when Hgcl₂ was applied afterincubation at 30?C for 18 hr. This increase in the enzyme activitywas followed by furano-terpene accumulation. Application ofcycloheximide to discs immediately after preparation completelyinhibited the increase in the enzyme activity when HgCl₂ wasapplied after incubation. In contrast, the increase was delayedfor about 4 hr, then the activity was enhanced, when CHI wasapplied after preliminary incubation. CHI completely inhibitedprotein synthesis when applied to the discs after the preliminaryincubation, as judged by the inhibition of the incorporationof ¹⁴C-leucine into protein and the inhibition of the increasein peroxidase activity which is synthesized de novo. These resultssuggest that the inactive precursor of HMG-CoA reductase issynthesized during the preliminary incubation in response onlyto wounding then it is converted into the active form aftertreatment with HgCl₂. (Received January 11, 1979; )     

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 synthesis in embryo axes of germinating pea seeds

RNA synthesis during germination was investigated by labelingpea embryo axes or seedling roots with radioactive uridine oradenosine. The results indicated that all RNA species of pre-rRNAs(ribosomal precursor RNAs), rRNAs, heterodisperse-type RNA and4–5S low molecular weight RNA were synthesized from the6th to 64th hour of the period examined. At the very early stageof germination, some conspicuous labeling of the heterodisperse-typeRNA was observed after pulse-labeling. There was no great differencein the labeling patterns of various RNA species with regardto other later stages. When embryo axes were labeled for 1 hrwith ³H-adenosine from the 16th hour, about 25% of the labeledwhole cell RNA was retained on the membrane filter. The ratioof labeled poly(A)-containing RNA, however, decreased as germinationproceeded. The poly (A)-containing RNA sedimented heterodisperselywith a mean value of about 20S in a sucrose density gradient;this size-distribution did not vary throughout germination. (Received January 16, 1979; )     

Effect of Abscisic Acid on Sorbitol Uptake in Growing Apple Fruits

Levels of abscisic acid (ABA) in the fruit flesh of developingapples (cv. Golden Delicious) were measured by electron capturegas chromatography. ABA content of the tissue, calculated ona fresh weight basis, decreased at a constant rate from 200µg g^–1 in young fruit to 10 µg g^–1 inolder fruit and then increased when the ripening process commenced.On a whole fruit basis, the ABA level increased during the initialphase of fruit growth, remained constant during the linear growthphase and increased again when fruits started to ripen. During fruit development the ABA content correlated with therate of sorbitol uptake, when measured after discs of fruittissue were incubated in [¹⁴C]sorbitol. Sorbitol uptake washigh during the initial growth phase and declined at a constantrate during fruit development. ABA present in the incubation medium stimulated sorbitol uptakeinto fruit tissue at concentrations higher than 10^–8 M,whereas indolyl-3-acetic acid had no effect on uptake. When comparing sorbitol uptake in different zones of young fruit,it was found that uptake was higher in discs of outer fruitlayers than in discs from inner fruit zones. Key words: Pyrus malus, Apple, Abscisic acid, Sorbitol     

Studies on enzymes involved in DNA synthesis and thymine nucleotide formation in potato tuber slices

Activity changes of several enzymes involved in DNA synthesiswere investigated in potato tuber tissue in which DNA synthesiswas induced by slicing. Nucleoside phosphotransferase activityincreased only slightly during aging of the tissue discs. Thymidinemonophosphate (TMP) kinase activity increased about 36% afteraging for 24 hr. Protein synthesis in an early stage of agingwas necessary for the activity increase. A 2.7-fold increasewas observed in DNA polymerase activity after aging for 36 hr.The activity increase was due to continuous synthesis of enzymeprotein. In vivo examination of TMP synthetase suggests thatits activity does not necessarily increase before full developmentof DNA synthesis. It was concluded that among the enzymes examined,TMP kinase activity may increase shortly after slicing to supporta massive supply of thymidine triphosphate and the increasedactivity of DNA polymerase may contribute to the active synthesisof DNA in aged discs. (Received February 18, 1977; )     

Appearance of newly formed mRNA and rRNA as ribonucleoprotein-particles in the cytoplasmic subribosomal fraction of pea embryos

Incorporation studies with ³H-uridine or ³H-adenosine showedthat germinating pea embryos synthesize all types of poly A(+)RNA, rRNA and 4–5S RNA at the early stage of germination.After the pulse labeling for 30 min, only heterodisperse RNAand 4–5S RNA appeared in the cytoplasm as labeled RNAspecies. At this time the radioactivity was associated withcytoplasmic structures heavier than 80S and RNP particles of68–70S, 52–55S, 36–38S and 20–22S whichare presumed to be free mRNP particles in plants. When the pulse-labeledembryos were incubated for a further 60 min in an isotope-freemedium, the labeled 17S and 25S rRNA emerged in the cytoplasm,together with labeled heterodisperse and 4–5S RNAs. Moreradioactivity accumulated in the regions of the polysome, 62–65Sand 38–42S particles. The results of analysis of RNAsextracted from the whole cytoplasm, polysome or subribosomalfractions indicated that small subunits of newly formed ribosomesappear more rapidly in the cytoplasm than new large subunits,which accumulate for a while as free particles in the cytoplasmthen are incorporated into polysomes. The actino-mycin treatmentwhich caused preferential inhibition of rRNA synthesis reducedthe accumulation of free, newly formed ribosome subunits andpartially permitted detection of the presumed mRNP particlesin the subribosomal region even after the chase treatment. (Received June 28, 1976; )     

Enhancement of DNA polymerase activity in potato tuber slices

DNA polymerase was extracted from potato (Solanum tuberosumL.) tuber discs and the temporal correlation of its activitychange to DNA synthesis in vivo was examined during aging ofthe discs. Most of the DNA polymerase was recovered as a boundform in the 18,000?g precipitate. Reaction with the bound-formenzyme was dependent on the presence of four deoxynucleosidetriphosphates, Mg²⁺, and a template. "Activated" DNA and heat-denaturedDNA, but not native DNA, were utilized as templates. The polymeraseactivity was sensitive to SH reagents. Fresh discs, which donot synthesize DNA in vivo, contained a significant amount ofDNA polymerase and its activity increased linearly with timeuntil 48 hr after slicing and became four times that of freshdiscs after 72 hr, whereas the activity of DNA synthesis invivo increased with time and decreased after reaching a maximumat 30 hr. Cycloheximide inhibited the enhancement of polymeraseactivity. DNA polymerase from aged and fresh discs had identicalrequirements for deoxynucleotides and a template in their reactions,sensitivity to SH reagent, and affinity to thymidine triphosphate. (Received February 18, 1977; )     

Multiple effects of the inhibitory protein of ethylene production on cellular metabolisms

The effects of an inhibitory protein of ethylene productionisolated from etiolated mung bean hypocotyls (Planta 113: 115,1973) were investigated. Etiolated mung bean hypocotyl segmentsincubated with IAA for 3 hr (1st incubation) to induce ethylene-producingactivity were incubated for 1 hr with IAA in the presence ofthe inhibitory protein and a radioactive material to measuremetabolic activity. Under the conditions where ethylene productionwas inhibited 80% or more by the protein, RNA synthesis, proteinsynthesis and phosphate uptake were suppressed 55–60,65–80, and 60–75%, respectively. Conversion of 1-¹⁴C-acetateto CO₂, lipid, basic and neutral fractions was also inhibited,but the degrees of inhibition were much less than those forthe other processes. When the segments pretreated with the inhibitoryprotein during the 1st incubation period were washed free ofthe protein and assayed for their metabolic activities, theinhibition of RNA and protein syntheses and of phosphate uptakewas partially restored, while ethylene-producing activity wasfully restored to the control level. Similar reversible inhibitoryeffects were also observed for those metabolic activities inthe tissue segments not treated with IAA, thus not producinginduced ethylene. Oxygen uptake and conversion of U-¹⁴C-glucoseto CO₂ were not affected by the inhibitory protein. The possibilitythat the inhibitory protein acts on cell surface membranes andthe modified membranes affect the regulatory mechanism of cellularmetabolism is discussed. ¹ This investigation was supported in part by grants from theMinistries of Education (B-248009), and of Agriculture and Forestryof Japan. (Received November 4, 1977; )     

Studies on chloroplast development in Chlamydomonas reinhardtii I. Effect of brief illumination on chlorophyll synthesis

When dark grown cells of Chlamydomonas reinhardtii y-1 mutantwere exposed to continuous light, an immediate transformationof small amounts of protochlorophyll(ide), which had been presentin the dark grown cells, to chlorophyll was observed. Afterthis, there was a slow accumulation of chlorophyll lasting for2.5-3 hr before the start of exponential synthesis. Initialaccumulation of chlorophyll was distinctly slower at a highlight intensity (13,000 lux) than it was at moderate intensitiesof light (2,000–5,000 lux). However, the exponential synthesisof chlorophyll started after the same 2.5–3 hr of illumination. A brief pre-illumination of cells followed by incubation indarkness was effective in promoting chlorophyll synthesis undersubsequent continuous illumination at high, as well as moderatelight intensities. Pretreatment alleviated retardation of theinitial chlorophyll accumulation by light of high intensity.The promoting effect of preillumination on chlorophyll synthesiswas sufficient, even when a light impulse as short as 10 secwas given. However, the effect was dependent on length of thedark period after the short pre-illumination. The full extentof this effect was observed when the dark period was about 2.5–3hr long. Further dark incubation gradually decreased the effect. On the basis of these findings, it is assumed that a factor(s)responsible for promotion of chlorophyll (or chloroplast) synthesisin the process of greening of dark grown cells is produced duringthe dark period after a brief pre-illumination, and that thefactor is turned over at a relatively fast rate. The possiblenature of the presumed factor is discussed in relation to chloroplastdevelopment. ¹Present address: Department of Biology, Faculty of Science,Kobe University, Nada-ku, Kobe, Japan. (Received August 18, 1970; )     

CHANGES IN ACTIVITIES OF 5-DEHYDKOQUINATE HYDRO-LYASE AND SHIKIMATE-NADP OXIDOREDUCTASE IN SLICED SWEET POTATO ROOTS

Sweet potato roots of variety Norin No. 1 were cut into discs(319 mm), which were incubated at 28–29 for 4 days.At 24-hour intervals, activities of 5-dehydroquinate hydro-lyaseand shikimate-NADP oxidoreductase in the buffer extracts ofdiscs were estimated. The activities of both enzymes were significantlydetected in the fresh root tissues. 5-Dehydroquinate hydro-lyaseactivity per fresh weight increased by three to four times within2 days after slicing, and decreased gradually. Shikimate-NADPoxidoreductase activity at the lst-day incubation period wasfound to be about three times as much as the activity foundin the fresh roots, and it remained for 4 days after slicing.The role of these enzymes in polyphenol biosynthesis in thesliced root tissues is discussed. An attempt to detect the enzymicconversion of dehydroquinic acid to quinic acid is also described. ¹This paper constitutes Part 55 of the Phytopathological Chemistryof Sweet Potato with Black Rot. ²Present address: Department of Biology, Tokyo MetropolitanUniversity, Tokyo. Nagoya     

RNA metabolism in apices of Pharbitis nil daring floral induction

RNA metabolism was studied in apices of Pharbitis nil duringand after floral induction. In continuous light ³H-uridine accumulatedin RNA at a constant rate over an 18 hr period. In darkness,however, the rate of accumulation of label into RNA was constantuntil the 10th hour at which time a rapid burst of accumulationoccurred, peaking at the 14th hour of darkness and followedby a net loss of label. The RNA involved in this burst is probablymRNA due to its size and poly(A) content. This phenomenon doesnot seem to be associated with floral induction, since the siteof perception is the apex, and it also occurs under conditionswhere floral initiation is inhibited by a brief light interruptionof the dark period. Immediately after floral induction by a16-hr dark period the rate of RNA synthesis was suppressed about14%. This suppression lasts for about 12 hr and was followedby a twofold increase in the rate of RNA synthesis, comparedto non-induced apices, at 64 hr after the beginning of the inductivedark period. These post-induction changes were found to occurin all RNA fractions. ¹Present address: Department of Radiation Biology and Biophysics,University of Rochester School of Medicine and Dentistry, Rochester,N.Y. 14642, U.S.A. (Received March 15, 1976; )     

Biosynthesis and stability of globin mRNA in cultured erythroleukemic friend cells

Biosynthesis and stability of the mRNA population in DMSO-induced Friend erythroleukemic cells were studied after labeling the RNA with ³H-uridine and then chasing it with nonlabeled uridine. Globin RNA metabolism was studied by hybridization to excess complementary DNA covalently coupled to oligo(dT)-cellulose. After a labeling period of 120 min, 2–4% of the poly(A)-containing labeled RNA was in globin RNA; it decayed with a half-life of 16–17 hr. The rest of the poly(A)-containing RNA was composed of two kinetic populations: 85–90% decayed with a half-life of about 3 hr, while 10% decayed with a half-life of about 37 hr. The portion of globin RNA in labeled poly(A)-containing RNA behaved in an unexpected fashion during the chase period. During the initial chase period, the percentage of globin RNA increased rapidly, reaching a maximum of about 15% at 20 hr, but if subsequently declined gradually.Based on these findings, a model was built that describes the changes in the proportion of globin mRNA in poly(A)-containing RNA during continuous synthesis and after chase of the labeled RNA. It appears that if the parameters described remain constant during the maturation of erythroblasts, then this model would not account for the almost exclusive presence of globin RNA in the reticulocyte. By far the most effective way to achieve this high level of globin RNA is the destabilization of the mRNA population which is more stable than globin RNA, and not the stabilization of globin RNA itself.     

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; )