Preformed mRNA and Light-induced Germination of Pine (Pinus thunbergii) Seed

The mRNAs were isolated from dry, dark-imbibed and light-imbibedpine (Pinus thunbergii) seeds, in which germination is inducedupon exposure to light, and their translational activities ina wheat embryo cell-free system were examined. A portion ofthe mRNA extracted from each type of seed appeared to be poly(A)⁺RNA.A significant translational activity was already present inthe RNA fraction isolated from the dry pine seeds. The preformedmRNA seems not to be translated in vivo during the dark-imbibition,since most of the in vivo protein synthesis did not occur untilthe seeds were exposed to light. The SDS polyacrylamide gelelectrophoregrams of the polypeptides synthesized in vitro inresponse to either the preformed mRNA or the mRNAs from thedark-imbibed or light-imbibed seeds were qualitatively identical;thus it seems that the preformed mRNA is conserved during darkimbibition, and that its translation is initiated after theexposure of the imbibed seeds to light. (Received August 10, 1981; Accepted May 15, 1982)     

Metabolic Changes in Axes of Germinating Vigna unguiculata Seeds as Related to Effects of Removal of Cotyledons

[¹⁴C]-Labeled amino acids and sucrose were fed to Vigna unguiculataseeds through cut-ends of cotyledons, and incorporations ofradioactivity into trichloroacetic acid- and 80% ethanol-insolublefractions of axes, respectively, were followed during 48 h ofthe post-imbibition development. The results of these studies,together with determinations of changes in dry weight and proteincontents after the onset of imbibition, indicated that the reservematerials stored in cotyledons were available for active growthof axes only after 12 h of post-imbibition. However, pulse-labelingexperiments, where [³H]-labeled leucine and uridine were feddirectly to axes attached to or detached from cotyledons, indicatedthat synthesis of protein and RNA in both axes was very pronouncedeven at earlier stages (2–8 h) of post-imbibition. Albuminand globulin proteins of axes disappeared most rapidly duringthe 6–12 h period of post-imbibition. Cycloheximide, -amanitinand cordycepin added to imbibing axes inhibited the degradationof major globulin proteins, whereas the inhibitors had littleeffect on the degradation of major albumin proteins. Both proteolyticand amylolytic activities were found to occur in embryonic axesof ‘dry’ seeds, and increased to higher levels asthe germination proceeded. Axes at early stages of germinationmay degrade the self-sustained reserve proteins and utilizethem for the synthesis of new proteins. (Received June 11, 1983; Accepted August 16, 1983)     

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     

Chromatin subunit structure in different tissues of higher plants

The basic chromatin structure of higher plants (Vicia faba andTrillium kamtschaticum) was examined biochemically. After digestionwith micrococcal nuclease, the chromatins of these species yieldedDNA-protein components which sedimented as discrete peaks at11S, 15S, 19S, and so on in a sucrose gradient. The buoyantdensity of Vicia chromatin subunits was about 1.44 g?cm^–3in CsCl. Polyacrylamide gel electrophoresis of histone fromthese subunits of Vicia and Trillium chromatins indicated thatthe 11S monomer contained very little histone H1 but a fullcomplement of all other histones, whereas the oligomers containedH1 as in the case of undigested chromatin. Therefore, the modeof organization of basic chromatin structure in higher plantsis identical with that reported with various other eukasyotes,although two plant histone components are different from thecorresponding mammalian histones, H2A and H2B, in molecularweight and amino acid composition. The results indicated alsothat chromosomes prepared from Trillium meiotic cells do notdiffer from chromatins of Trillium or Vicia somatic cells inthe sensitivity to nuclease digestion or in the size of theirsubunits. (Received May 19, 1978; )     

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; )     

The Messenger RNA Population in the Embryonic Axes of Phaseolus vulgaris During Development and Following Germination

Misra, S. and Bewley, J. D. 1985. The messenger RNA populationin the embryonic axes of Phaseolus vulgaris during developmentand following germination.—J. exp. Bot. 36: 1644–1652. Messenger RNAs were extracted from young, mid-maturation, late-maturation,mature-dry, and 20-h-germinated embryonic axes of Phaseolusvulgaris cv. Taylor's Horticultural. They were translated invitro in a rabbit reticulocyte lysate protein synthesizing system.Analysis of the translation products using two-dimensional polyacrylamidegel electrophoresis indicated that there were substantial changesin the messenger RNA populations of developing and germinatingaxes. The number of polypeptides synthesized increased sharplyat 20–22 d after pollination and then declined. Therewas a parallel increase and decrease in the Poly(A)⁺ contentof the seed axis. The analysis showed that certain messageswere present throughout development and were stored in maturedry seed. These messages were degraded upon subsequent rehydration.Some messages appeared during mid-maturation but declined duringlater stages of development and were absent from the matureseed. In the germinating seed a set of messages unique to germinationappeared. Key words: —Seed development, germination, mRNA, in vitro translation, Phaseolus vulgaris     

Transport and Metabolism of Dihydrozeatin Riboside in Germinating Lupin Seeds

[³H]-dihydrozeatin riboside was applied selectively to the embryonicaxes or to the cotyledons of germinating lupin (Lupinus luteusL. cv. Weiko III) seeds 6 h following the start of imbibition.There was little transport of dihydrozeatin riboside from embryoto cotyledons up to 6 h after the application, but a substantialamount of radioactivity had moved into the cotyledons at theend of the 10 h incubation period. However, there was no detectablemovement of [³H]-dihydrozeatin riboside from the cotyledonsto the embryonic axis. This indicated a highly polarized movementof cytokinins during the early stages of seed germination. Exogenouslyapplied [³H]-dihydrozeatin riboside was found to be very stable,both when applied to the embryonic axes and cotyledons of intactseed, or following excision, and there was little metabolismwith only small amounts of radioactivity found associated withdegradative metabolites. The embryonic axis of this specieshas recently been found to synthesize cytokinins within 12 hfrom the start of imbibition, and the results of this studyindicate that the embryo-derived cytokinin is probably transportedto the cotyledons where it accumulates and subsequently participatesin the control of cotyledon function. Key words: Lupinus luteus, cytokinin transport and metabolism, dihydrozeatin riboside, seed germination     

Changes in ubiquitin and ubiquitin-protein conjugates during seed formation and germination

Changes in both free ubiquitin and ubiquitin-protein conjugateswere followed in cotyledons of lupin (Lupinus albus L.) duringthe course of seed formation, from the flower to the dry seed,and during germination and seedling growth, from the dry seedto the senescing cotyledons. The observed levels of ubiquitinconjugates, detected by immunoblotting using antiubiquitin antibodiesand by autoradiography using ¹²⁵I-labelled ubiquitin, suggestan intense involvement of the ubiquitin-mediated proteolyticpathway during the highly regulated phases of seed formationand germination. High amounts of free ubiquitin are presentat all stages in all tissues examined. With the exception ofthe dry seed, the high molecular mass ubiquitin-protein conjugatesare also present at all stages. Higher amounts of these conjugateswere found during the initial stages of pod development andseed germination and during the most active phases of storageprotein deposition and degradation. Germination and seedlinggrowth in total darkness not only delays the degradation ofthe storage proteins, but also extends the period characterizedby the presence of a high amount of these conjugates. No suchconjugates were detected in the dry seeds, probably reflectingthe extremely low metabolic activity observed in these organs.A number of smaller molecular mass polypeptides were also detectedat different stages of seed development, germination and seedlinggrowth. Of particular interest is the abrupt accumulation ofan abundant 20 kDa polypeptide in the cotyledons during the4th day after imbibition, which is maintained in high amountsin these organs, rapidly declining after about 12–14 d.The pattern of accumulation of the 20 kDa polypeptide is controlledneither by light nor by the embryo axes, and large variationsin its concentration are observed during heat shock. Key words: Ubiquitin, ubiquitin-protein conjugates, seed storage proteins, protein synthesis, protein degradation     

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; )     

Natural Ageing: Poly(A) Polymerase in Germinating Embryos of Triticum durum Wheat

The viability of seeds is associated with ageing and storageconditions. A loss of viability is accompanied by slow germination,reduced growth, and a decline in protein and poly(A)⁺RNA synthesis.This paper reports on the activity of poly(A) polymerase indry and germinating embryos of Triticum durum Desf. cv. Cappellicaryopses of different ages and viability. The enzyme was presentas a single form during ageing and germination. The poly(A)polymerase was active at decreasing levels in all aged dry embryos,in parallel with loss of viability. Its activity strongly increasedduring the germination only in viable embryos. The observedincrease was due to de novo synthesis of the enzyme. Poly(A)polymerase synthesis was low during germination of less viableembryos and absent in older ones. Reduced poly(A) polymeraseactivity in dry or germinated wheat embryos may cause a shorteningof poly(A) chains in vitro and a decline in poly(A)⁺RNA synthesis.Copyright1995, 1999 Academic Press Triticum durum Desf. cv. Cappelli, wheat, embryo, natural ageing, poly(A) polymerase     

In vivo Studies on the Occurrence of Stored mRNA in Embryonic Axes of Vigna unguiculata Seeds

-Amanitin and cordycepin at various concentrations were testedfor their inhibitory effect on the fresh weight increase ofVigna unguiculata embryonic axes after the onset of imbibitionand on the incorporation rate of ³H-labeled leucine into proteinin axes of the 36–38 h stage. -Amanitin at 0.5–5µ/Kg/ml clearly exerted an inhibitory effect on both thefresh weight increase and the protein synthesis. This drug at1 µg/ml, however, showed no significant effect on theprotein synthesis at an early stage of imbibition (4 h), whereascycloheximide was a very potent inhibitor. By experiments inwhich ‘dry’ axes were allowed to imbibe 3H-lebeledadenosine solution for 4 and 12 h in the presence of -amanitin,it was found that poly A⁺RNA was newly synthesized to some extentin axes as early as 4 h after the onset of imbibition and thatthe drug effectively inhibited the poly A⁺RNA synthesis. Theresults may indicate the occurrence of stored mRNA in embryonicaxes of V. unguiculata seeds. (Received June 11, 1983; Accepted August 16, 1983)     

Studies on the Growth in Culture of Plant Cells: XXIII ISOLATION OF NUCLEI AND HISTONES FROM CULTURED CELLS OF ACER PSEUDOPLATANUS L.

A technique is described for the isolation of purified nucleifrom suspension culture cells of Acer pseudoplatanus. This involvesa grinding medium containing 70% (v/v) glycerol, 1 mM Mg²⁺,2 mM Ca²⁺, and Tris buffer at pH 7.8, prestorage and disruptionof the cells at –20 °C in a Potter-Elvehjem homogenizer,and purification by filtration and centrifugation in the presenceof Triton X-100. The nuclear yield is c. 25% as assessed bynuclear count or DNA estimation and the nuclei are active inthe RNA synthesizing system of Tautvydas (1971). When the histones of these nuclei are extracted in H₂SO₄ andprecipitated by ethanol, 113 µg histone is obtained perµg nuclear DNA and the histone fraction contains 22% basicamino acids and has a lysine: arginine ratio of 2.6. Acid-ureagel electrophoresis shows the presence of five major histones(H1, H2A, H2B, H3, and H4 in sequence from anode to cathode)having respectively molecular weights of 24 500, 13 500, 13300, 12 800, and 11 000. There is very good correspondence betweencalf thymus histones H3 (reduced form) and H4 and two of theseAcer histones. The other Acer histones differ from the calfthymus histones H1, H2A, H2B in molecular weight but can beprovisionally equated with these by a newly developed differentialstaining reaction. Calf thymus histone H2A appears to be lessrich in lysine than the corresponding Acer histone. Evidence from a pulse-chase experiment with (¹⁴C)lysine and[³H]tryptophan is in favour of the cytoplasmic synthesis ofthe histones.     

Total Uptake and Incorporation into DNA of 3H-Thymidine, 3H-Deoxyuridine and 3H-Thymine in the Primary Root of Vicia faba L.: I. INTACT ROOTS

Total uptake and incorporation of ³H-thymidine, ³H-thymine,and ³H-deoxyuridine into DNA have been investigated in the apical3 cm of the primary root of Vicia faba. Evidence has been obtainedthat endogenous TdR in these roots may be transported eitherapically or basally; apical movement being greater than movementfrom the apex towards the base of the root. The results havebeen discussed with respect to the possible distribution ofendogenous pools of thymidine, thymine, and deoxyuridine inthe primary root of V. faba.     

The Distribution of Nitrate Assimilation Between Root and Shoot in Vicia faba L.

Nitrate assimilation was examined in two cultivars (Banner Winterand Herz Freya) of Vicia faba L. supplied with a range of nitrateconcentrations. The distribution between root and shoot wasassessed. The cultivars showed responses to increased applied nitrateconcentration. Total plant dry weight and carbon content remainedconstant while shoot: root dry weight ratio, total plant nitrogen,total plant leaf area and specific leaf area (SLA) all increased.The proportion of total plant nitrate and nitrate reductase(NR) activity found in the shoot of both cultivars increasedwith applied nitrate concentrations as did NO₃^–: Kjeldahl-Nratios of xylem sap. The cultivars differed in that a greaterproportion of total plant NR activity occurred in the shootof cv. Herz Freya at all applied nitrate concentrations, andits xylem sap NO₃^–: Kjeldahl-N ratio and SLA were consistentlygreater. It is concluded that the distribution of nitrate assimilationbetween root and shoot of V. faba varies both with cultivarand with external nitrate concentration. Vicia faba L., field bean, nitrate assimilation, nitrate reductase, xylem sap analysis     

Effects of NaCl on Growth, Nitrogen Incorporation and Chemical Composition of Inoculated and NH4NO3 Fertilized Vicia faba (L. ) Plants

Vicia faba cv. Maris Bead was grown either on fixed nitrogenor on ammonium nitrate. After 4 weeks growth, nutrient solutionswere supplemented with 50, 75 and 100 mol m^–3 NaCl for15 d. Five harvests were made at weekly intervals, beginningat 4 weeks. Effects of salinity were directly related to dose,plant growth (fresh and dry weight) being depressed in bothN-fixing and N-fertilized plants. The number of nodules perplant and the proportion of those formed which developed intothe active nitrogen fixing state were depressed by salt stress.Increased size of nodules in salt-stressed plants only partlycompensated for the lower specific nitrogenase activity. Theeffects of salinity on plant nitrogen content were more pronouncedon N-flxing than on N-fertilized plants. The former took upmore Na⁺ and Cl^– than the latter: the implications ofthis and of ionic imbalance are discussed. Key words: Vicia faba, Growth, Salt stress, Nodulation     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Development of RNase activity in embryonic axes of germinating pea seeds

RNase activity in embryonic pea axes increased in parallel withthe rise of RNA synthesis as germination proceeded. The developmentof this enzymatic activity was modified antagonistically byapplication of GA₃ and ABA and inhibited severely by treatmentwith CH. Sedimentation analysis of ³H-adenosine-labeled RNAindicated that the synthesis of all types of RNA species isuniformly stimulated by GA₃ and inhibited by ABA. However, 5-FUtreatments, which severely inhibited the synthesis of rRNA,with a slight effect on that of mRNA, had no appreciable effecton the development of RNase activity in the axes. These resultsindicate that active RNA synthesis during germination is independentof the development of RNase activity and that the de novo synthesisof RNases may be controlled by the synthesis of their specificmRNAs. Among the three types of RNase (RNase I, II and III) detectedin the embryonic axes, RNase III showed a sharp increase inactivity with embryo growth and the activity of this enzymewas mainly associated with the endoplasmic reticulum. (Received June 5, 1978; )     

Determination of apoplastic K+ in intact leaves by ratio imaging of PBFI fluorescence

The tetraammonium salt of the K⁺ binding fluorescent dye benzofuranisophthalate (PBFI) was used to investigate the influence ofpotassium nutrition (0.1–2.1 mol m^–3) on apoplasticK⁺ inVicia faba leaves by means of ratio imaging. As a referencethe infiltration-centrifugation method was used. Both methodsreflected the influence of K⁺ supply on apoplastic K⁺ concentration.The abaxial leaf side revealed significantly higher K⁺ concentrations(20-25 mol m^–3) than the adaxial side (5–8 mol m^–3).Application of CCCP led to an immediate increase in apoplasticK⁺ demonstrating the reliability of the PBFI method. Key words: Vicia faba, leaf, apoplast, K⁺, PBFI, ratio imaging, ratiometric fluorescence microscopy     

Cell Morphogenesis and Macromolecule Synthesis during Phytochrome-controlled Germination of Spores of the Fern, Pteris vittata

The object of this study was to characterize the pattern ofcell morphogenesis and synthesis of nucleic acids and proteinsduring phytochrome-controlled germination of spores of the fern,Pteris vittata. Phytochrome activation and germination wereinitiated in fully imbibed spores by exposure to a saturatingdose of red light. At timed intervals thereafter, spores werefixed in acrolein and embedded in glycol methacrylate for examinationin the light microscope. The first sign of germination, visiblein sections of the spore 12 h after irradiation, was the hydrolysisof storage protein granules. This was followed by a migrationof the nucleus from its central location to one side of thespore. Subsequently, the protoplast enlarged at the site ofthe nucleus and appeared outside the exine as a papillate structure.An asymmetrical division of the protoplast gave rise to a smallcolourless rhizoid cell and a large, chloroplast-containingprotonemal cell. During the early phase of germination, DNAwas synthesized both in the nucleus and cytoplasm as judgedby autoradiography of [³H]thymidine incorporation. [³H]Uridine,a precursor of RNA synthesis, was incorporated into the nucleolusand the rest of the nuclear material of germinating spores.Protein synthesis monitored by [³H]leucine incorporation occurredboth in the nucleus and cytoplasm during the early stage ofgermination, although a strictly cytoplasmic protein synthesiswas observed later. Addition of cycloheximide completely inhibitedgermination of photoinduced spores and incorporation of labelledprecursors of macromolecule synthesis into cellular components.Actinomycin D was much less effective as an inhibitor of germinationand, even in high concentrations of the drug which effectivelyinhibited DNA and RNA synthesis in spores, proteolysis and proteinsynthesis appeared normal. These findings are discussed withrespect to the regulation of nucleic acid and protein synthesisduring spore germination and the role of phytochrome in theprocess.     

The Role of ABA in the Control of Apple Seed Dormancy Re-appraised by Combined Gas Chromatography-Mass Spectrometry

Measured by GC—MS²—SIR³, endogenous ABA⁴ in embryonicaxes of seeds of Malus pumila L. cv. Golden Delicious decreased8-fold and cotyledon ABA by only 60%, during 10–50 d ofstratification at 5 ?C, after ABA leaching during an initial24 h soaking. During stratification, the percentage germinationof embryos transferred to 17?C showed a significant linear dependenceon log_e of ABA levels in the axes at transfer. Between 50 and70 d, ABA levels increased markedly in axes and testa both ofstratified seeds and seeds allowed to re-dry at 17 ?C afterinitial soaking. The ability of fully stratified axes with elevatedendogenous ABA to germinate indicated that stratification haddecreased their ABA sensitivity. Changes in cotyledon ABA couldnot account for the promotory effect of cotyledons on germinationduring the first 30 d of stratification. Loss of testa inhibitionof germination during stratification was not linked with changesin testa ABA. Stratification markedly increased the sequestrationin the axes of exogenous ABA supplied via the cotyledons. Changesboth in axis ABA levels and sensitivity were thus correlatedwith dormancy release, but subject to modifying control by thecotyledons and testa not involving ABA. Rehydration of driedseeds affected axis ABA later during storage via mechanismsunconnected with dormancy. Key words: ABA, seed dormancy, stratification