An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

Rate of RNA synthesis and tRNA end-labeling during early development of Phaseolus

Summary Axes of Phaseolus vulgaris cease synthesis of RNA during the maturation stage of embryogeny. During the imbibition phase of germination RNA synthesis resumes after the axes reach a normal water content. In the first hour of inbibition a very low rate of incorporation of ³H-adenosine into is RNA found, and the primary site of incorporation is the-CCA end of tRNA. At later stages of germination tRNA end-labeling accounts for a minor fraction of adenosine incorporation. The rate of RNA synthesis increases after initiation of axis elongation to a maximal rate at 18 h of germination. ATP pool-size and specific activity vary over a several-fold range during development, an important consideration in determining the rate of RNA synthesis.Supported by a grant from the National Science Foundation (GB 8709) to M. E. Clutter and I. M. Sussex and by a National Science Foundation Predoctoral Fellowship (V. W.). Submitted to the Graduate School, Yale University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

AN AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS DURING POLLEN EMBRYOGENESIS IN HYOSCYAMUS NIGER (HENBANE)

RNA synthesis during pollen embryogenesis in cultured anther segments of Hyoscyamus niger (henbane) has been followed by autoradiography of ³H-uridine incorporation. Embryogenic divisions were initiated in binucleate pollen grains in which the generative nucleus or both generative and vegetative nuclei synthesized RNA. When the first haploid mitosis in culture resulted in pollen grains with two nearly identical nuclei, those in which both nuclei synthesized RNA became embryogenic. Binucleate pollen grains in which ³H-uridine incorporation was confined exclusively to the vegetative nucleus gradually became starch-filled and nonembryogenic. Based on the degree of involvement of the vegetative nucleus in embryoid formation, some differences were noted between the counts of autoradiographic silver grains over cells cut off by the generative and vegetative nuclei during progressive embryogenesis. The possible significance of RNA synthesis in the nuclei of binucleate pollen grains in determining the pathway of embryogenic divisions is discussed.     

Cell wall carbohydrates in Phycomyces blakesleeanus burgeff

The carbohydrate composition of the cell walls from spores, mycelium and sporangiophores of Phycomyces blakesleeanus was analyzed. Spore wall polysaccharides contained over 50% glucose, about 20% uronic acids, 10% mannose and 10% amino-sugars. During the growth of the hyphae amino-sugars became the main carbohydrate (45%); uronic acids contributed some 25%, glucose and fucose 10% and galactose nearly 6%. Sporangiophores contained almost 90% aminosugars and some 6% uronic acids. Traces of rhamnose were found in all wall preparations. A similar picture emerged from studies on the incorporation of [U-¹⁴C]-glucose into wall materials.Furthermore we looked for a GDP-fucose synthesizing system and found an increasing activity during early germination. This rise in activity was inhibited by cycloheximide but not by 5-fluorouracil.     

Ribosomal RNA Turnover in Contact Inhibited Cells

CONTACT inhibition of animal cell growth is accompanied by a decreased rate of incorporation of nucleosides into RNA^1–3. Contact inhibited cells, however, transport exogenously-supplied nucleosides more slowly than do rapidly growing cells^4,5, suggesting that the rate of incorporation of isotopically labelled precursors into total cellular RNA may be a poor measure of the absolute rate of RNA synthesis by these cells. Recently, Emerson⁶ determined the actual rates of synthesis of ribosomal RNA (rRNA) and of the rapidly labelled heterogeneous species (HnRNA) by labelling with ³H-adenosine and measuring both the specific activity of the ATP pool and the rate of incorporation of isotope into the various RNA species. He concluded that contact inhibited cells synthesize ribosomal precursor RNA two to four times more slowly than do rapidly growing cells, but that there is little if any reduction in the instantaneous rate of synthesis of HnRNA by the non-growing cells. We have independently reached the same conclusion from simultaneous measurements on the specific radioactivity of the UTP pool and the rate of ³H-uridine incorporation into RNAs (unpublished work of Edlin and myself). However, although synthesis of the 45S precursor to ribosomal RNA is reduced two to four times in contact inhibited cells, the rate of cell multiplication and the rate of rRNA accumulation are reduced ten times. This suggests either “wastage”⁷ of newly synthesized 45S rRNA precursor, or turnover of ribosomes in contact inhibited cells Two lines of evidence suggest that “wastage” of 45S RNA does not play a significant role in this system. (1) The rate of synthesis of 45S RNA in both growing and contact inhibited cells agrees well with that expected from the observed rates of synthesis of 28S and 18S RNAs (unpublished work of Edlin and myself). Emerson has made similar calculations⁶. (2) 45S RNA labelled with a 20 min pulse of ³H-uridine is converted in the presence of actinomycin D to 28S and 18S RNAs with the same efficiency (approximately 50%) in both growing and contact inhibited cells. These results indicate that, in order to maintain a balanced complement of ribosomal RNAs, contact inhibited cells must turn over their ribosomes. We present evidence here that rRNA is stable in rapidly growing chick cells, but begins to turn over with a half-life of approximately 35–45 h as cells approach confluence and become contact inhibited.     

Synthèse des RNA au cours de la germination du radis

RNA synthesis in radish is studied during the first stages of germination. The radish seeds allowed to germinate in the dark, on distilled water, synthesize ribosomal RNA and accumulate a particular RNA, not incorporated in ribosomes. The results of ³²P incorporation in RNA of radish seedlings indicate a progressive formation of ribosomal RNA. Two species of rapidly labelled RNA are synthesized. With labelling time, their chromatographic behaviour on MAK columus evolves, while their electrophoretic characteristics remain stable. It is assumed that these two species are involved in ribosome formation. In vivo experiments with chloramphenicol support this conclusion. RNA which accumulates during germination, could be a particular type of ribosomal RNA which could be enable, under the definite culture conditions, to enter into ribosomal structures.     

Energetic aspects of spore germination in filamentous fungi

The antifungal activity of substances interfering with the function and biogenesis of mitochondria was studied. Strict anaerobiosis, cyanide, azide, oligomycin, bongkrekic acid and ethidium bromide were found to prevent spore germination ofAspergillus niger andPenicillium italicum in liquid germination medium. The effect of azide, oligomycin and ethidium bromide was fungicidal. Cyanide and azide completely inhibited the incorporation of¹⁴C-leucine and¹⁴C-uracil into germinating conidia ofA. niger. Oligomycin and ethidium bromide reduced the extent of incorporation of both precursors in the first few hours of conidial germination and at later stages stopped it completely. The inhibition of both spore germination and macromolecules synthesis during the germination ofA. niger conidia were in relation to the specific inhibitory effect of the agents on respiratory activity of dormant conidia and mycelial cells. The results indicate that both the function of mitochondrial genetic and protein synthesizing systems and the function of oxidative phosphorylation are essential for normal spore germination and fungal growth.     

Genes expressed in the male gametophyte of flowering plants and their isolation

Recombinant cDNA libraries to poly(A)RNA isolated from mature pollen of Zea mays and Tradescantia paludosa have been constructed. Northern blot analyses indicate that several of the clones are unique to pollen and are not expressed in vegetative tissues. The majority, however, are expressed both in pollen and vegetative tissues. Southern hybridizations show that the pollen specific sequences in corn are present in one or a very few copies in the genome. By using several of the clones as probes, it was found that there are at least two different groups of mRNAs with respect to their synthesis. The mRNAs of the first group represented by the pollen specific clones are synthesized after microspore mitosis and increase in concentration up to maturity. The second group, exemplified by actin mRNA, begins to accumulate soon after meiosis, reaches its maximum by late pollen interphase, and decreases thereafter. Although the actin mRNA and the pollen specific mRNAs studied show very different patterns of initiation of synthesis and accumulation during pollen development, the rates of decline of these mRNAs during the first 60 minutes of germination and pollen tube growth in Tradescantia are similar and reflect the previously observed declines in rates of protein synthesis during this period.     

Relationship between uridine kinase activity and rate of incorporation of uridine into acid-soluble pool and into RNA during growth cycle of rat hepatoma cells

Uridine kinase activity measured in cell-free extracts of Novikoff rat hepatoma cells grown in suspension culture fluctuates about 10 fold during the growth cycle of the cells. Maximum specific activity (units/10⁶ cells) is observed early in the exponential phase and then decreases progressively until the stationary phase. The rate of incorporation of uridine into the acid-soluble pool by intact cells fluctuates in a similar manner and both the rate of uridine incorporation by intact cells and the uridine kinase actvity of the cells increase several fold before cell division commences following dilution of stationary phase cultures with freshmedium. Regardless of the stage of growth, uridine is rapidly phosphorylated to the triphosphate level by the cells. The rates of incorporation of uridine into the nucleotide pool and into RNA by intact cells fluctuate in a similar manner during the growth cycle. However, evidence is presented that indicates that alterations in the rate of incorporation of uridine into RNA are not simply due to changes in the rate of phosphorylation of uridine, but are regulated independently. Inhibition of protein synthesis by treating cells with puromycin or actidione causes a marked inhibition of incorporation of uridine into RNA, but has little effect on the phosphorylation of uridine to UTP for several hours. Thus the depression of incorporation of uridine into RNA probably reflects a decrease in the rate of RNA synthesis as a result of inhibition of protein synthesis. Inhibition of RNA synthesis by treating cells with actinomycin D does not affect the rate of conversion of uridine to UTP and thus results in the accumulation of labeled UTP in treated cells.     

The effect of synaptic stimulation on RNA and protein metabolism in the R2 soma of Aplysia

Excitatory synaptic stimulation of the R2 neuron in the abdominal ganglion of Aplysia californica causes an increased incorporation of ³Huridine into RNA. However, this could be the result of a change in precursor specific activity rather than an increase in RNA synthesis. We find that at low external uridine concentrations (1.5 μM) there is no increase in ³H-uridine incorporation correlated with synaptic stimulation. In addition, no change in incorporation of ³H-leucine into total protein or in the pattern of newly-synthesized proteins, resolved by electrophoresis on SDS-polyacrylamide gels, was detected with stimulation. Since the R2 neuron can be stimulated without a detectable change in RNA or protein synthesis, we conclude that the increase in incorporation observed at high external uridine concentrations (100 μM) could be caused by increased specific activity in a precursor pool rather than by an RNA synthesis change.     

Microcyst germination in the cellular slime mold, Polysphondylium pallidum: Effects of actinomycin D and cycloheximide on macromolecular synthesis and enzyme accumulation

Germination of microcysts of Polysphondylium pallidum is characterized by an immediate rapid increase in incorporation of [³H]leucine into protein which is cycloheximide-sensitive but unaffected by actinomycin D. Significant RNA synthesis, as measured by [³H]uridine incorporation, does not begin until approx. 2 h after the onset of germination. The increase in [³H]uridine incorporation is prevented by actinomycin D. Germination and the increase in alkaline phosphatase and β-glucosidase enzyme activities are prevented by cycloheximide but unaffected by actinomycin D. The data strongly imply the presence of stable RNA in dormant microcysts and indicate a requirement for a discrete period of protein synthesis for germination of microcysts of P. pallidum.     

RNA and Protein Synthesis in Germinating Pine Pollen

The results of autoradiographic experiments demonstrate that,as with the pollen of most other species, both the generativeand vegetative nuclei of Loblolly Pine (Pinus taeda) activelyengage in RNA synthesis from the very early stages of pollengermination. Unlike most other species, however, this newlysynthesized RNA includes rRNA. Evidence is provided for theimportance of this newly synthesized RNA in the process of continuedpollen tube growth. One and two-dimensional gel electrophoretic analysis revealsa number of both qualitative and quantitative differences amongthe proteins synthesized during the early stages of germinationand the later stages of pollen tube growth. One of the mostnotable of these is a 36 kD protein, the synthesis of whichpredominates during the later stages of pollen germination.A similar pattern of 36 kD protein synthesis is observed whenmRNA extracted from pollen at each of these stages is translatedin vitro. Key words: Pinus, pollen tube growth     

Reactivation of Ribonucleic Acid and Protein Synthesis During Germination of Blastocladiella Zoospores and the Role of the Ribosomal Nuclear Cap

Freshly harvested zoospores of Blastocladiella emersonii begin to germinate about 15 min after inoculation into a defined growth medium at a density of 10(6) zoospores per ml. Flagellum retraction accompanies encystment, and dispersal of the ribosomal nuclear cap takes place shortly thereafter. The primary rhizoid begins to emerge at 25 to 30 min and starts to branch at ca. 60 min. The first nuclear division occurs between 120 and 190 min. The dry weight per cell increases linearly after 60 min, whereas the deoxyribonucleic acid per cell doubles between 120 and 240 min. A linear increase in total ribonucleic acid (RNA) is detectable beginning at 40 to 45 min, and in total protein beginning at 80 min; neither process is interrupted during nuclear division. Encystment and nuclear cap disorganization are associated with a sharp rise in the rates of precursor incorporation into RNA and protein. Cycloheximide at 20 mug/ml prevents leucine incorporation at all stages and inhibits development beyond the earliest encystment stage. Actinomycin D at 25 mug to 50 mug/ml prevents uracil incorporation, but it has no effect on leucine incorporation or development until 40 to 45 min. At the latter stage, actinomycin D causes a sharp developmental arrest and begins to inhibit leucine incorporation. It is concluded that early protein synthesis must occur on the ribosomes formed during the prior growth phase and conserved through the zoospore stage in the nuclear cap. The results further indicate that this synthesis is dependent upon messenger RNA already present in the zoospore before germination.     

What the papers say: Cellular dedifferentiation and spore germination in Dictyostelium may utilize similar regulatory pathways

Cellular dedifferentiation is an important developmental response to perturbations in morphogenesis. In the cellular slime mold Dictyostelium discoideum this process gives cells the flexibility, when multicellular development is disrupted, to respond to nutrients and reinitiate vegetative growth. Recent studies in D. discoideum described by Soll and colleagues⁽¹⁾ show that genes previously thought to be expressed only during spore germination are also expressed during induced dedifferentiation, suggesting that similar molecular mechanisms are involved in these two developmental processes. It should now be possible to determine whether the developmental programs that control dedifferentiation during spore germination also control conversion of cell types in the multicellular organism.     

Mitochondrial DNA synthesis during fungal spore germination

Summary Germinating spores of the fungus Botryodiplodia theobromae incorporated guanine-8-C¹⁴ into both the nuclear DNA and mitochondrial DNA fractions. Ethidium bromide inhibited the synthesis of mitochondrial DNA without having a significant effect on nuclear DNA synthesis or on the rate and extent of spore germination. Rates of leucine and uracil incorporation and of oxygen uptake were not significantly affected by ethidium bromide until germination was nearly completed. Mitochondrial DNA synthesis is apparently not required for germination of the spores of B. theobromae but is probably essential to continued vegetative growth.Abbreviations DNA deoxyribonucleic acid - mit-DNA mitochondrial DNA - nuc-DNA nuclear DNA - RNA ribonucleic acid - EB ethidium bromide - Tris tris (hydroxymethyl)aminomethane Published with the approval of the Director as Paper No. 3331, Journal Series, Nebraska Agricultural Experiment Station. Research reported was conducted under Project No. 21-17. Paper No. 7877, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Differential effects of actinomycin d and cordycepin in lettuce seed germination and RNA synthesis

Intact lettuce seed germination was inhibited by cordycepin but not by actinomycin D; however, when seeds were clipped at the cotyledonary end, actinomycin D partially inhibited germination. Uptake studies with intact seeds using ³H-actinomycin D showed that it was unable to reach the embryo prior to radical protrusion. ³H-Cordycepin uptake studies using intact seeds showed that cordycepin was able to reach the embryo during the first 3 hours of incubation and at subsequent times. The pericarp and endosperm offered resistance to penetration of cordycepin into the embryo. In contrast to actinomycin D, cordycepin markedly inhibited ³H-uridine incorporation into RNA of intact seeds during the first 10 and 12 hours of incubation. About 60% of ³H-adenosine incorporation into poly A-RNA was inhibited by cordycepin during 12 hours of incubation, whereas actinomycin D had little effect. RNA synthesis appears to be essential for seed germination.     

Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex

Summary Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells ofZea mays — a species in which endomitosis occurs — andTulipa kaufmanniana — in which this process does not occur. InTulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. InZea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with³H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone.³H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments inZea mays and decreases slightly inTulipa kaufmanniana.It is argued that the differences between the incorporation of³H uridine and that or³H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

Carbon catabolism and synthesis of macromolecules during spore germination of Microsporum gypseum

Either d- or l-leucine (10(-3)m) and unsaturated long-chain fatty acids such as oleic, linoleic, and arachidonic (10(-4)m) significantly stimulated macroconidia germination of Microsporum gypseum. Saturated long-chain fatty acids did not affect germination, whereas saturated short-chain fatty acids such as caprylic, hexanoic, and butyric were completely inhibitory. Germination was followed by an increase in endogenous respiration and a decrease in dry weight of approximately 5% at 4 hr. Endogenous fatty acids and soluble carbohydrates were utilized significantly during germination. Tritiated leucine, uridine, and thymidine were incorporated respectively into protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) fractions within the first 5 min of germination. Incorporation of oleic-1-C(14) into RNA and protein was significantly increased after germ tube development. Net synthesis of RNA and protein started prior to germ tube protrusion. Increase in DNA could be detected only later. A significant increase in RNA and protein during the 4th hr of germination was correlated with vegetative development. Inhibition of respiration and incorporation of leucine-H(3) and uridine-H(3) into corresponding macromolecules by dl-fluorophenylalanine and phenethyl alcohol started before germ tube appearance. Griseofulvin significantly inhibited incorporation of uridine-H(3) and thymidine-H(3), but not of leucine-H(3). This inhibition occurred only after initial vegetative development. In contrast to the two other inhibitors, which substantially inhibited germination, griseofulvin only slightly retarded the period of germination and did not affect respiration.     

GIBBERELLIC ACID-INDUCED GERMINATION OF SPORES OF ANEMIA PHYLLITIDIS: NUCLEIC ACID AND PROTEIN SYNTHESIS DURING GERMINATION

The distribution and synthesis of nucleic acids and proteins during gibberellic acid-induced germination of spores of Anemia phyllitidis were studied in order to relate biochemical activity with morphogenetic aspects of germination. Germination is accompanied by the hydrolysis of storage protein granules and the localized appearance of cytoplasmic RNA, protein, and insoluble carbohydrates in a small area adjoining the spore wall and surrounding the nucleus. The protoplast of the spore enlarges in this region, the spore wall breaks and a protonemal cell is formed which contains many chloroplasts. A second division in the spore at right angles to the first yields a rhizoid cell. Autoradiography of ³H-thymidine incorporation has shown that DNA is synthesized both in the nucleus and in the immediately surrounding cytoplasm of the germinating spore until some time after the first division, although a strictly nuclear DNA synthesis is observed later. Synthesis of RNA and proteins is limited to the presumptive regions of the germinating spore which become the protonema and rhizoid, shifting to specific sites in these cells as germination proceeds. The nucleus of the spore continues to be biosynthetically active long after it ceases to divide.