Untersuchungen über RNA- und proteinbiosynthesen in aktivierten und temperaturblockiertenAgrostemma githago-samen: einfluss von benzylaminopurin

Embryos ofAgrostemma githago seeds blocked by higher temperature are suitable objects for the study of the regulation of gen expression independent of transeriptional processes. Germination of afterripened seeds can be prevented by imbibing at 30°C, whereas at 20°C germination will be completed after 30 h of soaking (BORRISS 1956). In temperature-blocked embryos RNA is synthesized with undiminished intensity, whereas DNA and protein syntheses are remarkably reduced. Equal amounts of poly(A)-RNA (probably mRNA), enriched by affinity chromatography on oligo(dT)-ceIlulose columns, are detected in blocked and activated embryos. We suggest that in blocked embryos not synthesis itself, but the availability of mRNA to polysomal fraction is reduced. Benzylaminopurine is able to break secondary dormancy ofAgrostemma githago seeds. In benzylaminopurine-treated seeds there is only a slight increase of the incorporation of¹⁴C-L-leu-cine in protein.     

Adenylate metabolism of embryonic axes from deteriorated soybean seeds

RNA and protein syntheses in axes excised from dry soybean (Glycine max L.) seeds at different levels of deterioration were assayed. Low rates of protein synthesis in slightly deteriorated seeds were not due to losses in ribosomal or soluble fraction activities. However, the lowered rates of RNA and protein syntheses of deteriorated seeds were associated with reduced ATP content of the tissues. Adenine and adenosine conversions to ATP were reduced in deteriorated axes, and these reductions were reflected in reduced incorporation of these compounds into RNA.     

Protein and nucleic acid synthesis during imbibition of dormant and after-ripened Vaccaria pyramidata seeds.

The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [³H]thymidine incorporation does not start until the second period of the imbibition phase. [³H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [³H]uracil and l-[¹⁴C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [³H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [³H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds.     

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     

Untersuchungen über die Regulation der DNS-Synthese während des Aktivitätswechsels vonAgrostemma githago-Samen

The relationships between DNA synthesis and germination capacity ofAgrostemma seeds have been studied. Protein synthesis and RNA synthesis are activated at the very beginning of imbibition, whereas DNA synthesis starts in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30° C), or aged seeds with a low germination capacity are characterized by a remarkably reduced protein synthesis. DNA synthesis is also reduced. The inhibition of protein-synthesis ofAgrostemma embryos fed with cycloheximid or actinomycin D causes a depression of DNA synthesis. These results indicate that the initiation of DNA synthesis of imbibingAgrostemma seeds depends on the synthesis of special proteins. Abscisic acid inhibits growth as well as DNA synthesis of isolatedAgrostemma embryos. Mitomycin inhibits germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also show a reduced incorporation of³H-thymidine in DNA. We suggest that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, is involved in the mechanism of afterripening of theAgrostemma seeds.     

An ATP-synthesizing system in seeds

Using onion seed powder, a semi-in vitro system for ATP synthesis in seeds has been developed. The system requires AMP, phosphoenolpyruvate (PEP) and orthophosphate with apparent Km values of 0.8, 1.5 and 3.0 mM, respectively. ATP synthesis is pH-dependent with a sharp optimum at pH 6.4, it exhibits linearity with time up to 40 min, and with a seed powder concentration between 25 and 150 mg ml^-1. The system is stimulated by low concentrations (<25 mM) of K⁺ and Mg²⁺ but is inhibited by higher concentrations of K⁺ and Mg²⁺ as well as by low concentrations of Li⁺, Na⁺ and especially Ca²⁺. The maximal rate is about 5 pmol min^-1 mg seed powder^-1 in dry onion seeds. During seed imbibition the rate of activity increases by about 120% after 3 h, reaching a plateau which is steady up to 18 h, when the radicle emerges. A comparison of the ATP content in seeds during the early period of imbibition with the capacity of ATP synthesis at this stage reveals that the described system could provide, during germination, 100 times more ATP than that found in imbibed seeds. The system is shown to be present in ten different types of seeds.     

The cytoplasmic distribution and characterization of poly(A)+RNA in oocytes and embryos of Drosophilia.

Using the presence of poly(A) tracts as a marker for mRNA, we have examined the distribution of this class of RNA between polysomes and free RNP particles. This has been done in mature oocytes and in embryos aged for various times from fertilization through to hatching of a larva. The proportion of ribosomes that are in polysomes to those that are not has been calculated. In mature oocytes, 58% of the poly(A)⁺ RNA and 72% of the ribosomes are not in polysomes. By 1 hr, this drops to 51% of the poly(A)⁺ RNA and 48% of the ribosomes. By 7 hr, a plateau is reached: 30% of each are not in polysomes. The poly(A)⁺ RNA in the cytoplasm of oocytes and 1-hr embryos is found in particles with an average size of 50S and a range of 30–70S. The poly(A)⁺ RNA ranges in size from 7 to 40S, with an average size of 22S. The polyA from this RNA is 50–200 nucleotides long with an average of 115 nucleotides. These data have allowed us to calculate that 1–2% of the total RNA is poly(A)⁺ RNA.     

Differences in Patterns of Newly Synthesized Proteins in Imbibing Embryos of After-ripened and Aged Seeds of Agrostemma githago and Comparison of Protein Synthesis In Vivo and In Vitro

Patterns of newly synthesized proteins in imbibing after-ripenedyoung and aged Agrostemma embryos show differences in the dynamicsof cotyledons and axes. In the course of imbibition the terminationof some syntheses in the embryonal parts of aged seeds is delayed.As was shown by in vitro translation, mRNAs coding for the synthesisof some proteins are still present in the embryos when proteintranslation has already finished. Key words: Protein pattern, in vivo protein synthesis, in vitro protein synthesis, after-ripened embryos, aged embryos, Agrostemma githago     

Proteinbiosynthesen in dormanten und nachgereiften embryonen und samen von Agrostemma githago

Seed germination of Agrostemma githago is prevented by inhibitors of protein and RNA synthesis. Thus protein as well as RNA synthesis are essential prerequisites for germination. Early protein synthesis of Agrostemnia embryos can be completely inhibited by cycloheximide and cordycepin. During the aging of seeds there is a considerable decrease in germination capacity and protein synthesis. In dormant and afterripened embryos of Agrostemma githago¹⁴C-leucine and ¹⁴C-uracil are incorporated in protein and RNA respectively with nearly the same intensity, whereas RNA and protein synthesis of dormant seeds and embryos starts earlier than in those subjected to afterripening. ³H-uracil-labelled RNA from dormant and afterripened embryos are able to hybridize on oligo-dT-cellulose to the same extent. There is a similarity in the protein pattern of dormant and afterripened embryos revealed by electrophoresis in polyacrylamide gels of double-labelled proteins. According to these results dormancy of Agrostemma githago is not caused by a general but by a specific metabolic block.     

Stimulation of synthesis and translational activity of polyadenylated messenger RNA in wounded potato tubers by 2, 4-dichlorophenoxyacetic acid

Mechanical wounding of potato tubers induced a rapid synthesis of RNA in the wounded tissues. Both total and polyadenylated RNA increased with time after wounding. Treatment of wounded tissues with the synthetic hormone 2,4-dichlorophenoxyacetic acid (2,4-D, 10^?4 M) further stimulated their syntheses. The poly (A) ⁺RNA from hormone treated tissues was more active in in vitro protein synthesis. The in vitro translation of poly (A) ⁺RNA from both hormone treated and untreated tissues was inhibited by 7-methylguanosin-5′ phosphate, while 7-methylguanosine had no effect, suggesting that both poly (A) ⁺RNAs contained a blocked 5′-cap structure and that the cap structure was important for in vitro protein synthesis.     

Rates of synthesis of major classes of RNA in Drosophila embryos.

We have been successful in labeling to high specific activity (3 × 10⁵ dpm/μg) the RNA synthesized by large numbers of Drosophila embryos. Embryos of various developmental stages were rendered permeable with octane and labeled with [³H]uridine for 1 hr. At each stage the total dpm incorporated into RNA and the specific activity of the UTP pool were measured and used to calculate the absolute rate of RNA synthesis per embryo. This rate increases during embryonic development, from 1 pmole UTP/hr at 2 hr after oviposition to 6 pmoles UTP/hr at 15 hr. The rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNAs were determined by analyzing the fractionated RNAs from each stage by sucrose gradient sedimentation. There is a significant activation of nuclear RNA synthesis at the blastoderm stage (approximately 2 hr after oviposition). After blastoderm, the rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNA per embryo increase continuously; the rate of synthesis of each of these classes per nucleus, however, remains fairly constant. After making corrections for turnover during the labeling period, we find that the rates of synthesis of the major classes of RNA per nucleus at the gastrula stage are: cytoplasmic poly(A)⁺ RNA, 0.06 fg/nucleus-min; hnRNA, 0.86 fg/nucleus-min; and ribosomal RNA, 0.46 fg/nucleus-min. These rates are compared to rates of RNA synthesis in sea urchin embryos.     

Nucleic acid and protein synthesis and loss of vigour in germinating wheat embryos

A study has been made of the RNA and protein synthesising systems of wheat embryos isolated from seed lots having high viability but differing in vigour. The rate of RNA and protein synthesis in wheat embryos during the early hours of germination is related to the vigour of the seed lot. The imposition of a stress factor, in the nature of a sub-optimal germination temperature, during germination of isolated wheat embryos magnifies the differences in rates of protein and RNA synthesis between high and low vigour seed. Using cell-free protein synthesising systems it has been demonstrated that an important difference between high and low vigour embryos lies in the relative levels of messenger RNA in the embryo. High vigour embryos contain relatively higher levels of poly A⁺-RNA (i.e. potential mRNA species) than lower vigour embryos and furthermore the level of poly A⁺-RNA in high vigour embryos increases during early germination whilst in lower vigour embryos the level decreases. The difference in poly A⁺-RNA levels accounts, at least partially, for the differences in rates of protein synthesis observed between embryos from high and low vigour wheat seed during early germination at both optimal and sub-optimal germination temperatures.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - poly A⁺-RNA polyadenylated RNA - GM germination medium - PMS post-mitochondrial supernatant fraction     

Modulation of poly(A)+ RNA levels in fungal-infected wheat embryos through the selective inactivation of RNA polymerase II and poly(A) polymerase

Infection of germinating wheat embryos by a fungal pathogen (Drechslera sorokiana) drastically lowered (70–73%) the relative abundance of poly(A)⁺ RNA. This was paralleled by a significant loss in the activities of RNA polymerase II (60–70%) and poly(A) polymerase (80–85%) enzymes. The inhibition of RNA polymerase II (60–65%) and poly(A) polymerase (70–85%) activities was also witnessed by the in vitro addition of the fungal extract to the enzyme preparations isolated from healthy embryos. The fungal extract showed negligible phosphatase and nuclease activities. This ruled out the possibility of rapid degradation of the labelled substrate [³H]ATP, primer RNA, or even the labelled reaction products under our assay conditions. The inhibitory effect of the fungal extract could be alleviated by fractionating the treated enzyme preparation by phosphocellulose chromatography. This indicated that the fungal extract was directly responsible for the inactivation of the polymerases in a reversible manner. The inhibitory function of the fungal extract was destroyed by treatment with pronase, but not with RNAase A and RNAase Ti. Poly(A) ‘tails’ were enzymatically excised from ³²P-labelled poly(A)⁺ RNA and fractionated on acrylamide gels for autoradiographic analysis. The lengths of the ³²P-labelled poly(A) ‘tails’ in control and infected embryos turned out to be identical (64 nucleotides). Our results suggest that the relative abundance of poly(A)⁺ RNA is diminished in fungal-infected wheat embryos through the selective inactivation of RNA polymerase II and poly(A) polymerase enzymes.     

Correlation between seed longevity and RNA integrity in the embryos of rice seeds

The mature embryos of rice seeds contain translatable mRNAs required for the initial phase of germination. To clarify the relationship between seed longevity and RNA integrity in embryos, germinability and stability of embryonic RNAs were analyzed using the seeds of japonica rice cultivars subjected to controlled deterioration treatment (CDT) or long periods of storage. Degradation of RNA from embryos of a japonica rice cultivar “Nipponbare” was induced by CDT before the decline of the germination rate and we observed a positive relationship between seed germinability and integrity of embryonic RNAs. Moreover, this relationship was confirmed in the experiments using aged seeds from the “Nipponbare”, “Sasanishiki” and “Koshihikari” rice cultivars. In addition, the RNA integrity number (RIN) values, calculated using electrophoresis data and Agilent Bioanalyzer software, had a positive correlation with germinability (R²=0.75). Therefore, the stability of embryonic RNAs required for germination is involved in maintaining seed longevity over time and RIN values can serve as a quantitative indicator to evaluate germinability in rice.     

Macromolecular synthesis during plant embryogeny: rates of RNA synthesis in Phaseolus coccineus embryos and suspensors

A comparative study of RNA metabolism as an indicator of major changes of tissue organization, cell number, and physiology in the two developmentally and cytologically distinct parts of the bean embryo, the organogenetic part and the suspensor, was carried out. The metabolism of RNA was determined separately for these two parts of embryos removed aseptically from seeds at different times during embryogeny and incubated in culture medium containing ³H-adenosine. Equilibration of ATP in the nucleotide pool, ATP pool size and specific activity, total RNA content, rate of RNA synthesis in culture, rate of RNA synthesis and specific activity during embryogeny, and total protein content were determined. Synthetic activity of the suspensor was highest early in development and then declined, whereas synthetic activity of the organogenetic part increased throughout development. These changes may reflect developmental and functional differences in the two parts of the embryo.     

Relative amounts of newly synthesized poly(A)+ and poly(A)- messenger RNA during development of Xenopus laevis

The relative amounts of newly synthesized poly(A)⁺ and poly(A)^? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)⁺ and poly(A)^? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)⁺ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)^? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)^? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)^? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)^? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)⁺ and poly(A)^? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 10⁵. The poly(A) segment of poly(A)⁺ mRNA is about 125 nucleotides long. Analysis of the poly(A)^? mRNA fraction has shown that it lacks poly(A)₁₂₅.     

VARIABILITY IN RATIOS OF PHYTOPLANKTON CARBON AND RNA TO ATP AND CHLOROPHYLL A IN BATCH AND CONTINUOUS CULTURES1,2

The feasibility of estimating phytoplankton carbon and RNA concentrations from measurements of ATP and chlorophyll a (chl a) concentrations was studied using chemostat populations of the marine diatom Thalassiosira weissflogii (Grunow) Fryxell & Hasle (= T. fluviatilis Hustedt). C:ATP and RNA:ATP ratios were studied for six additional marine species in batch culture representing five classes of phytoplankton. Statistical analyses revealed that both the growth rate and the factor limiting growth (NO₃^-, NH₄⁺, PO₄^3- or light) could alter C:ATP, RNA: ATP, C:chl a and RNA:chl a ratios by amounts which were large compared to measurement error. An analysis of variance of the batch culture results indicated that both species and the source of inorganic nitrogen (NO₃^-, or NH₄⁺) had a significant effect on C:ATP and RNA:ATP ratios. Light had less of an influence on C:ATP and RNA:ATP ratios than on C:chl a and RNA:chl a ratios, and for this reason we feel that phytoplankton C and RNA concentrations can be estimated with greater reliability from ATP than from chl a measurements. The range of C:ATP and RNA:ATP values found for T. weissflogii under a variety of growth conditions was similar to that for the six additional species grown in batch culture, suggesting that this range of values is indicative of the extremes likely to occur in living cells. Our results and additional data in the literature indicate that phytoplankton C and RNA concentrations can be estimated to within a factor of two by multiplying ATP concentrations by 311 and 35, respectively, in N limited systems, and by 341 and 36, respectively in PO₄^3- limited systems.     

Nucleic Acids Synthesis of Nuclear Polyhedrosis Virus in Cultured Embryonic Cells of Silkworm

Embryos of the silkworm, Bombyx mori L., were dispersed by trypsin and the dissociated cells were cultured for infection with nuclear polyhedrosis virus (NPV) of the silkworm. The monolayer and suspension cultures were infected with NPV. RNA and DNA syntheses in the normal and NPV-infected cells were measured by incorporation of ³²P into RNA and DNA fractions. RNA and DNA syntheses in the cells after infection significantly increased over those in control cells (mock infection). The effects of actinomycin D, chloramphenicol and mitomycin C on RNA and DNA syntheses in infected cells were examined. The syntheses were inhibited by the antibiotics. It was suggested that the cellular DNA synthesis was inhibited by the viral infection, because the mitomycin C-resistant DNA synthesis was found in the normal cells but not in the infected cells treated with mitomycin C. The rate of DNA synthesis induced by NPV was immediately dropped to that of control cells by addition of chloramphenicol, while the RNA synthesis induced by NPV was not affected for 6 hr after the addition of chloramphenicol. If the antibiotic did not affect the size of precursor pools, this event suggested that the RNA polymerase concerned with viral RNA synthesis was more stable than the DNA polymerase participating in the viral DNA synthesis. The viral DNA as templates for RNA and DNA syntheses was decomposed by mitomycin C.