Preformed and newly synthesized messenger RNA in germinating wheat embryos

In 6 h germinated wheat (Triticum aestivum L. cv. Cama) embryos, more than half of the messenger RNAs are actively involved in translation. Neither preformed nor newly synthesized poly A⁺-RNA is translated preferentially. Germination in the presence of cordycepin showed that the half-life of the templates is about 2 h and that the newly synthesized messengers are essential to support protein synthesis in the embryo from the first hours of germination. Most of the messenger RNAs in 6 h germinated embryos are newly synthesized. The polypeptides coded for by either the endogenous messenger ribonucleoproteins or purified poly A⁺-RNA from both dry and germinated embryos are qualitatively identical; minor quantitative differences can however be observed.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - mRNP messenger ribonucleoprotein - poly A⁺-RNA polyadenylic acid containing RNA - PB polysome buffer - GM germination medium     

Amino acid pools and protein synthesis in germinating maize embryos

Amino acid pools were analyzed in maize (Zea mays L.) axes germinated for 0, 6 and 24 h. Proteins synthesized at early (0–6 h) and late (18–24 h) stages were characterized by gel electrophoresis and fluorography after either ¹⁴C-leucine or ¹⁴C-lysine pulses. An increase of amino acid incorporation after 18–24 h was observed, as well as changes in the protein patterns of the corresponding fluorographs. Analysis of the endogenous amino acid pools showed major changes in contents of proline, alanine, isoleucine, valine, leucine and lysine. A selective increase of lysine incorporation into proteins during the late stage was detected.Under the Academic Colaboration Program between the Universidad Nacional Autónoma de México and the Colegio de Postgraduados, Chapingo, México.     

Protein synthesis patterns in barley embryos during germination

Summary The incorporation pattern of [¹⁴C] amino-acid into protein during the first 8 h of germination in isolated barley embryos (Hordeum vulgare) is described. Two maxima were recognised. The first, at 4 h, was entirely accounted for by scutellum activity and the second, at 8 h, coincided with active radicle elongation. An intervening minimum was situated at 5.5 h. The first peak was insensitive to actinomycin-D but the second showed a partial inhibition by this compound. Only slight changes in enzyme activity were associated with these periods of increased synthesis. Incorporation of [17-¹⁴C] kaurenoic acid into compounds co-chromatographing with gibberellins was followed over the same period in both embryos and scutella and high activity was found after only 2–4 h. It is concluded that, on the basis of protein synthetic activity, the scutellum is the most probable source of the initial gibberellin stimulus.     

Differential metabolism of sodium azide in maize callus and germinating embryos

Sodium azide is a potent mutagen of maize (Zea mays L.) kernels that may have potential as a point mutagen for inducing biochemical mutations in maize tissue cultures. Azide mutagenicity was evaluated in friable, embryogenic maize callus and a nonregenerable maize suspension culture by determining the number of resistant variant cell lines able to grow on media containing inhibitory concentrations of lysine plus threonine (LT). The number of LT-resistant variants selected from either culture type did not increase in response to azide treatment. In addition, there was no increase in somatic mutations in more than 100 plants regenerated from azide treated LT-resistant lines. The levels of mutagenic metabolite of azide (presumably azidoalanine), were determined by bioassay in the two azide-treated maize callus types and compared to levels of mutagenic metabolite in embryos isolated from azide-treated kernels. The two types of maize tissue cultures and isolated embryos contained similar levels of mutagenic metabolite 4 h after azide treatment indicating similar uptake and conversion of azide to mutagenic metabolite in the three tissues. Mutagenic metabolite in azide-treated embryos did not significantly decrease after 40 h. However, mutagenic metabolite levels in both azide-treated tissue cultures decreased to near background levels within 20 h providing evidence for rapid metabolism of the azide mutagenic metabolite. The lack of evidence for azide mutagenicity in maize callus and its known potent mutagenicity in kernels appears to be associated with specific differences in azide metabolism between callus tissues and kernel embryos.     

Protein breakdown and protease properties of germinating maize endosperm

Protein breakdown during germination of maize at 28° is closely correlated with the appearance of protease activity. In the first 2 days of germination, a slight disaggregation of only G₃ glutelins into more simple elements (albumin-globulins) can be observed. Between 2 and 2.5 days, there is extensive breakdown of all protein fractions, the rate of which coincides with the rate of appearance of proteolytic activity. After 2.5 days these phenomena slow down and the bulk of the endosperm proteins disappears. Three acid proteases in endosperm extracts of germinated grain (P₁₁, P₂₁ and P₂₂) have been isolated by affinity chromatography and gel filtration, and partially characterized. P₁₁ (MW 40 000) which is present in the ungerminated grain, cannot hydrolyse prolamins and is insensitive to reducing agents. P₂₁ (MW 36 000) and P₂₂ (MW 12 000), which appear on day 3 of germination, can degrade prolamins in vitro. Reducing agents enhance their activity and prevent their aggregation or denaturation. Comparative assays with different substrates suggest our enzyme preparations are principally endotype proteases with little contaminating carboxypeptidase activity.     

Maternal messenger RNA and histone synthesis in embryos of the surf clam Spisula solidissima.

Messenger RNA has been isolated from the postribosomal supernatant of Spisula solidissima eggs. This mRNA directs the synthesis of several proteins when added to the ascites or wheat germ cell free system. No histone except F1 is coded for by Spisula egg mRNA, in contrast to what has been reported previously for sea urchin egg mRNA. In sea urchin eggs histone mRNA is among the abundant species of maternal mRNA.Histones have been prepared from Spisula embryos at different development stages and histone synthesis followed by incubation with (¹⁴C)lysine. The analysis by electrophoresis on acrylamide gels indicates that the pattern of synthesis of histones changes during development and that a new histone F1 fraction is actively synthesized from the 32–64 cells stage. In earlier embryos a different F1 histone is synthesized and the mRNA for this protein may be the only histone mRNA present in eggs.     

RNA synthesis in germinating red bean seeds

Nucleic acids from ³²P-labelled germinating red bean seeds wereinvestigated by means of MAK column chromatography. 1) In cotyledons,synthesis of D-RNA occurred in the early stages of germination,3 to 24 hr after the onset of imbibition. ³²P was also incorporatedinto rRNA continuously at rather a moderate rate. DNA-RNA hybridizationexperiments revealed that the proportion of heterogeneous RNA(D-RNA) to rRNA decreased gradually. Nucleotide analysis suggestedthat tRNA was synthesized de novo, and that its CCA-end exchangewas remarkable at early stages of germination. 2) In embryos,however, the incorporation of ³²P into rRNA was very much greaterthan into D-RNA, and the exchange reaction at CCA-end of tRNAwas not detected. The role of D-RNA, found in cotyledons inthe initial stages of germination, was discussed. ¹Present address: Research Institute for Biochemical Regulation,Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya,Japan. (Received May 10, 1972; )     

Stored polyadenylated RNA and loss of vigour in germinating wheat embryos

Loss of vigour in wheat seed is associated with lesions affecting the rate of disappearance of stored poly A⁺ RNA (presumptive mRNA) in the germinating embryo when germination takes place at a sub-optimal temperature. During germination in the presence of α-amanitin and consequent of de novo polyA⁺ RNA biosynthesis, the wheat embryo can degrade up to 70% of the stored poly A⁺ RNA of the quiescent embryo before any significant reduction in the rate of protein biosynthesis in the embryo becomes apparent. It is possible that two subpopulations of poly A⁺ RNA species exist in wheat embryos during early germination, one population being degraded rapidly upon rehydration of the embryo whilst the other population supports protein biosynthesis in the initial germination stages prior to degradation.     

Lectin synthesis in developing and germinating wheat and rye embryos

Wheat (Triticum aestivum L.) and rye (Secale cereale L.) lectins are specifically synthesized during seed formation. They accumulate exponentially in the primary axes in a period coinciding with the development of this complex organ. Since the specific lectin content also increases dramatically, there is apparently an outburst of lectin synthesis during the development of the primary axes. Germinating embryos also synthesize some lectin. The fortunate availability of a highly specific procedure for the isolation of cereal lectins enabled us to follow the kinetics of their synthesis during early germination. Stored mRNAs appear to be involved in this residual lectin synthesis.     

Onset of deoxyribonucleic Acid synthesis in germinating wheat embryos

Germinating wheat embryos (Triticum vulgare var. Florence) synthesize proteins before the onset of DNA synthesis. The onset of DNA replication occurs at about 15 hours of germination and was shown to depend on proteins synthesized before 9 hours of germination with the use of blasticidin S, a specific inhibitor of protein synthesis. A 10-fold increase in the activity of DNA-dependent DNA polymerase was found in extracts derived from germinated embryos, as compared to the activity found in extracts from ungerminated embryos.     

Selective inhibition by arabinosylcytosine of oligo(U)-containing RNA synthesis in germinating wheat embryos

Arabinosylcytosine (araC) was found to influence the pattern of RNA labelling in early wheat embryos. Although the rate of uridine-¹⁴C incorporation into total RNA remained unchanged, specific radioactivity of an oligo(U)-containing subclass of nonpolyadenylated RNA was markedly lower (50%) in araC-treated (1.5 mM, 1 h) than in control embryos. The inhibitory effect was stronger for nuclear than for cytoplasmic subpopulation of these RNA species. h labelling of all other RNA fractions, including bulk nonpolyadenylated RNA as well as poly(A)-containing RNA of both nuclear and cytoplasmic origins, did not respond to the presence of araC.     

Protein synthesis in germinating Saccharomyces cerevisiae ascospores

The uptake and incorporation of macromolecular precursors in germinating Saccharomyces cerevisiae ascospores were investigated. Addition of cycloheximide at various times during germination revealed that protein synthesis can occur within 20 min after the spores are shifted to glucose-containing media. The time of initiation of uptake and incorporation of several amino acids differed; this can be attributed to differing amino acid pool levels in the spores, as well as differing transport activities. Two-dimensional gel electrophoresis of proteins labeled with [35S]methionine for various 20-min periods after germination began showed at least one protein whose synthesis begins well after the bulk of the proteins.     

Protein synthesis patterns following stage-specific heat shock in early Drosophila embryos

Summary Very short heat shocks are administered to carefully staged early embryos of Drosophila melanogaster, and the effects on protein synthesis pattern investigated. A shock as short as 2 min will induce the heat shock response (reduction of normal protein synthesis, increased synthesis of the heat shock proteins) in syncytial blastoderm or later stages. Thus the initial events of the heat shock response must occur within 2 min, and not reverse upon rapid return to 22° C. A low level of synthesis of the 70 kDa heat shock protein is sometimes visible in unshocked animals, but may be induced by the labeling procedure. Survival following a short shock is not strictly correlated with a high level of heat shock response. Pre-blastoderm embryos do not produce significant heat shock protein, but survive a 2 min 43°C heat shock better than do heat shock response competent blastoderm embryos. The protein synthesis pattern prior to the blastoderm stage is very stable, possibly enhancing survival following a short shock. Shocks of 3 min or longer are more detrimental to pre-blastoderm embryos than to later stages, confirming the role of the heat shock response in survival following a longer shock. Stage-specific developmental defects (phenocopies) may be induced by heat shock at the blastoderm or later stages. Induction of these defects may require disruption of the normal protein synthesis pattern. Use of very short heat shocks to induce the heat shock response will be valuable in identifying the precise time at which a specific defect can be induced.