Germinating Wheat Embryos Research Articles

Deoxyribonucleic acid synthesis and deoxyribonucleic acid-polymerase activity during early germination of wheat embryos at high and low viability

3H-thymidine incorporation and DNA-polymerase activity during early hours of wheat embryo germination at two viability levels have been studied. The patterns of two biosynthetic activities, as well as the dependence of DNA synthesis on protein synthesis, indicated the presence of a delay in the early phase of imbibition of the aged embryos with respect to viable germs.

RNA Synthesis in Germinating Embryonic Axes of Soybean and Wheat

The rate of synthesis of RNA during early germination of wheat and soybean embryos was investigated by ascertaining the incorporation of radioactive uridine into RNA. In wheat embryos, where the lag period preceding rapid growth is 5.5 hours, there is a 2-fold increase in RNA synthesis between 1.5 and 5.5 hours, with half of the increase occurring by 3.5 hours. In soybean axes, where the lag period is 9.5 hours, the increased rate of RNA synthesis is 5.5-fold between 1.5 and 9.5 hours, with three fourths of this increase occurring after 4 hours.Analysis of the ratio of radioactivity incorporated into the 18S and 26S rRNAs of the germinating embryos provided a further measure of the increased rates of RNA synthesis. With wheat embryos, the 26S/18S ratio increased from 1.0 at 1.5 hours to 1.5 at 3.5 hours, while with the soybean axes, distinct ribosomal patterns were obtained only after 4 hours and the 26S/18S rRNA ratio increased from 0.4 at 4 hours to 1.0 at 9 hours. The extent of methylation of the rRNA synthesized at 4 and 9 hours in the soybean axes was similar, indicating that the methylating capacity of the axes is probably not rate limiting to rRNA synthesis. In both seed embryo systems the level of UTP increased 2 to 3-fold during the lag phase of germination. With wheat embryos, the time course of the increase in UTP correlated approximately with the change in the rate of RNA synthesis. With the soybean axes, however, the increase in the rate of RNA synthesis occurred predominantly after the rise in the level of UTP.

An inhibitor of poly(adenosine diphosphate ribose) glycohydrolase found in vivo in germinating wheat embryos [proceedings

An inhibitor of poly(adenosine diphosphate ribose) glycohydrolase found in vivo in germinating wheat embryos [proceedings

Isolation of a single-stranded extrachromosomal DNA from germinating wheat embryos.

Newly-synthesized cytoplasmic non-mitochondrial DNA was isolated from wheat embryos which had been germinated in the presence of [14C]-thymidine for a time period not long enough to trigger the first post-dormant round of the nuclear DNA replication. This extrachromosomal DNA fraction consisted of linear single-stranded polydeoxyribonucleotide chains, corresponding in size to approximately 1.1 X 10(6) daltons, and amounted to about 0.5% of the total cellular DNA content. It is suggested that the appearance of the newly-synthesized polydeoxyribonucleotide chains in the cytoplasm may be a physiological signal for the initiation of the nuclear DNA replication in germinating wheat embryo cells.

Germination-induced Changes in Chromosomal Proteins of Spring and Winter Wheat Embryos

The template activity of chromatin from winter wheat embryos gradually increased during germination and was regulated with some nonhistone proteins different from the two major ones, molecular weight 39k and 59k polypeptides, previously reported.To clarify chromosomal proteins which are involved in regulation of template activity of chromatin, we studied the quantitative and qualitative changes in chromosomal proteins. Differences in acid-soluble and acid-insoluble proteins between chromatins from wheat germ and embryos germinated for various times were visualized by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.Nonhistone proteins of 39k, 41k, and 50k molecular weights were specifically present in wheat germ and in 24- or 48-hour germinated wheat embryos, thereafter greatly reduced or finally disappeared. In contrast, nonhistone protein of 37k was absent in germ and in embryos germinated for 24 hours and appeared after 48 hours of germination. Thereafter it was present in abundant amounts in 96-hour germinated winter wheat embryos and in 72-hour germinated spring embryos, corresponding to 7 and 10% of total nonhistone proteins, respectively. Histone H1, especially H1d, was slightly reduced after 48-hour germination, as much as basic nonhistone proteins having electrophoretic mobilities between H1d and H2B. Further-more, similarity and diversity of chromosomal proteins between spring and winter wheat embryos are shown in this study. A subspecies of histone H1c of spring wheat had faster electrophoretic mobility than that of winter wheat.

Analysis of NAD + in picomole amounts with sodium [ 32P]pyrophosphate and NAD-pyrophosphorylase

A new method is described for the determination of NAD + in picomole amounts. An enzymatic coupling system of NAD-pyrophosphorylase and hexokinase is used to convert sodium [ 32P]pyrophosphate and NAD + to [ 32P]ADP, glucose 6-[ 32P]phosphate, and NMN. The key step in this analysis is the selective adsorption of the reaction product [ 32P]ADP, onto activated charcoal with a solution of 1 m K 2HPO 4:10% trichloroacetic acid (1:3, v/v, pH 2). The range of concentrations of NAD + that can be measured is 1–200 pmol. The simplicity of the method allows as many as 180 samples to be assayed in 4–5 h. This procedure has been used to quantitate NAD + in crude extracts of germinating wheat embryos.

Purine salvage in cotyledons of germinating lupin seeds

Purine metabolism is much less elucidated in plants [ 1,2] than in animal tissues. In contrast to animal tissues, in plants adenosine is hydrolyzed to adenine and ribose by adenosine nucleosidase (EC 3.2.2.7.) found in leaves of various species [3-71, as well as in the cotyledons of yellow lupin seedlings [8]. For the cotyledons, the highest adenosine nucleosidase activity has been observed on day 4-5 of seed germination [9]. However, as Brown found [ IO,1 l] in legume seeds, adenine does not accumulate in their cotyledons at any stage of germination. This could suggest that the activity of purine salvage pathways is relatively high. The aim of this work was to study changes in the activity of enzymes of the purine salvage pathways in cotyledons of germinating lupin seeds. In animals, two ways of purine salvage are postulated: (i) The one-step way catalyzed by purine phosphoribosyltransferases (EC 2.4.2.7. and EC 2.4.2.8.); (ii) The two-step way dependent on purine nucleoside phosphorylase (EC 2.4.2.1.) and nucleoside kinases (EC 2.7.1.20 and 2.7.1.73.) or nucleoside phosphotransferase (EC 2.7.1.77). Some of these enzymic activities have been studied in germinating wheat embryos, but only up to the second day of germination [ 121.

Factors affecting the onset of deoxyribonucleic acid synthesis during wheat embryo germination. Study of the changes in DNA polymerases A, B and C and the pool of DNA precursors

DNA synthesis starts about 12 h after water imbibition in wheat embryos. We have determined that noticeable amounts of labelled thymidine are found inside the embryo only after 6 hr of germination. DNA polymerase C from ungerminated wheat embryos decreased markedly in activity during the first hours of germination, whereas the activities of DNA polymerases A and B increased, having a maximum at about 15 h or germination. Serological evidence has suggested a clear antigenic relationship between DNA polymerases A and C. Although the pool of ATP increases rapidly after water imbibition, the increase in the pool of dNTP species was much slower.

Dependence of protein synthesis on RNA synthesis during the early hours of germination of wheat embryos

WHEAT EMBRYO germination is characterised by an increase in fresh weight during the first 40 min of imbibition, followed by a lag period of 5 h and finally, a phase of cell elongation during which fresh weight increases at a sustained rate1. An essential step in seed germination is de novo synthesis of proteins, but for the first 40 min it is mediated by preformed mRNA transcribed during embryogenesis2 Previous reports have shown that although there is a 2.5–3-fold increase in the relative rate of protein synthesis during the lag period (40 min–5.5 h)3, there is no appreciable change in the rate of RNA synthesis4–7 With the recently developed sensitive assays for ribonucleoside triphosphates8,9, the rate of RNA synthesis and its mode of regulation in early germination of wheat embryos was reexamined10 In contrast to previous findings4–7, our results clearly show a two- to threefold increase in the rate of RNA synthesis during the lag period of wheat embryo germination This increase in RNA synthesis could conceivably account for the threefold increase in the rate of protein synthesis4 and raises the possibility that protein synthesis may depend on RNA synthesis after the first 40 min of germination We describe here experiments undertaken in wheat (Triticum aestivum L.) embryos to study the effect of inhibition of RNA synthesis on the rate of protein synthesis. Our data suggest that protein synthesis depends on RNA synthesis, and that the substrate level of mRNA is not a rate-limiting determinant for protein synthesis during the early hours of germination.

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.

Rapid Increase in UDP-Glucose during Early Wheat Embryo Germination

The cellular content of UDP-glucose in isolated wheat (Triticum aestivum L.) embryo increases 8-fold during the first 40 minutes of imbibition. An additional 3-fold increase in the amount of UDP-glucose was observed in the next 5 hours of germination. This communication also describes a unique, quantitative method to achieve a high sensitivity in a direct determination of UDP-glucose with Na [(32)P]pyrophosphate and UDP-glucose pyrophosphorylase. The sensitivity of the assay for UDP-glucose is 10 picomoles.

Proteins bound to poly(A) sequences of polyribosomes from germinating wheat embryos

A preparation of purified polyribosomes was isolated from wheat embryos germinating for 1 h with [ 3H]adenosine. Treatment of polyribosomes with pancreatic ribonuclease leads to the liberation of particles containing poly(A) sequences. Poly(A)-containing complexes show a heterogeneous distribution in the region 8–15 S. The ability of 8–15 S material to be absorbed to membrane filters under the conditions of low ionic strength and its sensitivity to pronase indicate that poly(A) sequences in polyribosomes are associated with protein. Analysis of this protein by electrophoresis in a polyacrylamide gel demonstrates the presence of two polypeptides with the molecular weights equal to 86 000 and 52 000.

Age-dependent DNA labeling and deoxyribonucleotide synthesis in wheat seeds

Utilisation of ribonucleosides as precursors of DNA biosynthesis was studied in germinating wheat embryos because the reductive pathway leading to deoxyribonucleotides is very difficult to demonstrate in extracts of higher plants in vitro. [5- 3H]Cytidine and [6- 3H]uridine are incorporated into wheat DNA (RNA-free) via ribonucleotide reduction without intermediate scission of the glycosidic bond. This reaction is observed at 20–30 h after the onset of germination only in aged (2–4-year-old) seeds while the embryos isolated from fresh grains show very little cytidine incorporation; in contrast, thymidine incorporation into DNA between 10 and 18 h of germination is not age dependent. Fresh wheat contains a soluble, heat-stable inhibitor fraction, most probably a modified oligonucleotide, which efficiently prevents cytidine incorporation when added to old embryos together with the labeled nucleoside. This material also inhibits purified Escherichia coli ribonucleotide reductase and is thought to be part of the control system for ribonucleotide reduction in wheat; it may gradually decay during storage of the seeds. Dry wheat embryos do not contain deoxyribonucleoside triphosphates. Pool sizes of dATP and dTTP in germinating embryos were found to reach 1 pmol/μg DNA at 10–15 h of germination (i.e. before ribonucleotide reduction) and were independent of the age of seeds. These data suggest that wheat contains other preformed dexoyribonucleoside derivatives which are phosphorylated at an early time and can initially sustain DNA synthesis. Induction of measurable ribonucleotide reductase activity in fresh winter wheat was for the first time accomplished by 15 days of vernalization of the seeds at +2°C.

Preformed Messenger RNAs and Early Wheat Embryo Germination

Wheat (Triticum aestivum L.) embryo homogenates have been fractionated into three cell fractions from which RNA was extracted and assayed for mRNA content by in vitro translation and by [(3)H]polyuridylic acid hybridization. In dry embryos the preformed mRNAs are distributed equally between a rapidly sedimenting "pellet" fraction and a cytoplasmic "ribosomal/subribosomal" fraction. During germination 25 to 40% of the total mRNA becomes polyribosomal. The remaining 60 to 75% is retained in the pellet and ribosomal/subribosomal fractions.To compare the nucleotide sequences of the different mRNA fractions, cDNAs were transcribed from polyribosomal (A+) RNA of 40-minute imbibed embryos and from total A(+) RNA of dry embryos, and the ability of these cDNAs to hybridize with the more prevalent classes of mRNA from the different cell fractions was analyzed. The results suggest that there is no significant difference between the preformed mRNAs that move into polyribosomes and those remaining in the nonpolyribosomal fractions. In addition there appears to be no difference between the mRNAs of polyribosomes from embryos germinated for 45 minutes and 5 hours. Between 5 hours and 2 days, however, there is a considerable change in the mRNA composition of the embryos. We conclude for the prevalent classes of message, that the preformed mRNAs of the wheat embryo are not involved in temporal regulation of early development but that they function primarily to allow a rapid resumption of growth upon exposure of the embryo to water.

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [(14)C]acetate and [(35)S]sulphate to 2-3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [(35)S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.

Regulation of RNA synthesis in early germination of isolated wheat (Triticum aestivum L) embryo

THE change from dormancy to germination of seeds requires water and an appropriate temperature. Wheat embryo germination is characterised by an initial increase in fresh weight, followed by a 5-h lag1. Although RNA synthesis is one of the earliest biologically measurable activities in wheat embryo germination2,3, its mode of regulation has not been established. Reports3–6 have suggested that the rate of synthesis does not change during the first 6 h of germination. But no adjustment was made for the specific activities of ribonucleoside triphosphate, UTP. This would have led to a misinterpretation of incorporation data when there was either a change in the concentration of the nucleotide or a difference in the rate of phosphorylation from nucleoside to nucleotide as germination progressed. To investigate further whether there is a change in the rate of RNA synthesis in the germination phase, we have used recentlydeveloped enzyme assays7–9 to measure picomole changes in purine pyrimidine ribonucleotide levels. We found a threefold increase in the rate of RNA synthesis of wheat embryos germinated for 40 min–5.5 h. We used the reaction catalysed by uridine 5′-diphosphoglucose pyrophosphorylase to determine the content of UTP. The key step in this analysis is the selective adsorption of the reaction product, UDP-14C-glucose, on to activated charcoal in the presence of 0.8 M Trizma base9.

Analysis of glucose 1-phosphate at the picomole level with [ 14C]UTP and uridine 5′-diphosphoglucose pyrophosphorylase

A new sensitive method is described for glucose 1-phosphate analysis. The key reaction is the pyrophosphorolysis of UDP-glucose catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase. The reaction product, [ 14C]UDP-glucose, is separated from [ 14C]UTP by adsorbing [ 14C]UTP selectively onto polyethyleneimine cellulose or by separating both labeled compounds on one-dimensional polyethyleneimine thin-layer chromatograms. The sensitivity of the method for glucose 1-phosphate analysis is 5 pmol. The method has been successfully employed to monitor the level of glucose 1-phosphate in early germination of wheat embryos.

A rapid quantitative method for measuring UTP, UDP, and UMP in the picomole range

A procedure for the determination of picomole amounts of uracil nucleotides is described. The key reaction is the condensation of UTP and [ 14C]glucose 1-phosphate catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase yielding UDP-[ 14C]glucose. The product is determined by selective adsorption onto charcoal in the presence of 0.8 m Trizma Base. UDP is measured as UTP after its conversion in an incubation with excess ATP and nucleoside diphosphate kinase. Similarly, UMP is analyzed after it is converted to UDP by nucleoside monophosphate kinase. The uracil nucleotide content of germinated wheat embryos had been determined with this method.

Isolation and characterization of 32P-labeled mitochondrial and cytosol ribosomal RNA from germinating wheat embryos

With the aim of preparing highly labeled material, the incorporation of [ 32P]-orthophosphate into mitochondral and cytosol ribosomal RNA was examined in germinating wheat embryos. Nucleic acids were extracted from mitochondria and from post-mitochondrial supernatant (cytosol) prepared from homogenates of viable embryos (8 g) imbibed for 24 h in a medium containing [ 32P]orthophosphate (100 mCi). High-molecular-weight ribosomal RNA was selectively precipitated in the presence of 3 M NaCl and was further resolved on sucrose density gradients and polyacrylamide gels. Both the mitochondrial and cytosol NaCl-insoluble RNA fractions were found to contain two major radioactive components, corresponding to the large (26 S) and small (18 S) rRNA species. On non-denaturing gels, these species had apparent molecular weights of 1.3 and 0.67 million daltons (cytosol) and 1.3 and 0.75 million daltons (mitochondrial). The individual, purified [ 32P]rRNA components (isolated from sucrose gradients) had specific activities of 2–3 · 10 6 cpm A 260 unit, and were suitable for analysis of nucleotide composition and sequence. By hydrolyzing the individual [ 32P]RNA specimens with purified snake venom phosphodiesterase and resolving the products by two-dimensional paper chromatography, it was possible to determine the specific activities (cpm/μmol) of the four major 5′-nucleotide constituents. The results indicated that there had been no differential 32P-labeling of the nuclear and mitochondrial pools of ribonucleoside 5′-triphosphates (rNTP) during the 24 h imbibition period; however, as previously observed in this system (Lau, R.Y., Kennedy, T.D. and Lane, B.G. (1974) Can. J. Biochem. 52, 1110–1123), there had been unequal 32P-labeling of the individual rNTPs in both the mitochondria and nucleus. The relative specific activities of the 5′-nucleotide constituents of the mitochondrial and cytosol rRNA species were essentially the same, and in the order pA ⋍ pU > pG > pC . By making suitable corrections for these differences in specific activity, the nucleotide composition of each of the [ 32P]rRNA specimens could be calculated. Only minor differences in the proportions of the four major 5′-nucleotide constituents and in the overall G + C content were apparent when the cytosol rRNA components were compared with their mitochondrial counterparts. However, the mitochondrial RNA species were found to have a markedly lower content of modified nucleoside constituents (pseudouridine and O 2′-methylnucleosides). These data provide further evidence for the existence of distinctive species of high-molecular-weight ribosomal RNA in wheat embryo mitochondria, and demonstrate the utility of the wheat embryo system for the production of isotopically labeled, higher plant mitochondrial RNAs.

Regulation of o-diphenolase activity by auxin and abscisic acid in germinating wheat embryos

Abstract During germination o -diphenolase activity increased several fold ( x 13) in wheat embryos. The enzyme activity and the number of multiple forms wer