Rye Embryos Research Articles

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Metabolism of aged seeds. Changes in rye embryo polyribosomes during seed aging

Germination, total dehydrogenase activity, ribosomal proteins and ribonucleic acids of embryos obtained from winter rye harvested in different years and of distinctly differing viability were studied. Diminishing viability was accompanied by declines in the amount of ribosome-bound mRNA and a drop in the intensity of its synthesis during germination. Electrophoretic analysis of control embryo (fully viable) ribosome proteins revealed 55 different bands: 10 acidic and 45 basic. The embryos of aged seeds had 43 different bands of which 8 were acidic, 35 basic and almost all of which had an altered electrophoretic mobility as compared with the control sample. As aging progressed, the percentages of lysine, histidine and arginine increased while those of tyrosine, methionine, proline and serine decreased.

The metabolism of aged seeds. The involvement of the pentose phosphate pathway in respiration in germinating rye grains of various ages

Winter rye grains from different harvest years (having distinctly different viabilities) were studied in terms of germination, total dehydrogenase activity and growth analysis. Reduction of seed vigor and viability was accompanied by a decrease in the intensity of embryo respiration during germination. The use of (1-<sup>14</sup>C) glucose and (6-<sup>14</sup>C) glucose showed that germinating rye embryos catabolize glucose through glycolysis, Krebs cycle and the pentose phosphate pathway. It was also shown that the C<sub>6</sub>/C<sub>1</sub> ratio in respiring embryos initially increased during the first 48 hours of germination, then dropped, which suggests a mounting contribution of the PP pathway to the overall cotabolism. The oxidation of glucose in, embryos from the most deteriorated grains proceeded through glycolysis and Krebs cycle only.

Development of rye embryo. I. Preliminary investigations on ultrastructure of 7-day-old proembryo

Ultrastructural examination of 7-day-old rye proembryos did not reveal distinct differences in the cell structure of its particular parts. It was noted, however, that in the cells of the apical part vacuoles and multivesicular bodies were more numerous than, in the basal part. These specific features of the cells of the apical part of the proembryo (together with data of other authors) seem to indicate an early stage of development of the embryonal scutellum.

Studies on DNA integrity and DNA repair in germinating embryos of rye (Secale cereale).

ABSTRACT Experimental evidence is presented to show that single-stranded breaks occur in nuclear DNA during ageing of rye embryos in the dry state. Total DNA is retained and on imbibition unscheduled DNA synthesis occurs which can be attributed to DNA repair before the onset of semi-conservative DNA replication.

Expression analysis of somatic embryogenesis-related SERK, LEC1, VP1 and NiR ortologues in rye (Secale cereale L.)

The genetic basis of the regeneration process in cultured immature embryos of rye (Secale cereale L.) was analyzed. The experiments were designed to reveal differences between the in vitro culture responses of two inbred lines: L318 (a high regeneration ability) and L9 (a low potential for regeneration). The rye ortologues of plant genes previously recognized as crucial for somatic embryogenesis and morphogenesis in vitro were identified. Using oligonucleotide primers designed to conserved regions of the genes Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon 1 (LEC1), Viviparous 1 (VP1) and NiR (encoding ferredoxin-nitrite reductase), it was possible to amplify specific homologous sequences from rye RNA by RT-PCR. The transcript levels of these genes were then measured during the in vitro culture of zygotic embryos, and the sites of expression localized. The expression profiles of these genes indicate that their function is likely to be correlated with the in vitro response of rye. In line L9, increased expression of the rye SERK ortologue was observed at most stages during the culture of immature embryos. The suppression of ScSERK expression appears to start after the induction of somatic embryogenesis and lasts up to plant regeneration. The rye ortologues of the LEC1 and VP1 genes may function in a complimentary manner and have a negative effect on the production of the embryogenic callus. The expression of the rye NiR ortologue during in vitro culture reveals its importance in the process of plant regeneration.

Genetically directed differential subtraction chain products related to in vitro response of immature embryos of rye (Secale cereale L.): isolation, characterization, and expression analysis

Winter rye (Secale cereale L.) is known to be recalcitrant to tissue culture response (TCR). Moreover, the mechanisms controlling TCR are poorly recognized. In the present study, a Genetically Directed Differential Subtraction Chain (GDDSC) strategy was used to isolate genomic regions associated with TCR. Two pairs of bulks, R-NR and E > 90–E < 25, were prepared, and for each pair, the bulk was used both as a tester and as a driver. After eight rounds of subtraction, 45 unique GDDSC products were obtained. To verify the connection between GDDSCs and TCR two approaches were applied: Real-Time RT-PCR analysis and genetic mapping. The expression profiles of four out of six investigated products agrees with the phenotype and subtraction direction. Two from the developed GDDSC-SCAR markers showed polymorphism in lines L9 and L318, the parental components of a mapping population. The polymorphic SCAR GDDSC markers were mapped on the rye chromosome 4R (SCAR-GDDSC NR 440BamHI) and 6R (SCAR-GDDSC E < 25 340BamHI). The marker E < 25_340B9 was localized in the border region of QTL for embryogenic callus production.

Antioxidant activity of rye caryopses and embryos extracts

Phenolic compounds were extracted with 80% methanol from caryopses and embryos of rye (cv. Dańkowskie Złote and Amilo). In all extracts, reducing power, scavenging effect on DPPH radical, and antioxidant activity in a &amp;beta;-carotene-linoleate model system were examined. The highest content of total phenolic compounds was noted in the extract from caryopses of Amilo (7.93 mg/g of extract). UV spectra of all extracts were characterised by maxima originated from phenolic acids (320, 326 and 328 nm), and by maxima at shorter wavelengths (272 and 274 nm) attributed to other phenolic compounds. All extracts showed a good antioxidant activity in a &amp;beta;-carotene-linoleate model system. This activity was similar to that reported before in leguminous seeds extracts. The antioxidant activities of the extracts from the caryopses of Dańkowskie Złote and the embryos of both cultivars were very similar, especially during the second part of the incubation period. The extract of Amilo embryos showed a slightly weaker antioxidative effect. The weak antiradical effects in the experiments with DPPH radical and a weak reducing power were characteristic for all the extracts investigated. &amp;nbsp;

Callus formation and plant regeneration from immature and mature embryos of rye (Secale cereale L.)

An efficient protocol was developed to regenerate entire plants from immature embryos of elite genotypes of rye as a prerequisite to plant transformation. Three winter genotypes and one spring genotype were tested using both immature and mature embryos as explants. Four types of callus initiation media and five kinds of regeneration media were tested in all possible combinations. Immature embryos gave much higher levels of plant regeneration than mature embryos, but mature embryos could be induced to regenerate plants for all genotypes and media tested, although at low levels. A minimum stage of embryo development must be reached before embryos can be cultured successfully. Genotypic effects were less pronounced than those reported for inbred cereal species such as wheat and barley, but there was an effect of genotype on percentage of callus formation. There was a significant interaction between genotype and initiation media. Composition of the initiation media affected both the percentage of callus formation from embryos and subsequent frequencies of plant regeneration. Composition of the regeneration media had no effect on level of plant regeneration. Immature embryos of all genotypes tested could be induced to produce 90–100% callus on appropriate initiation media and all regenerated shoots from approximately one-half to three-quarters of the calluses produced.

In vitro translation of messenger RNA in a rabbit reticulocyte lysate cell-free system.

The identification of specific messenger RNA molecules and the characterization of the proteins encoded by them, has been greatly assisted by the development of in vitro translation systems. These cell-free extracts comprise the cellular components necessary for protein synthesis, i.e., ribosomes, tRNA, rRNA, amino acids, initiation, elongation and termination factors, and the energy-generating system (1). Heterologous mRNAs are faithfully and efficiently translated in extracts of HeLa cells (2), Krebs II ascites tumor cells (2), mouse L cells (2), rat and mouse liver cells (3), Chinese hamster ovary (CHO) cells (2), and rabbit reticulocyte lysates (2,4), in addition to those of rye embryo (5) and wheat germ (6). Translation in cell-free systems is simpler and more rapid (60 min vs 24 h) than the in vivo translation system using Xenopus oocytes.

The requirement for DNA repair in desiccation tolerance of germinating embryos

AbstractIt is proposed that desiccation tolerance in the embryo of seeds depends upon the capacity to repair damage to genomic DNA when the desiccated embryo is rehydrated. From a study of imbibed and hydrated embryos of rye (Secale cereale) and wild oat (Avena fatua) evidence is provided that it is neither the extent of water uptake by the cells, the ensuing stability of the DNA to desiccation, nor the onset of S-phase DNA synthesis in the first cell cycle of germination that determines whether the desiccated embryo will survive. It is shown that when α- and β-polymerases of DNA repair are inhibited by aphidicolin and dideoxythymidine-5'-triphosphate, respectively, a γ-irradiation-induced DNA fragmentation cannot be fully repaired. It is shown that in hydrated embryos, at a stage when desiccation tolerance is lost, embryo cells still repair irradiation-induced damage, but this repaired DNA is unstable to desiccation and cannot be rerepaired when water is again made available. The failure to re-repair on rehydration appears to be critical to embryo survival and successful germination.

In vitro translation of messenger RNA in a rabbit reticulocyte lysate cell-free system.

The identification of specific messenger RNA molecules and the characterization of the proteins encoded by them, has been greatly assisted by the development of in vitro translation systems. These cell-free extracts comprise the cellular components necessary for protein synthesis, i.e., ribosomes, tRNA, rRNA, amino acids, initiation, elongation and termination factors, and the energy-generating system (1). Heterologous mRNAs are faithfully and efficiently translated in extracts of HeLa cells (2), Krebs II ascites tumor cells (2), mouse L cells (2), rat and mouse liver cells (3), Chinese hamster ovary (CHO) cells (2), and rabbit reticulocyte lysates (2,4), in addition to those of rye embryo (5) and wheat germ (6). Translation in cell-free systems is simpler and more rapid (60 min vs 24 h) than the in vivo translation system using Xenopus oocytes.

Function of DNA synthesis and DNA repair in the survival of embryos during early germination and in dormancy

AbstractDNA synthesis following the addition of water to excised embryos of non-dormant rye and to embryos of non-dormant and dormant genetic lines of Avena fatua has been examined. All the samples exhibit an early unscheduled DNA synthesis, have similar responses to DNA polymerase inhibitors and a similar increase in DNA ligase function for the first 24 h of imbibition, irrespective of whether they germinate or remain dormant. A β-polymerase-mediated DNA repair activity is indicated immediately upon imbibition with the stable incorporation of 3H-methyl-thymidine into high molecular weight DNA. Following γ-irradiation of dry or imbibed embryos, inhibitor results suggest the appearance of an additional α- or δ-polymerase activity in the ensuing repair. Abscisic acid permits the early unirradiated repair synthesis, but like aphidicolin, it inhibits replicative DNA synthesis and partly inhibits the post-irradiation incorporation of thymidine. DNA synthesis takes place continuously throughout dormancy of imbibed embryos: precursor incorporation into DNA cannot be chased in the short term and occurs in the absence of an S-phase or endoreduplication of nuclear DNA. It is proposed that dormant imbibed embryos maintain the integrity of the genome by a continuous but slow replacement of DNA steered by a non-amplifying and modified form of replicative DNA synthesis and that abscisic acid may play a determining role in this process.

A large DNA repeat of the dispersion pattern common to wheat and rye genomes

A Hind III-generated fragment of wheat embryo nuclear DNA has been cloned and sequenced. The cloned fragment corresponds to a 1241 bp long, moderately repeated (60,000 copies/genome) segment of the genomic DNA. The repeat is AT-rich (67%), contains an open reading frame for 151 amino acids and several nucleotide blocks resembling the consensus domain of autonomously replicating sequences. Southern blot hybridization analyses indicate that the repeat is scattered through the wheat genome. A sequence homologous to this repeat occurs also in rye embryo nuclear DNA where it shows the same dispersion pattern as that observed for the wheat repeat.

Rye–wheat hybrids: the production of wheat chromosome additions to rye

To produce rye–wheat addition lines, 21-chromosome rye–wheat hybrids were produced by crossing tetraploid triticale with diploid rye. The subsequent selfing of the hybrids lead to monosomic and double monosomic wheat additions to rye. Screening of the progeny was carried out using the C-banding technique. The wheat additions can be fertile. Wheat chromosome 6B was the chromosome most frequently added to rye. Ribosomal spacer probe pTa250.4 was used to confirm the results obtained by C-banding for the 6B wheat additions to rye. Embryos of the 21-chromosome rye–wheat hybrids showed a good potential for propagating more plantlets after they had been transferred to artificial medium.Key words: rye–wheat addition lines.

An embryo‐specific protein of barley (Hordeum vulgare)

An immunological approach has been used to identify embryo-specific products that can be used as molecular markers of embryogenesis. Immunoadsorption of antisera to remove antigens common to embryos, meristematic cells and callus, revealed one major embryo-specific antigen, a polypeptide of 17 kDa. The antigen appeared at mid-stages of zygotic embryo formation and remained at similar levels up to six days post-germination of the seedling. The polypeptide could not be detected by protein staining, suggesting it is a non-abundant product. Appearance of the antigen could be induced by culture of zygotic embryos in vitro on abscisic acid (1 microM) or mannitol (9% mass/vol.). Cross-reactive products of near-identical molecular mass were observed in embryos of wheat, rye and oats but not distantly related cereals, nor embryos from dicotyledonous species. The timing of the appearance of the antigen was different in embryos formed from microspores during anther culture in vitro. In the cultured material, the 17-kDa polypeptide preceded the appearance of morphologically distinct embryonic structure.

Metabolic labelling of polyisoprenoids using tritiated water

Procedures are described for the metabolic labelling of polyisoprenoids using tritiated water. Unlike other precursors, tritiated water equilibrates rapidly with tissue water to generate a pool which remains constant for the duration of most experiments. The problems of differences in precursor pool size between tissues and physiological states are thus avoided. Results obtained for the metabolic labelling of dolichol and dolichyl phosphate (Dol-P) in soybean embryos and mice, both of which contain primarily dolichol, are presented. The methodology for soybeans was applied to rye which contains nearly equal amounts of dolichol and polyprenol. These alcohols (and presumably their phosphorylated forms) can also be metabolically labelled using tritiated water. The rye embryo provides an excellent system for studies on the α-saturation reaction responsible for the formation of dolichol intermediates from corresponding polyprenol forms.

DNA ligase in repair and replication in the embryos of rye, Secale cereale

Fragmentation of nuclear DNA and loss of DNA integrity, occur progressively in the embryos of dry seeds. On imbibition, a loss of ability to repair DNA and an extended delay to DNA replication reflect the extent of genetic impairment. Changes in DNA ligase activity are central to these events. Evidence indicates that a single species of DNA ligase is involved.

Analysis of the DNA synthesised during early germination of rye embryos using BND-cellulose chromatography

BND-cellulose chromatography has been used to determine the type of DNA synthesis occurring during the early germination of rye embryos, defined in this context as from the time of imbibition to 8 h later. The approach taken for this study is to follow the type of labelled DNA obtained through the columns after pulse-labelling of embryos for 10, 20 and 30 min at different times of imbibition. High viable and low viable embryos have been compared. 3 types of synthesis can be distinguished. (1) A repair type observed in γ-irradiated embryos imbibed for 50 min. (2) A replicative synthesis is evident in high viable embryos imbibed for 8 h. (3) A third type can be recognized in non-irradiated high and low viable embryos, imbibed for up to 2 h. This has certain characteristics of semiconservative DNA synthesis but is insensitive to abscisic acid and probably obscures any repair synthesis that could be taking place during that period. The nature of this third type of DNA synthesis is discussed.

Identification of Homologous Globulins from Embryos of Wheat, Barley, Rye and Oats

Identification of Homologous Globulins from Embryos of Wheat, Barley, Rye and Oats