RNases and nucleases in embryos and endosperms from naturally aged wheat seeds stored in different conditions

Temperature and moisture content are particularly important factors influencing the longevity of seeds, and therefore the ageing of seeds is closely tied to storage conditions. The ageing process is characterised by many physiological and biochemical changes: membranes tend to leak, enzymes lose catalytic activity, and chromosomes accumulate mutations. Since viability loss is also associated with the breakdown of nucleic acids, the aim of the study was to determine whether the damage induced by ageing could be associated with changes in the activity of RNases and nucleases in embryos and endosperms of differently stored wheat seeds. In order to better characterise seed conditions, the damage to membranes during seed ageing was evaluated by measuring the conductivity of the soaking solution during imbibition, and by using the Evans Blue colorant; lipid peroxidation was also recorded. RNases and nucleases were studied by SDS-PAGE and activity staining. Ageing of seeds stored in a dry state involved a progressive loss of membrane integrity, which increased with the degree of ageing, while lipid peroxidation remained unchanged. Changes in nucleolytic enzyme activity were recorded in embryos: a decrease in RNases and an increase in nucleases. In the endosperm compartment there were no significant differences in ribonuclease and nuclease patterns during seed ageing. Moreover, neutral RNases were absent in endosperms of dry seeds and were activated following imbibition. Present studies reveal that embryos and endosperms have different enzymatic patterns, thus highlighting that the two seed compartments age independently. A different nucleolytic pattern was present in seeds of comparable viability and membrane damage, which were stored differently, and nuclease metabolism was subject to regulation according to both ageing and the length of the storage period.     

Glucose metabolism of embryos and endosperms from deteriorating barley and wheat seeds

Changes in glucose utilization into CO₂ and ethanol-insoluble material were followed in whole seeds, embryos, and endosperms of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) which had reached different levels of deterioration through accelerated aging treatments. Excised embryos from deteriorated wheat seeds had reduced respiration and glucose utilization into ethanol-insoluble material but not into CO₂. These treatments had no effect on respiration of excised endosperms, although they reduced utilization of glucose into ethanol-insoluble material and CO₂. Changes in metabolic activity of whole seeds in response to deterioration treatments are difficult to interpret because they represent the sum of the changes that take place in the embryos and endosperms. Changes in respiration and glucose utilization in these two tissues neither proceed at the same rate nor go in the same direction during deterioration.     

Poly(A)-containing RNA from germinating wheat embryos

Rapidly labelled mRNAs were isolated from informosomes and polyribosomes of imbibed wheat embryos. The distribution of poly(A) sequences in these fractions were studied by poly(U) Sepharose chromatography. It was shown that informosomes contain 11% polyadenylated mRNA while polyribosomes--38%. This fact suggests the important role of poly(A) sequences for translation of mRNA.     

A quick method to isolate RNA from wheat and other carbohydrate-rich seeds

No reports on isolating RNA from carbohydrate-rich wheat seeds have been published. Because of the presence of carbohydrates, published protocols yield small amounts of poor quality RNA. Extracting seeds in a buffer (pH 9, 150 mM NaCl, 1% sarcosyl) ensured maximum RNA solubility and the removal of most interfering substances. Extracted RNA was purified using a guanidine hydrochloride-based buffer system. This protocol yields up to 148 μg of RNA from 100 mg of tissue in 3.5 h. An A₂₆₀/A₂₈₀ ratio of 1.85 indicates RNA purity. Isolated RNA was amenable to downstream applications such as differential display. The developed method was extended to other carbohydrate-rich seeds, such as barley and maize, with success.     

On the content of protein and nucleic acids in organs of chick embryos of different age

In 10-, 14-and 18-days-old chick embryos, concentrations of total protein and nucleic acids (NA) were measured using spectrophotometry in liver, brain, red gastrocnemial and white pectoral muscles and in chorioallantois. It was shown that the initial protein and NA concentration was the highest in liver and decreased markedly in the above-mentioned organs. At the second half of embryogenesis, concentration of organ protein increased 4 times in muscles, 2 times in brain, and 1.4 times in liver. This indicates a rise of cell density in the organs or of the intracellular protein concentration. The protein/NA ratio reflecting the cell size or concentration of their protein practically did not change in liver, but increased 2.3 and 2.5 times in pectoral and gastrocnemial muscles, respectively, and 2 times in brain. In chorioallantois the maximal protein concentration was observed in then 14-day old embryos, with the protein/NA ratio remaining practically unchanged.     

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     

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.     

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.     

Changes in hybridizable RNA in winter wheat embryos during germination and vernalization

DNA-RNA hybridization-competition experiments were performedto analyze RNA heterogeneity in wheat embryos during germinationand vernalization. A significant difference was found betweenRNA populations of 24-hr and 3-day germinated embryos, whileminor differences were detected between 3- and 5-day germinatedembryo RNAs and between 20- and 40-day cold-treated embryo RNAs.RNA populations in 30-day cold-treated embryos were significantlydifferent from those in 50-day ones. The RNA species presentin 50-day cold-treated embryos were not similar to those in3- and 5-day germinated embryos. A great portion of the RNAspecies in 3-day germinated embryos was found in 30-day cold-treatedembryos. These results suggested that some new RNA species aresynthesized in wheat embryos during 30 to 50 days of cold treatment. ¹ Present address: Department of Biochemistry, Faculty of PharmaceuticalSciences, Higashi Nippon Gakuen University, Ishikari-Tobetsu,Hokkaido 061–02, Japan. (Received February 21, 1977; )     

Hybridization properties of non-polyadenylated oligo(U)-containing RNA from germinating wheat embryos

• 1.1. Total cytoplasmic RNA of germinating wheat embryos was fractionated by affinity chromatography and separated into non-polyadenylated oligo(U)-containing RNA (A(−)U(+)RNA) and polyadenylated oligo(U)-lacking RNA (A(+)U(−)RNA).
• 2.2. The reassociation kinetics of ³²P-labelled complementary DNA (cDNA) reverse-transcribed from A(−)U(+)RNA shows that this RNA fraction is transcribed from unique DNA sequences of the genome similarly as typical mRNA.
• 3.3. Cross hybridization experiments show no significant sequence homology between the two RNA fractions. Therefore it is concluded that non-polyadenylated oligo(U)-containing RNA of wheat embryo may represent a discrete class of mRNA.

     

"Cap" sequences in poly(A)-rich RNA from dry wheat embryos

Although template-active RNA in dry seeds and embryos has attracted widespread interest, there have been no published reports about 5'-terminal "capping" sequences in such RNA. Boro[3H]hydride labeling of periodate-oxidized termini and high performance liquid chromatography of cap oligonucleotides have been used to compare terminal sequences in poly(A)-rich RNA from dry and germinating embryos. As is the case in germinating embryos, poly(A)-rich RNA from dry embryos contains only "type 0" cap sequences, i.e., m7G(5')ppp(5')N, in which m7G is the 7-methylguanosine cap and N is any of the classical ribonucleosides: adenosine (A), guanosine (G), cytidine (C),a nd uridine (U). Striking differences between the cell-free translational capacities of bulk messenger RNA (mRNA) populations from dry and germinating embryos are not reflected in signal differences in their proportions of "type 0" cap structures: in general, there is approximately 40% m7G(5')ppp(5')A, with roughly equivalent amounts of m7G(5')ppp(5')G and m7G(5')ppp(5')C accounting for most of the remaining sequences. The findings with mRNA from dry plant embryos serve to emphasize interesting differences between patterns of methylation in the capped and uncapped RNA molecules in higher plants and animals; the differences have not been previously noted in the literature and are the subject of brief comment in this paper.     

Intracellular levels of polyadenylated RNA and loss of vigour in germinating wheat embryos

Polyadenylated-RNA (Poly(A)⁺RNA) levels have been studied during the germination of wheat embryos of high viability but differing vigour. In high-vigour embryos imbibed at 20°C the level of poly(A)⁺RNA falls dramatically over the first hour of imbibition, then remains constant up to 3 h of imbibition before increasing rapidly to a level similar to that found in the quiescent state by 7 h of imbibition. Median-vigour embryos imbibed at 20°C show similar changes in poly(A)⁺RNA content but the initial decrease and subsequent increase in poly(A)⁺RNA levels are less marked. On imbibition at 10°C, the poly(A)⁺RNA content in high-vigour embryos decreases to a lesser extent during the first hour than at 20°C and the level increases more slowly over the next 6 h than during the same time period at 20°C. The level of poly(A)⁺RNA in medianvigour embryos remains constant over the first 4 h of germination and then falls to a level of about half that found in quiescent high-vigour embryos. Polyacrylamide gel electrophoresis of total-RNA samples shows that the polyadenylic acid (poly(A)) sequences occur in RNA species ranging in size from 35-7S. Polyacrylamide gel electrophoresis of isolated poly(A) sequences demonstrates the presence of two size classes of poly(A) in quiescent embryos, but at 20°C a more heterodisperse pattern appears by 2 h of imbibition. At 10°C, two size classes of poly(A) persist throughout the period studied in both high- and median-vigour embryos, although in median-vigour embryos the ratio of larger: smaller poly(A)-tail sizes decreases more rapidly than in high-vigour embryos.Abbreviations Poly(A) polyadenylic acid - poly(U) polyuridylic acid - poly(A)⁺RNA polyadenylated RNA     

Changes in nicotinamide nucleotide content and glucose metabolism in wheat embryos during vernalization

Contents of nicotinamide nucleotides, NAD, NADP, NADH₂ and NADPH₂,in wheat embryos were determined during normal germination andcold treatment. They increased markedly in the early stage ofgermination then decreased later. During cold treatment, thesenucleotides increased initially as in normal germination, butdid not decrease in the later stages. The C₆/C₁ ratios during germination decreased gradually withaging, but after about the fourth day increased. This suggeststhat the pentose phosphate pathway operates actively. Duringcold treatment, the C₆/C₁ ratio decreased slightly with aging.This suggests that the EMP pathway operates predominantly inthe glucose metabolism. Based on these results, the correlationbetween NADPH₂ and glucose metabolism during cold treatmentwas discussed. Embryos which had absorbed labelled glucose were fractionatedinto several chemical components. The radioisotope accumulatedmainly in the sugar fraction, with small amounts in the organicacid, amino acid and nucleic acid fractions. Changes in thecontent of each component showed that sugar, organic acid andamino acid increased during cold treatment. ¹ Present address: Department of Biochemistry, Faculty of PharmaceuticalScience, Higashi Nippon Gaguen University, Ishikari-Tobetsu,Hokkaido 061-02, Japan. ² Present address: Hokkaido Forest Products Research Institute,Asahikawa 070, Japan. ³ Present address: Kakuto 534, Komae City, Tokyo 182, Japan. (Received November 1, 1976; )     

Deoxyribonucleic Acid polymerase from wheat embryos

A soluble DNA-dependent DNA polymerase was extracted from wheat embryos. In vitro, the incorporation of radioactive thymidine triphosphate into acid-insoluble material is dependent upon the presence of the enzyme, all four deoxyribonucleotide triphosphates, Mg²⁺, and a DNA template. Incorporation occurs on native, alkali-denatured, and strictly double-stranded DNA. The in vitro synthesized product is a polydeoxynucleotide with a chain length shorter than the template; it has the same buoyant density as wheat embryo DNA when this DNA is used as template; and it forms a double-stranded complex with the DNA template. These data suggest that the in vitro DNA synthesis catalyzed by proteins extracted from wheat embryos occurs in a semiconservative way.     

DNA primase activity from wheat embryos

DNA primase is a recently discovered enzyme capable of synthesizing short primers involved in the initiation of DNA replication.Partially purified preparations from 4 h germinated wheat embryos or commercial wheat germ are able to catalyze the ribonucleoside triphosphate dependent synthesis of DNA with poly dT and M₁₃ single stranded DNA as templates. DNA synthesis is completely dependent on the presence of template and primase. Primase activity from wheat embryos has a molecular weight of about 110000 and a sedimentation coefficient of 5S. The enzyme activity is not inhibited by -amanitin (1 mg/ml) or aphidicolin when the latter is assayed with endogeneous plant DNA polymerase activity. Alkaline hydrolysis of the product synthesized in the presence of [-³²P]dATP and poly dT generates [³²P]-labeled 3(2)AMP showing that a ribo-deoxynucleotide linkage is formed. The size of the oligoribonucleotide primer varies from 2 to 15 residues. Most of the wheat DNA polymerase activity can be eliminated by phosphocellulose chromatography, since the bulk of plant DNA primase is not retained by this resin. Nevertheless, a small but significant amoung of DNA polymerase activity is found associated with DNA primase. By using different inhibitors of DNA polymerase different templates, we have found good indications that DNA polymerase A (-like) is associated with the DNA primase. Moreover, when the previously purified DNA polymerases from wheat embryos (2) were assayed in the presence of primase activity, only DNA polymerase A was able to stimulate DNA synthesis.