Germinating Wheat Embryos Research Articles

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 α-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 [α-(32)P]dATP and poly dT generates [(32)P]-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 (γ-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.

The growth-related 28-kilodalton protein in germinating wheat. Use of peptide mapping to identify cryptic forms in cell-free extracts and protein-synthesizing systems

Characteristic peptide maps have been prepared and used to identify cryptic forms of the methionine-labeled 28-kilodalton (28-kd) protein (the G protein) which marks onset of growth in germinating wheat embryos. Peptide mapping of material eluted from areas detected as "bands" in autoradiograms of one-dimensional Laemmli-type gels and from areas detected as "spots" in autoradiograms of two-dimensional O'Farrell-type gels has led to identification of a counterpart to G among methionine-labeled products of cell-free synthesis. Identification of its cell-free counterpart has made it possible to show that translatable mRNA for the G protein, like G itself, only begins to appear between 5 and 10 h after onset of water uptake by dry wheat embryos. Peptide mapping of methionine- or proline-labeled material eluted from an area detected as a conspicuous "band" in autoradiograms of one-dimensional Laemmli-type gels has uncovered the existence of an unusual (apparently complexed) form of G in extracts of germinating but not dry wheat embryos. Unique among the hundreds of prominent proteins made by germinating wheat embryos, G exists as part of a thermolabile "150-kd complex" which is refractory to dismutation in a reducing environment containing sodium dodecyl sulphate (SDS). Presence or absence of the G complex constitutes the only conspicuous difference between SDS–polyacrylamide distributions of newly synthesized proteins derived from unheated as opposed to heated extracts of germinating embryos. Analysis of extracts prepared at different times after onset of water imbibition by dry wheat embryos shows that time course of emergence of the G complex, detected in unheated extracts, and of the G protein, detected in heated extracts, is the same. By fractional electroelution and silver staining of protein, it has been possible to show that G may be the principal if not the only protein component of the G complex.

Subunit Structure Differences in RNA Polymerase II Purified from Ungerminated versus Germinated Wheat Embryos

DNA-dependent RNA polymerase II (RNAP II) was purified from wheat embryos germinated for 0, 12, 24, and 36 hours and examined with several polyacrylamide gel electrophoretic systems. A changing electrophoretic pattern of RNAP II was observed on nondenaturing polyacrylamide gels. Subunit structure analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that from ungerminated embryos, RNAP IIA was almost exclusively obtained which has a subunit structure identical to that established for wheat germ RNAP II previously (Jendrisak, Burgess 1977 Biochemistry 16: 1959-1964). Twelve polypeptides with molecular weights x 10(-3) of 220, 140, 42, 40, 27, 25, 21, 20, 17.8, 17.0, 16.3, and 16.0 were routinely found to be associated with the purified enzyme. From embryos germinated for 36 hours, RNAP IIB was almost exclusively obtained which has a largest subunit of 180,000 mol wt instead of 220,000. From embryos germinated for 24 hours, an approximately equimolar mixture of RNAP IIA and IIB was obtained. Peptide maps of the 220,000 and 180,000 mol wt polypeptides of RNAP IIA and IIB were virtually identical, indicative of a precursor-product relationship for the two polypeptides. In addition to these results, SDS-PAGE indicated that the stoichiometry of the 27,000 mol wt polypeptide increased at the expense of the 25,000 mol wt polypeptide during germination and concomitantly with the appearance of the 180,000 molecular weight polypeptide. No modifications (e.g. gain, loss, or altered mobilities on analytical gels) in any of the other RNAP II subunits were observed in enzyme purified from embryos after various times of germination as determined by a variety of electrophoretic analyses under denaturing conditions.

Transcriptional requirement for the induction of acid phosphatase activity in germinating excised wheat embryos ( Triticum aestivum)

About 5–6-fold stimulation of acid phosphatase activity was observed in excised wheat embryos germinated for 48 h. This increase in acid phosphatase activity was inhibited by cycloheximide (CHI) (20 μg/ml) and 5 amino acid analogues (1 mM each), thus indicating the requirement of de novo protein synthesis. Strong inhibition of enzyme activity by cordycepin (500 μM) in germinating wheat embryos excluded the presence of a stored mRNA for acid phosphatase, although stored mRNA for this enzyme has been reported in the embryo-less half-seeds of wheat. Thus it appears that different mechanisms operate for the regulation of acid phosphatase activity in embryo and endosperm of wheat grains. Exogenous application of gibberellic acid (GA 3) (10 μM) increased 24–37% acid phosphatase activity in germinated wheat embryos (48 h). The hormone (GA 3) also induced a marked change in the isoenzymes of acid phosphatase. Both inhibitors of RNA and protein syntheses virtually abolished the activity of GA 3-induced isoenzymes of acid phosphatase. Abscisic acid (ABA) (10 μM) alone and in the presence of GA 3 (10 μM) substantially decreased the acid phosphatase activity in germinated wheat embryos. Cycocel (1 mM), an inhibitor of GA 3 biosynthesis, decreased acid phosphatase activity (53% inhibition) in wheat embryos and this inhibition was partially counteracted by the addition of GA 3 (100 μM).

De Novo Synthesis of 3′-Nucleotidase in Germinating Wheat Embryo

The enzyme 3'-AMP nucleotidase was purified 2,500- to 5,000-fold from extracts of an acetone powder of wheat (Triticum aestivum) embryonic axes germinated for 40 hours. Sodium dodecyl sulfate acrylamide gel electrophoresis and chromatography on Biogel-P100 indicate that the enzyme is monomeric with a molecular weight of 39,000. Extracts of embryos germinated up to 6 hours have only 1% of the 40-hour level of enzyme activity. To see if the increase to 40 hours represents de novo synthesis, extracts were compared for their ability to react with a rabbit antibody prepared against the enzyme. In immunodiffusion tests, 40-hour extracts showed a strong precipitin line coincident with that of the purified enzyme, whereas no precipitation was observed with 1-hour extracts. When the enzyme present in 40-hour extracts was partially inactivated by EDTA, it still blocked the ability of the antibody to inhibit enzyme activity. Extracts of 1-hour embryos, in contrast, were not able to block the inhibitory activity of the antibody. Embryos allowed to take up (35)SO(4) between 40 and 46 hours of germination synthesized (35)S-labeled 3'-nucleotidase. In contrast, no radioactive protein synthesized by embryos during the first 6 hours of germination coincided on gel electrophoresis with the enzyme. These results indicate that the increase in 3'-nucleotidase activity is a consequence of de novo synthesis of the enzyme.

Synthesis of poly(a) polymerase from conserved messenger RNA in germinating excised embryos of wheat

A 3.5–6.0-fold stimulation of poly(A) polymerase activity was observed in excised wheat embryos germinated for 48 hr. Addition of primer RNA to the enzyme assay mixture was necessary for the incorporation of [ 3H]AMP into the acid-precipitable polyadenylate product. Administration of six amino acid analogues (1 mM each) or cycloheximide (10 μg/ml) to the germinating embryos resulted in 77–82 % inhibition of poly(A) polymerase activity. The inhibitory response, elicited by the analogues, was substantially counteracted by the simultaneous addition of the corresponding six amino acids (2 mM each). This indicated that de novo protein synthesis was necessary for the enhancement of poly(A) polymerase activity. Cordycepin, a potent inhibitor of transcription, failed to block poly(A) polymerase activity; instead, the drug invariably brought about a significant stimulation ( ca 1.7–4.0-fold of the enzyme activity. Cordycepin, however, inhibited acid phosphatase activity by 77% in germinating wheat embryos. Actinomycin D also failed to inhibit poly(A) polymerase activity in germinating wheat embryos. The lack of inhibition of poly(A) polymerase by transcriptional inhibitors during early germination suggested that the enzyme was translated from its conserved mRNA, already stored in the dry wheat embryos.

Nucleotide Levels and Loss of Vigour and Viability in Germinating Wheat Embryos

Nucleotide Levels and Loss of Vigour and Viability in Germinating Wheat Embryos

Role of Nitrate in the Induction of Nitrite Reductase Activity during Wheat Seed Germination

Nitrate could not induce NR activity for the first 20 h during the early phase of wheat embryo germination, in spite of considerable accumulation of nitrate inside the tissue. Nitrate, however, induced NR without a lag phase in seeds germinated in water for 24, 32 and 48 h. NiR induction by NO- 3 was found to be independent of NR induction, since NiR activity was observed during the lag phase of NR synthesis and in the presence of tungstate-an inhibitor of NR synthesis. Studies with inhibitors of transcription and translation suggest that nitrate acts at the translation level for the induction of NiR during early germination of wheat embryos.

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.

RHYTHMICITY IN NITRATE REDUCTASE ACTIVITY IN WHEAT EMBRYOS DURING GERMINATION

SUMMARYNitrate reductase (NR) activity, induced by germination in KNO3 showed a strong rhythmicity during the early stages of germination of wheat embryos. Peaks of activity occurred approximately every 12 h. There was no relationship between the amounts of nitrate in the embryos and enzyme activity. The fluctuations in enzyme activity did not appear to be due to reversible inactivation of the enzyme. Some evidence suggests that the fluctuations in enzyme activity may result from a more rapid degradation of enzyme during the decreasing phase of the rhythm.

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.

Mineral Ion Transport in the Embryos of Germinating Wheat (Triticum aestivum)

The germinating wheat embryo contains two mineral ion pumps. One is a H+/K+ antiport system located in the scutellum and the other is a H+/anion symport, or possibly an anion/OH+ antiport system located in the embryo axis. The scutellum pump closely resembles that found in other plant tissues; its affinity constant for K+ is about 0-03 mM and its activity is energy dependent. The embryo axis pump is able to transport K+ in place of H+ and appears to be a general monovalent cation/anion symport system. The scutellum pumping activity is stimulated by GA and the GA action is inhibited by ABA. GA stimulates H+ more than K+ transport, electrochemical neutrality always being maintained by anion transport. In contrast to reports about IAA-stimulated ion transport in other plant tissues, there is no evidence that the GA action in the scutellum is dependent upon active protein synthesis.

Germination of Wheat Embryos and the Transport of Amino Acids into a Protein Synthesis Precursor Pool

Wheat (Triticum aestivum L. var. Lew) embryonic axes take up externally supplied radioactive amino acid (from a solution greater than 2 millimolar) such that the specific radioactivity of the total internal amino acid rapidly reaches that of the external solution. Nevertheless, incorporation of radioactive amino acid into protein increases steadily as the concentration of external amino acid is increased, indicating that the amino acid that is precursor to protein synthesis is not in equilibrium with the total internal amino acid pool. When the external source of amino acid is removed, incorporation of radiolabeled amino acid into protein continues at a rate comparable to that of embryos maintained in the radioactive solution. In explanation of these data, it is suggested that there are two separate cytoplasmic pools of amino acids, one a protein synthesis precursor pool, and the second, an expandable pool into which exogenous radioactive amino acids are taken up. The protein synthesis pool is fed at a limited rate from the expandable pool and at a far greater rate from an endogenous source. As a consequence, the specific activity of the amino acid that is the precursor for protein synthesis is considerably below that of the total internal pool and is determined by the rate of movement into the protein synthesis pool from the expanded radioactive cytoplasmic pool.The rate of movement of amino acids from the expandable pool into the protein synthesis pool increases approximately 5-fold during the initial 4.5 hours of embryo germination. When this change is considered in analyzing the relative rates of protein synthesis, there is probably no more than a 2-fold increase in protein synthetic capacity between embryos germinated for 1.5 and 4.5 hours. The leveling off of the change in transport capacity after 4.5 hours suggests that the earlier increase in the rate of this process may be a necessary step before the embryos can begin to accelerate their growth rate.

Synthesis and turnover of proteins and mRNA in germinating wheat embryos.

The most prominent methionine-labeled protein made when cell-free systems are programmed with bulk mRNA from dry wheat embryos has been identified with what may be the most abundant protein in dry wheat embryos. The protein has been brought to purity and has a distinctive amino acid composition, Gly and Glx accounting for almost 40% of the total amino acids. Designated E because of its conspicuous association with early inhibition of dry wheat embryos, the protein and its mRNA are abundant during the "early" phase (0--1 h) of postimbibition development, and easily detected during "lag" phase (1--5 h), but they are almost totally degraded soon after entry into the "growth" phase of development, by about 10 h postimbibition. The most prominent methionine-labeled protein peculiar to the cell-free translational capacity of bulk mRNA from "growth" phase embryos is not detected as a product of in vivo synthesis. Its electrophoretic properties and its time course of emergence, after 5 h postimbibition development, suggest that this major product of cell-free synthesis may be an in vitro counterpart to a prominent methionine-labeled protein made only in vivo, by "growth" phase embryos. Designated G because of its conspicuous association with "growth" phase development, the cell-free product does not comigrate with any prominent dye-stained band in electrophoretic distributions of wheat proteins. The suspected cellular counterpart to G, also, does not comigrate with a prominent dye-stained wheat protein during electrophoresis, and although found in particulate as well as soluble fractions of wheat embryo homogenates it is not concentrated in either nuclei or mitochondria, as isolated.

The Effect of Fusicoccin and Monovalent Cations on the Viability of Wheat Seeds

Fusicoccin (FC), K+ and Na+ restore the viability of aged wheat seeds and isolated wheat embryos. We studied the effect of FC and K+ on the evolution of the proton extrusion activity and net K+ uptake during the early phases of germination of wheat embryos isolated from unaged and aged seeds. Our data show that in this material, germination is also characterized by the activation of the proton-extruding activity and by the leakage and subsequent re-absorption of K+. The percentage of germination appeared to be correlated with the amount of proton extrusion and K+ net uptake. In aged embryos the positive effects of FC and K+ on germination were correlated with a marked increase in both proton extrusion and K+ net uptake. These findings suggest that: (a) in wheat seeds, germination is also correlated to the activation of a mechanism linked to H+/K+ exchange; (b) the damage of this mechanism is, under our conditions, responsible for the loss of viability.

Rapid appearance of newly-synthesized poly(A)-containing RNA in the cytoplasm of germinating wheat embryos

Isolated wheat embryos were germinated for short time periods (up to 4 h) in the presence of [2- 14C]uridine to label newly-synthesized RNA species. Total nuclear and total cytoplasmic RNA fractions as well as poly(A)-containing nuclear and cytoplasmic RNA preparations were then obtained and their radioactivities compared. In spite of the fact that there was no preferential labelling of the nuclear poly(A)-containing RNA(A(+)RNA), specific radioactivity of the cytoplasmic A(+)RNA in 1 h-germinated embryos was much higher than that of the total cytoplasmic RNA. In order embryos, the proportions changed in the favour of non-polyadenylated RNA. Electrophoretic pattern and ability to support amino acid incorporation in vitro indicated a messenger-like nature for the cytoplasmic A(+)RNA.

Comparative study of levels of secondary processing in bulk mRNA from dry and germinating wheat embryos.

There has been no previous study of levels of secondary processing in the template-active RNA of dry seeds and embryos. Such information is needed to evaluate the role of transcription in changing the cell-free translational capacity of bulk RNA during early imbibition of water by dry wheat embryos [J. Biol. Chem. 255, 5969-5970 (1980)]. Although it probably contains a higher proportion of "hidden breaks' than bulk mRNA from imbibing embryos, bulk mRNA in dry wheat embryos is also "capped' by 7-methylguanosine at 5' termini, devoid of unmethylated "caps', methylated internally (N6-methyladenosine) and polyadenylated at 3' termini. In contrast to other developing systems, there is no evidence that there are signal changes in levels of secondary processing in the bulk mRNA populations which support change in cell-free translational capacity of RNA 1-5 h postimbibition of dry wheat embryos. Change in the pattern of protein synthesis 5-24 h postimbibition also takes place without signal changes in levels of secondary processing in template-active RNA. The analytical data are evaluated and discussed in terms of difficulties allied with comparative analyses of poly(A)-rich RNA from dry and imbibing embryos, the former being refractory to, and the latter being most easily analyzed by, labeling in vivo.

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.

Oligo(U)- and poly(U)-containing RNA of wheat embryo

A UMP-rich RNA fraction was separated from the bulk cellular RNA by affinity chromatography of wheat embryo total RNA. The obtained preparation was heterogeneous and contained polyribonucleotide chain segments which were resistant to RNAase T 1 and consisted mainly of UMP residues (87 mol%). The UMP-rich segments were of various sizes, including large oligonucleotides and polynucleotides (up to approx. 150 nucleotides in length). The oligo(U)- and poly(U)-containing RNA fraction occurred in a low amount (approx. 1% of total RNA) both in dry and germinating wheat embryos. However, at the onset of germination, labelled precursors were preferentially incorporated into the UMP-rich RNA species. The early-synthesized RNA appeared and underwent a considerable degradation within the cell nuclei. It is assumed that both delayed maturation of structural gene transcripts and rapid transcription of regulatory gene units during initial germination stages contribute to the transient abundance of newly made UMP-rich RNA in the early wheat embryos.

The use of alpha-amanitin to inhibit in vivo RNA synthesis and germination in wheat embryos.

Low concentrations (0.10 to 1.0 microgram/ml) of alpha-amanitin inhibit wheat embryo germination. Labeled RNA, synthesized in vivo by embryos imbibed in the presence of [3H]uridine and various concentrations of alpha-amanitin, was analyzed by oligo(dT)-cellulose chromatograhy and by gel electrophoresis (acrylamide and agarose) coupled with fluorography. Low concentrations of alpha-amanitin (0.10 to 1.0 microgram/ml) strongly and selectively inhibited in vivo poly(A) + RNA synthesis in a manner which closely paralleled alpha-amanitin inhibition of purified RNA polymerase II. These results suggest that de novo mRNA transcription is required for germination. Higher concentrations of alpha-amanitin inhibited in vivo 5 S rRNA and tRNA synthesis in a manner which closely paralleled alpha-amanitin inhibition of purified RNA polymerase III. High concentrations of alpha-amanitin also inhibited accumulation of radioactivity into the rRNA precursor as well as into mature 25 S and 18 S rRNAs in a manner which also closely paralleled alpha-amanitin inhibition of RNA polymerase III. The discrepancy of the in vivo inhibitory effect of high alpha-amanitin concentrations on rRNA synthesis versus a lack of effect on purified RNA polymerase I, which presumably transcribes these genes, can be explained if continued transcription of the large rRNA precursor by RNA polymerase I requires ongoing transcription by RNA polymerase III, or if there is degradation (wastage) of rRNA precursor and/or processed products in the absence of transcription by RNA polymerase III.