Germinating Wheat Embryos Research Articles

Characterization of 45S ribonucleoprotein particles detected in wheat embryo cytoplasm

Relatively homogeneous 45S ribonucleoprotein particles containing considerable amounts of newly-synthesized rapidly labelled RNA were detected in cytoplasmic extract from germinating wheat embryos. These particles have the buoyant density in CsCl of about 1.50 g/cm 3 and apparently are not represented by free cytoplasmic informosomes. The isolated particles contain 17S rRNA, rapidly-labeled heterogeneous (6–16S) RNA with strong messenger activity and several low molecular weight RNAs among which the 5.3S RNA (of about 135 nt) is the most abundant. According to 2D-PAGE two groups of polypeptides can be revealed in the particles. Polypeptides of the first group (10–45 kDa) correspond to structural ribosomal proteins. The second group consists of higher molecular weight polypeptides (30–100 kDa) and may include polypeptides of mRNP and translation initiation factors. The 45S particles may represent a native [mRNP × 40S RS × eIFs × Met-tRNA i × GTP] complexes accumulating in vivo as a translation initiation intermediate.

Evidence to show the phosphoprotein nature of poly(A) polymerase in germinated wheat embryos: enzyme regulation through phosphorylation

Poly(A) polymerase from wheat embryos was shown to be a phosphoprotein by labelling the enzyme in vivo with [ 32P]orthophosphate. Excised germinated wheat embryos (48 h) were incubated (12 h) in a sterile solution of sucrose (2%) containing [ 32P]orthophosphate (25 mCi) for labelling phosphoproteins. Poly(A) polymerase was then purified by ATP-Sepharose affinity chromatography. The ATP-Sepharose fraction showed a single protein-stained band on native PAGE that matched with the activity peak of the unlabelled enzyme. This proved the electrophoretic homogeneity of the enzyme preparation. Serial scanning of the parallel unstained gel showed a single 32P-labelled radioactive peak that matched the protein-stained band. Analysis of 32P-labelled purified poly(A) polymerase on SDS-PAGE also showed a single radioactive band that coincided with the protein-stained band (65 kDa) of a monomeric poly(A) polymerase. Chemical characterization of the 32P-labelled purified poly(A) polymerase by acid hydrolysis showed two radioactive spots by paper chromatography that matched authentic phosphoserine and phosphothreonine. These results suggested that wheat embryo poly(A) polymerase is phosphorylated in vivo at the serine and threonine residues. A significant amount of dephosphorylation (∼ 74%) of poly(A) polymerase was achieved by treating the 32P-labelled purified enzyme with alkaline phosphatase. Addition of alkaline phosphatase along with sodium ortho-vanadate, however, showed a meagre dephosphorylation (∼8%) of 32P-labelled poly(A) polymerase. This is because ortho-vanadate virtually blocked the activity of phosphatase (∼90%) and thus prevented the dephosphorylation of poly(A) polymerase. This enabled us to compare the biological activity of phosphorylated and substantially dephosphorylated forms of poly(A) polymerase. Thus, purified poly(A) polymerase was dephosphorylated with alkaline phosphatase and thereafter, phosphatase activity was blocked in situ by the addition of sodium ortho-vanadate. We observed a substantial decrease (∼ 60%) in poly(A) polymerase activity following treatment with alkaline phosphatase. However, poly(A) polymerase activity was not inhibited to any significant level by alkaline phosphatase in the simultaneous presence of sodium ortho-vanadate. We consider that the partially dephosphorylated form of poly(A) polymerase is less active than the phosphorylated form in wheat embryos.

Purification of an endoproteinase that digests the wheat ‘Em’ protein in vitro, and determination of its cleavage sites

Germinating wheat embryos contain two endoproteolytic activities which digest the prominent ‘Em’ polypeptide. These are easily assayed in clarified embryonic homogenates and are distinguishable by the pattern of their peptide products and by their different pH optima. One activity has a pH optimum of 4.0; the second activity is a cysteine endoproteinase with a preference for the ‘Em’ protein as its substrate. It is maximally active between pH 5.5 and 6 at 25°C. Analysis of the early cleavage products of the cysteine proteinase indicates scissile bonds between residues Glu 32-Ala 33 and Asn 36-Leu 37 in the ‘Em’ polypeptide. This endoproteinase has been purified and identified as a single polypeptide species of ca. 38,000 kDa.

Selective proteolysis of the wheat Em polypeptide: Identification of an endopeptidase activity in germinating wheat embryos

The ‘Em’ polypeptide is the most abundant cytosolic polypeptide in mature wheat embryos. It is selectively and completely degraded within the first 24 h of germination. Extracts from germinated embryos contain endopeptidase activities which degrade the Em polypeptide. These are separable into a major and minor component by ion-exchange chromatography and the use of inhibitors shows the major component to be a cysteine proteinase. This activity shows a strong preference for the Em polypeptide as a substrate, being inactive against polypeptides which are not developmentally regulated and showing only low activity towards developmentally related, but otherwise nonhomologous ‘dehydrin’ polypeptides.

The Effect of Salinity Stress upon Protein Synthesis of Germinating Wheat Embryos

Two differently salt-sensitive wheat genotypes were imbibed in 0·4 M NaCl for 72 h or, alternatively, for 48 h and then transferred to water. Seed germination, fresh weight and protein synthesis in embryos were determined. The following differences were found in the synthesis of in vivo [35S]methionine-labelled proteins during salt imbibition: (a) a general decrease or disappearance of polypeptides specific to the radicle emergence phase in the salt-sensitive genotype; (b) a new synthesis of polypeptides which are not found during water imbibition and are common to both genotypes; (c) a differential synthesis of polypeptides that are unique to each cultivar. Upon return to water, salt-induced proteins ceased to be synthesized while proteins associated with an advanced germination phase were actively produced. These results suggest that the expression of 'salt stress' proteins is related to the adaptation process of seeds to salinity as well as to the genetic constitution of a selected salt-tolerant genotype.

Germin, a protein marker of early plant development, is an oxalate oxidase

Germin is a homopentameric glycoprotein, the synthesis of which coincides with the onset of growth in germinating wheat embryos. There have been detailed studies of germin structure, biosynthesis, homology with other proteins, and of its value as a marker of wheat development. Germin isoforms associated with the apoplast have been speculated to have a role in embryo hydration during maturation and germination. Antigenically related isoforms of germin are present during germination in all of the economically important cereals studied, and the amounts of germin-like proteins and coding elements have been found to undergo conspicuous change when salt-tolerant higher plants are subjected to salt stress. In this report, we describe how circumstantial evidence arising from unrelated studies of barley oxalate oxidase and its coding elements have led to definitive evidence that the germin isoform made during wheat germination is an oxalate oxidase. Establishment of links between oxalate degradation, cereal germination, and salt tolerance has significant implications for a broad range of studies related to development and adaptation in higher plants. Roles for germin in cell wall biochemistry and tissue remodeling are discussed, with special emphasis on the generation of hydrogen peroxide during germin-induced oxidation of oxalate.

Purification and characterization of poly(A) polymerase from germinated wheat embryos: enzyme glycosylation

Wheat embryo poly(A) polymerase was purified (1348-fold) to electrophoretic homogeneity by adenosine triphosphate(ATP)-Sepharose and concanavalin A(Con A)-agarose affinity chromatography. The purified polymerase was devoid of cryptic nuclease activity after Con A-agarose affinity chromatography. Thus the Con A-agarose fraction showed a linear increase in poly(A) polymerase activity as a function of time up to 90 min. Fractionation of purified enzyme on native PAGE showed a single protein stained band that coincided with the activity peak of poly(A) polymerase. The molecular weight of poly(A) polymerase was 65 000–70 000 as determined by molecular sieve chromatography. A single subunit of purified poly(A) polymerase (mol. wt., 64 000) was observed on SDS-PAGE. This proved the monomeric nature of the enzyme. The binding of poly(A) polymerase to Con A-agarose and its elution with α-methyl mannopyranoside suggested its glycoprotein nature. The apparent K m of poly(A) polymerase for ATP was 86 μM. The 3H-labelled reaction product of purified enzyme binds to oligo(dT)-cellulose affinity matrix. In addition, the putative poly(A) product was not hydrolysed by RNase A and RNase T i. Thus, it was proved that poly(A) polymerase catalyzes the synthesis of poly(A) sequences.

Germin isoforms are discrete temporal markers of wheat development

Nascent synthesis and accumulation of germin and its mRNA mark the onset of renewed growth when wheat embryos are germinated in water. Germin is a water-soluble, pepsin-resistant protein that is not found in immature embryos, or in mature embryos before their germination. An antiserum was raised by injecting rabbits with germin that was freed of other proteins by pepsinization and gel filtration. The antiserum has been used to detect, in extracts of mature embryos from dry, ungerminated wheat grains, a protein that is antigenically related to germin. The antigenically related protein has been named pseudogermin. Pseudogermin accumulates, maximally, between 20-25-days postanthesis, then declines appreciably in amount by 30-days postanthesis, in soluble extracts of immature embryos from several wheat varieties. The antiserum was also used to identify germin and pseudogermin among the proteins extracted from cell walls and to bind immunogold to cell walls preparatory to visualizing freeze-cleaved embryos by scanning electron microscopy. Wall-associated germin accounts for about 40% of the total germin in germinating wheat embryos. Appearance of germin in the apoplast is the most conspicuous germination-related change in the distribution of cell-wall proteins. It seems that germin may act at the level of the apoplast and that pseudogermin may subsume the role of germin at low water potentials during embryogenesis. The N-terminal eicosapeptide sequences in germin and pseudogermin are very similar but SDS/PAGE analysis detects discrete differences between the mobilities of their constituent monomers as well as gross differences between the stabilities of the parent oligomers. Like germin, pseudogermin is a water-soluble, pepsin-resistant protein, but pseudogermin has unprecedented disulphide-independent thermostability properties that have never been previously reported for a water-soluble oligomeric protein. Polysaccharides that co-purify with otherwise pure specimens of germin (and pseudogermin) have been isolated for analysis and shown to be highly substituted glucuronogalactoarabinoxylans. The possible biological significance of selective and tenacious association between germin and glucuronogalactoarabinoxylans is discussed in relation to cell expansion during embryogenic and germinative development of wheat, as are some peculiarities of amino-acid sequence that suggest a possible relation between germin and a proton-specific ion pump: gastric ATPase.

Synthesis of extrachromosomal DNA and telomere-related sequences in germinating wheat embryos

AbstractNuclear DNA of wheat embryos was probed with a synthetic oligonucleotide (C3TA3C3TA3) to assess changes in the length of telomeres during storage and germination. Chromosomal DNA of dry embryos, especially those derived from long-term-stored caryopses, was considerably depleted of telomeric repeats, but, shortly after the start of imbibition, the chromosomal DNA became rich in sequences hybridizing to the telomeric probe. Simultaneously, newly synthesized telomere-related sequences appeared in an extrachromosomal form that could be separated from bulk nuclear DNA by velocity and isopycnic centrifugations. It is concluded that, in addition to typical repair events, rapid synthesis of telomeric repeats occurs in wheat embryos at the onset of germination.

Regulation of protein synthesis in germinating wheat embryos under polyethylene glycol and salt stress

AbstractWheat seeds were imbibed in solutions of polyethylene glycol (PEG) and NaCl, and embryo water content and incorporation of [35S]methionine into proteins were determined. Both osmotica reduced water uptake and protein synthesis, though these were immediately resumed upon removal of stress. Results ofin vivolabelling of newly synthesized proteins showed differences in the synthesis of many polypeptides when embryos that had imbibed in water passed from the radicle pre-emergence phase to early growth. The synthesis of a group of proteins associated with radicle emergence was reduced during imbibition in PEG or NaCl. All the major proteins of the pre-germination phase were produced in PEG-treated embryos, while some new polypeptides (‘salt stress’ proteins?) were actively synthesized in salt-treated embryos. Upon removal of PEG or NaCl, synthesis of proteins common to the phase of early embryo growth increased, while specific saltimbibition proteins disappeared. These findings are consistent with the hypothesis that biochemical processes leading to radicle emergence can be affected by osmotic stress and are likely to be damaged by severe stress.

Isolation of DNA polymerase alpha from germinated wheat embryos

DNA polymerase alpha from germinated wheat embryos was purified by ammonium sulphate fractionation, chromatography on DEAE-Toyopearl, followed by phosphocellulose and heparin Sepharose columns. The specific activity of the purified enzyme was more than 60,000 units/mg. It belongs to the alpha-type according to the large molecular mass, high sensitivity to NEM, aphidicoline, 200 mM KCl, low sensitivity to ethidium bromide and the absence of inhibition by ddTTP. DNA polymerase alpha consists of four subunits as shown by SDS-PAGE and seems to be homogeneous under non-denaturing conditions.

Water Uptake and Protein Synthesis in Germinating Wheat Embryos under the Osmotic Stress of Polyethylene Glycol

The experiments described were designed to investigate the relationship in wheat seeds between osmotic stress imposed by aqueous polyethylene-glycol solutions, embryo water uptake and the rate of protein synthesis as determined by [ 3 H]leucine incorporation into embryo tissues (as a measure of in vivo protein synthesis)

Cellular desiccation and hydration: developmentally regulated proteins, and the maturation and germination of seed embryos

Little more than a decade ago, 2-dimensional mapping of proteins and biochemical study of their allied coding elements (mRNA and DNA) were first used to probe possible changes in the embryo during seed germination. Because specification was of primary importance, our attention was initially directed toward the characterization of individual proteins and coding elements which, in preliminary surveys of the germinating wheat embryo, were found to be conspicuously subject to developmental regulation. Three of the proteins have become subjects of comprehensive investigations in this and other laboratories: the Em protein, the Ec protein, and germin. The Em and Ec proteins are encoded by the conserved mRNA 'stored' in the mature embryos of dry, field-ripened seeds but germin is encoded by the nascent mRNA formed after mature embryos are germinated in water. The Ec protein is the only bona fide Zn metallothionein yet found in higher plants. Studies of their biology and molecular biology suggest that the Em protein has a role in hormone-mediated (abscisic acid) cellular desiccation and that germin has a role in hormone-mediated (auxin) cellular hydration. It is projected that further studies of the Em protein may help elucidate the molecular basis for a loss of dessication tolerance during germination, and that further studies of germin may help elucidate the molecular basis of plant cell enlargement.

Post-transcriptional regulation of S-adenosylmethionine synthetase from its stored mRNA in germinated wheat embryos

About 2-3-fold stimulation of S-adenosylmethionine synthetase was witnessed in germinated wheat embryos (48 h). The enhancement of enzyme activity was significantly inhibited by cycloheximide and amino acid analogues. Simultaneous addition of corresponding amino acids alleviated the inhibitory effect of amino acid analogues. Conclusive proof for the de novo synthesis of S-adenosylmethionine synthetase was obtained by labelling this enzyme with [35SO4]2- in vivo. Thus de novo enzyme synthesis seemed necessary for the rise in activity of AdoMet synthetase in wheat embryos. Curiously, blocking of transcription with cordycepin failed to repress the de novo synthesis of AdoMet synthetase in germinated wheat embryos. We envisage the presence of stored mRNA for AdoMet synthetase in wheat embryos. Thus the regulation of this enzyme occurs at the post-transcriptional level. L-Methionine, which is one of the substrates of AdoMet synthetase, stimulated the enzyme activity (2-2.4-fold) over that observed in control germinated embryos. L-Methionine promotes increased de novo synthesis of AdoMet synthetase. Preincubation of enzyme fraction with L-Methionine failed to activate or stabilize the activity of AdoMet synthetase. Three isozymes of AdoMet synthetase were physically separated by DE-52 ion-exchange chromatography. One of the isozymes of AdoMet synthetase has been purified (1529-fold) to electrophoretic homogeneity by resorting to phenyl Sepharose and ATP Sepharose affinity chromatography. The purified enzyme catalyzed the synthesis of S-adenosylmethionine and also exhibited tripolyphosphatase activity. The reaction product of the purified enzyme was chemically and enzymatically characterized as S-adenosylmethionine. The molecular weight of the native enzyme is 174,000 and that of its subunit is 84,000 as determined on SDS-PAGE. Thus the native enzyme seems to be dimeric in nature.

Covalently bonded and adventitious glycans in germin.

Germin was previously shown to contain covalently bonded and adventitious glycans. The object of the present investigation was to characterize the two types of glycan. The presence of N- but not O-glycans in germin is indicated by the biosynthesis of altered forms, including an unglycosylated form of germin when wheat embryos are germinated in the presence of tunicamycin. After treating the doublet of germin pentamers (G and G') from normally germinated embryos with beta-N-acetylglucosaminidase, G is converted to a form that co-migrates with G' during electrophoresis in sodium dodecyl sulfate-polyacrylamide, but G' is unaffected. This suggests that the N-glycans in G contain antennary N-acetylglucosamine but that those in G' do not. This conclusion has been confirmed and elaborated by doubly labeling G and G' in vivo with [3H]glucosamine and [35S]methionine, and by characterizing sugar-labeled glycopeptides from G and G' by gel filtration, before and after their degradation by exoglycosidases. In the context of proven structures for the complex N-glycans in other plant glycoproteins, the findings, when combined with monosaccharide analyses of G and G', permit plausible speculation about the structure of the single N-glycan that is likely present in each G monomer (GlcNAc2(Man)2(Man-Xyl)(GlcNAc)(GlcNAc-Fuc] and G' monomer ((Man)2(Man-Xyl)(GlcNAc)(GlcNAc-Fuc)). The adventitious glycans, which can be removed by phenolic extraction of germin, have a composition similar to that expected for the characteristic hemicelluloses and pectins in monocot cell walls. The possible significance of this finding is discussed in relation to our continuing efforts to define the biochemical involvements of germin. In allied studies, affinity of its N-linked glycans for concanavalin A has been used to concentrate small amounts of germin from large volumes of wheat extract and to fractionate germin from tunicamycin-treated and normally germinated wheat embryos.

Transient Expression of Exogenous DNA in Intact, Viable Wheat Embryos Following Particle Bombardment

Expression of foreign DNA has been detected in intact, germinating wheat embryos (Triticum aestivum L.) following bombardment with tungsten particles complexed with a reporter gene encoding the bacterial enzyme β-glucuronidase. Expression was detected in situ in individual cells and groups of cells, by supplying the germinating embryos with the chromogenic substrate of the GUS enzyme, «X-gluc»

Polypeptide Structure of Germin as Deduced from cDNA Sequencing

Synthesis of a relatively rare glycoprotein (germin) signals the onset of growth in germinating wheat embryos. Germin mRNA (1075 nucleotide residues) has an 85-residue 5'-untranslated sequence, a 69-residue sequence that can encode a 23-residue signal-peptide sequence, a 603-residue sequence that can encode a 201-residue mature-protein sequence, and a 318-residue 3'-untranslated sequence that begins with a UAA-terminator codon, ends with a 63-residue polyadenylate tract, and has three polyadenylation (and other, related) signals (AAUAAN etc.). One polyadenylation signal is just 9 nucleotides from the polyadenylation site, the shortest stretch of nucleotides yet found between polyadenylation signal and site in any animal or plant mRNA. The mature-protein coding sequence in germin mRNA contains an unusually high proportion (87%) of G + C in the third positions of its codons. The amino acid sequence of germin does not have extensive internal homologies or repetitions, and it is not characterized by regions of unusually high charge density, as is nucleoplasmin, another water-soluble homopentameric protein with otherwise closely related structural properties. Germin does, however, contain a stretch of 34 uncharged amino acid residues and these may possibly mediate its homopentameric structure and its remarkable resistance to enzymic proteolysis. In view of a possible association of germin with cellular membranes, the most interesting relatedness of the germin sequence to the sequences of other proteins is an 80% homology between a decapeptide sequence in mature germin and a decapeptide sequence in Escherichia coli glycerol-3-phosphate acyltransferase. The relation of germin-gene structure to overall gene regulation during early plant growth is discussed.

Evidence for the de novo synthesis of poly(A) polymerase in germinated wheat embryos

A linear rise in the activity of poly(A) polymerase occurs following germination of excised wheat embryos. A roughly three-fold increase in poly(A) polymerase activity was paralleled by a rise (2.4-fold) in the relative abundance of poly(A) +RNA isolated from 72 hr germinated embryos over that at six hr after imbibition. Administration of cycloheximide (20 μg/ml) to germinated embryos significantly inhibited (80%) poly(A) polymerase activity with a dramatic decline (77 %) in the levels of total poly(A) +RNA. This indicated that the relative abundance of poly(A) +RNA is regulated by the modulation of poly(A) polymerase activity. The enhancement of poly(A) polymerase activity following embryo germination is primarily achieved through de novo synthesis of the enzyme. This has been conclusively shown by in vivo labelling of the newly synthesized total proteins with 35SO 4 2- in wheat embryos and ultimately recovering the label in the purified preparation of poly(A) polymerase. Fractionation of purified poly(A) polymerase on native polyacrylamide gels revealed a single protein band, thereby establishing the electrophoretic homogeneity of the enzyme preparation. Autoradiography of this gel showed a single radioactive band which corresponded with the protein band of the purified poly(A) polymerase. Further characterization of the purified labelled poly(A) polymerase was obtained by acid hydrolysis of the enzyme protein followed by the chromatographic separation of the 35S labelled amino acids. Autoradiography of the chromatogram revealed the presence of the label in the cysteine residues of poly(A) polymerase. These studies clearly indicated that de novo synthesis of poly(A) polymerase indeed occurs during growth of wheat embryos.

The effect of potassium on germinating wheat embryos at different viability levels

The effect of potassium administration in early germination of wheat (Triticum durum L., cv. Appulo) was studied by incubating isolated embryos at different viability levels with 1 mM KC1. The stimulation of germination and growth in aged embryos by K+ was accompanied by a significant increase in (3H) leucine incorporation into protein and in phospholipid synthesis as evidenced by TLC analysis of embryo phospholipids extracted after 24 h of imbibition. The metabolic improvement mediated by the cation would in turn repair aging damage occurring at subcellular sites including membranes.

Germin. Molecular cloning of cDNA that selects germin mRNA from bulk wheat mRNA.

(1) Bulk mRNA from germinated wheat embryos was denatured with methylmercury and subjected to electrophoresis in agarose gel to obtain a fraction of mRNA that was modestly enriched with respect to its complement of translatable germin mRNA. This fraction of mRNA was used as a source of primary templates for preparing a cDNA library. (2) Escherichia coli JM101 was transfected with recombinant pUC8 plasmids containing cDNA inserts. Colonies of transformed bacteria (ca. 4 x 10(3)) were differentially screened by hybridizing them with cDNA probes that were prepared from RNA populations containing different proportions of translatable germin mRNA. (3) A 160 base pair (bp) cDNA, which hybridized more strongly to the probe made from the RNA population containing the greater proportion of translatable germin mRNA in colony hybridizations, also hybridized more strongly to the RNA population containing the greater proportion of translatable germin mRNA when it was used as a probe for Northern analysis. (4) As judged by peptide mapping of a protein made by cell-free translation, the 160-bp cDNA selected virtually pure germin mRNA from the bulk mRNA of germinated wheat embryos when it was used in "hybrid release" experiments. The same 160-bp cDNA was used to select a "full length" germin cDNA from a library prepared by the Gubler-Hoffman method.