Isolation and characterization of oat globulin messenger RNA

When polyadenylated RNA, isolated from membrane-bound polysomes extracted from developing oat (Avena sativa L.) seeds, was translated in vitro in the rabbit reticulocyte system, two polypeptides of about 58 and 60 kilodaltons were immunoprecipitated by anti-oat globulin antibody. No electrophoretic bands corresponding to the 40 and 20 kilodalton polypeptides of oat globulin were present. However, when in vivo labeled extracts were immunoprecipitated with anti-oat globulin antibody, three groups of polypeptides (60, 40, and 20 kilodaltons) were present. It therefore seems probable that the two large polypeptides (58 and 60 kilodaltons) were precursors of the 40 and 20 kilodalton polypeptides. When the polyadenylated RNA coding for these polypeptides was size fractionated on a sucrose density gradient, it sedimented near the 18S region of the gradient. Translation of the RNA from the gradient fractions and immunoprecipitation of translation products indicated that the template for the 58 to 60 kilodalton `putative' precursors of oat globulin was probably the RNA which was approximately 18S in size.     

Ribosome-binding sites on chloroplastrbcL andpsbA mRNAs and light-induced initiation of D1 translation

Chloroplast ribosome-binding sites were identified on the plastidrbcL andpsbA mRNAs using toeprint analysis. TherbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis ofrbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site.Escherichia coli 30S ribosomes generated similar toeprint signals when mixed withrbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. ThepsbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associatedpsbA mRNA revealed ribosomes bound to the initiator region.E. coli 30S ribosomes did not associate with thepsbA translation initiation region.E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation onpsbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associatedpsbA mRNA and the abundance of initiation complexes bound topsbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.     

Storage Protein Synthesis during Oat (Avena sativa L.) Seed Development

Oat (Avena sativa L.) seeds harvested at 2-day intervals from anthesis to maturity were tested for their ability to incorporate [³⁵S]sulfate into protein. Incorporation of [³⁵S]sulfate into TCA-insoluble material began 2 to 4 days postanthesis (DPA), reached a peak 14 to 16 DPA, and was barely detectable by 24 DPA. Incorporation of label into globulin was parallel to total protein accumulation, and averaged about 85% of the total protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein extracted from developing seeds indicated that some polypeptides coinciding with the α and β globulin subunits were present 2 to 4 DPA, but the full complement of globulin polypeptides was not present until 10 DPA. Immunoprecipitation of in vivo labeled seed extracts showed that globulin polypeptides and the 59 kilodalton precursor were present at early stages of development (4 DPA). Quantitation of dot blot analysis, using an oat globulin cDNA clone as a probe, indicated that one species of oat globulin mRNA was most abundant 15 DPA, which is during the peak time of storage protein synthesis.     

Cotyledonary mRNA Species and Germinability of Immature Vigna unguiculata Seeds

We have defined four stages in the development of cowpea seeds:I(9–11 days after flowering), II (13–15), HI (17–19)and IV (22–24). Poly A⁺ RNA fractions were prepared fromcotyledons of developing (stages I–IV) and germinating(0, 12, 24 and 48 h after imbibition) seeds. Poly A⁺ RNAs fromstages I–III exhibited high translation activities witha maximum at stage II, and the activity was markedly reducedat stage IV. In cotyledons of germinating seeds, the translationactivity was low until 12 h after the onset of imbibition butrose thereafter. Analysis of in vitro translation products withSDS-polyacrylamide gel electrophoresis and fluorography showedthat the abundant mRNA population underwent a distinct changebetween stages II and III of seed development. Since the mRNApopulation at stage III was very similar to that of stage IV(mature seeds), it appears that, as far as mRNA species areconcerned, the prerequisites for germination are fully availablein the developing seeds by stage III. This assumption was supportedby the fact that immature seeds at stage III exhibited highgermination rates and normal axial growth and produced -amylaseat levels similar to those produced by mature seeds. Severalpolypeptides which have been regarded as translation productsof stored mRNA (poly A⁺ RNA from dry seeds) were detected atearlier stages of germination. (Received September 29, 1988; Accepted January 25, 1989)     

In situ localization of faba bean and oat legumin-type proteins in transgenic tobacco seeds by a highly sensitive immunological tissue print technique

We have used a highly sensitive immunological tissue print technique to study cell- and tissue-specific expression of heterologous genes in transgenic plants. Primary polyclonal antibodies, raised against legumin of faba bean (Vicia faba L.) and 12S globulin of oat (Avena sativa L.) were used to localize these proteins in transgenic tobacco seeds in a streptavidin-alkaline phosphatase assay in combination with biotinylated secondary antibodies producing a higher sensitivity (by several amplification steps) of the assay. Both storage protein genes were found to be expressed in a specific pattern. While legumin is preferentially accumulated in certain parts of the embryo, the oat legumin-type globulin is restricted to the endosperm. The applied technique is highly sensitive with a resolution power down to the singlecell level and allows rapid screening of large numbers of samples.     

Sequence and expression of the mRNA encoding the chymotrypsin inhibitor in winged bean (Psophocarpus tetragonolobus (L.) DC.)

The accumulation of the Kunitz-type chymotrypsin inhibitor WCI-3 in winged bean seeds is controlled developmentally. In vitro translation experiments showed that the WCI-3 mRNA was present in 35- and 40-day-old immature seeds after flowering. The size of the in vitro translation product is about 2 000 Da larger than that of the mature WCI-3 protein. The WCI-3 cDNA clones were isolated from a gtll cDNA library of 35-day-old immature seeds by immunoscreening. A nearly full-length cDNA clone was obtained containing an open reading frame of 207 amino acid residues. The deduced sequence of the 183 carboxy terminal amino acids coincides precisely with the amino acid sequence determined for purified WCI-3. The amino terminal extension of 24 residues has the characteristics of a signal peptide. Northern hybridization analysis of total poly(A)⁺ RNA showed that the WCI-3 mRNA is approximately 900 nucleotides long and accumulates in 35- and 40-day-old but not in 30-day-old immature seeds.     

A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues

We introduced the oat adc cDNA into rice under the control of the constitutive maize ubiquitin 1 promoter. We studied molecularly and biochemically sixteen independent transgenic plant lines. Significant increases in mRNA levels, ADC enzyme activity and polyamines were measured in transgenic callus. These increases were not maintained in vegetative tissue or seeds in regenerated plants, with the exception of one lineage. This particular lineage showed very significant increases in putrescine preferentially in seeds (up to 10 times compared to wild type and controls transformed with the hpt selectable marker alone). We have demonstrated that in cereals such as rice, over-expression of the oat adc cDNA results in increased accumulation of polyamines at different stages of development. We have also demonstrated that strong constitutive promoters, such as the maize ubiquitin 1 promoter, are sufficient to facilitate heritable high-level polyamine accumulation in seed. Our results demonstrate that by screening adequate numbers of independently derived transgenic plants, it is possible to identify those individuals which express a desired phenotype or genotype.     

In vitro synthesis of the Bowman-Birk and related soybean protease inhibitors

Poly (A)⁺ RNAs from immature soybean seeds were size fractionated in denaturing sucrose gradients to identify mRNA that directs the cell-free synthesis of the Bowman-Birk protease inhibitor and the related inhibitors PI I–IV. Polypeptides synthesized in vitro were labeled with (³⁵S)-cysteine and (³H)-serine and detected by immunoprecipitation with anti Bowman-Birk and anti PI I–IV sera. Immunoprecipitates of the translation products comigrated on SDS-polyacrylamide gels with the dimeric or trimeric aggregates of the authentic inhibitor proteins, which self-associate under certain conditions. Further evidence that these immunoprecipitates contained authentic polypeptides corresponding to the Bowman-Birk or PI IV inhibitor was shown by sequential amino acid analyses of peptides generated by cleavage with cyanogen bromide.     

In vitro translation of mRNA from Nostoc commune (Cyanobacteria)

RNA pools were extracted from cells of Nostoc commune UTEX 584 in exponential growth (liquid cultures) and from cells which had been immobilized and dried rapidly at -99.5 MPa. Levels of incorporation of ³⁵S-methionine, five- to sixfold higher than the endogenous level, were obtained after in vitro translation of the RNA preparations in a heterologous S30 cell-free system purified from Escherichia coli Q13. The levels of incorporation, obtained with a homologous N. commune UTEX 584 S30 system, were much lower. The requirement for magnesium in the heterologous system was 15–21 mM, translation of N. commune UTEX 584 RNA was inhibited when the RNA concentration was greater than 0.3 mg ml^–1, and translation was stimulated significantly by the presence of ammonium chloride. Few qualitative differences were observed between the pattern of proteins (SDS-PAGE) obtained after translation of the RNA pools from cells in exponential growth, and from those cells subjected to immobilization and rapid drying. The data suggest that short-term desiccation of N. commune UTEX 584 does not have a marked selective effect on the composition of the mRNA pool. In contrast, preparations of RNA from field materials of Nostoc commune HUN (desiccated for 5 years) were unable to drive high rates of translation in any of the systems tested and optimized for use in this study.     

Evidence for translational control of storage protein biosynthesis during embryogenesis ofAvena sativa L. (oat endosperm)

Oat polysomes direct the synthesisin vitro of a large number of products, the majority of which are the salt-soluble globulins (1,3,10,11,21). Total RNA or poly A⁺ RNA isolated from these polysomes directs the synthesis of the same number and types of products; however, the amount of globulins synthesized no longer represents the major products; rather, there is a decreased level of globulins and an increased amount of the other products synthesizedin vitro (6, 18). These results imply that the translational control can dictate final product levels. Reconstruction experiments using oat poly A⁺ mRNA and polysomal factors that are made free of endogenous RNA by nuclease digestion demonstrate that these factors do influence the translational specificity of oat globulin mRNA relative to other mRNAs. It is suggested that translational control is partially responsible for the levels of globulin in the mature grain.     

The expression of dehydrin proteins in desiccation-sensitive (recalcitrant) seeds of temperate trees

Proteins that have homology with dehydrins have been identified immunologically in the desiccationsensitive (recalcitrant) seeds of English oak (Quercus robur L.), European chestnut (Castanea sativa L.), horse chestnut (Aesculus hippocastanum L.), sycamore (Acer psuedoplatanus L.) and silver maple (Acer saccharinum L.), and in the desiccation-tolerant seeds of Norway maple (Acer platanoides L.). The mRNA for a late embryogenesis abundant (LEA) protein (dehydrin) was also detected by Northern blotting, using a cDNA clone (D11) from cotton embryos, in the recalcitrant and orthodox seeds. Medium-stringency washing was required to detect this hybridization. InQ. robur the amount of dehydrin protein increased during seed development, andLEA mRNA was induced by limited desiccation and by abscisic acid. Confirmation of the presence of dehydrin mRNA in matureQ. robur andC. sativa seeds was obtained by in-vitro translation of the extracted polyadenylated RNA followed by analysis of the immunoprecipitation products. Thus the presence of dehydrin proteins is not sufficient to confer desiccation tolerance on truly recalcitrant seeds, nor can their presence or absence be used as clear criteria for identification of recalcitrant seeds.     

Germination stimulation in wild oats (Avena fatua L.) by synthetic strigol analogs and gibberellic acid

At concentrations of 0.01–1 mM, five synthetic multiring analogs of strigol were effective germination stimulants of intact and dehulled wild oat (Avena fatua L.) seeds. The effect was concentration-dependent and equaled or exceeded that produced by equimolar gibberellic acid (GA₃). The most effective strigol analog treatments induced 55–80% germination within 7 days in intact wild oat seeds and resulted in 63–86% germination and normal seedling growth over 14 days. Intact wild oat controls germinated 14% after 14 days. The stimulation of wild oat germination by these synthetic strigol analogs demonstrates that these compounds, initially developed as germination stimulants for the seeds of the parasitic weed, witchweed (Striga asiatica L. Kuntz.), have bioregulatory activity in dormant seeds of monocots, as well as dicots. None of the compounds tested significantly affected the germination of nondormant cultivated oat seeds (Avena sativa L.). The commonly used dispersal agent, Tween 20 (0.1%), was found to inhibit germination of cultivated oats, alone and in the presence of 2% acetone.Names of companies or commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned.     

Cell-free translation of exogenous mRNA in extracts from dry pea primary axes

Extracts from the primary axes of dry pea (Pisum sativum L.) seeds are able to perform an initiation-dependent translation of exogenous mRNA. SDS polyacrylamide gel electrophoresis of the products synthesized under direction of alfalfa mosaic virus RNA (AMV-RNA) and tobacco mosaic virus RNA (TMV-RNA) shows that the fidelity of translation in this pea system is at least as high as in a wheat embryo cell-free protein synthesizing system. The endogenous messengers are also efficiently translated in extracts from the primary axes of pea seeds. The direct translation of these messengers in a homologous cell-free system may be of interest for a study of the products coded for by the long-lived messengers present in this plant.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - SDS sodium dodecyl sulphate - mRNP messenger ribonucleoprotein - AMV-RNA alfalfa mosaic virus RNA - TMV-RNA tobacco mosaic virus RNA - ATA aurin tricarboxylic acid - TCA trichloroacetic acid - S.A. specific activity     

Isolation and characterization of cDNAs encoding oat 12S globulin mRNAs

Summary A cDNA library was made from poly(A⁺) RNA isolated from developing oat seeds, and oat globulin cDNA clones were identified by hybridization with synthetic oligonucleotides. Globulin clones were characterized by restriction enzyme mapping and cross-hybridization analysis. Based on these comparisons, four classes of globulin clones were distinguished. These clones hybridized to multiple DNA fragments in restriction enzyme digests of oat genomic DNA, indicating that the genes exist in a multigene family. The nucleotide sequence of one of the globulin cDNA clones was determined. The amino acid sequence derived from the DNA sequence verified its identity as an oat globulin and confirmed that the protein is synthesized as a precursor similar to legume 11S storage globulins. The basic polypeptide encoded at the 3 end of the mRNA was found to be homologous to the basic polypeptides of other 11S seed globulins.Abbreviations ds double stranded - kb kilobase Author to whom correspondence should be addressed. Journal paper number 10460 of the Purdue Agricultural Experimental Station.     

Preparation of a cell-free translation system from PC12 cells

The postmitochondrial fraction (S10) contains the cellular components essential for translation, and a high-salt wash (HSW) of the ribosomes is enriched in eukaryotic initiation factors. This report describes the preparation of a cell-free translation system utilizing an S10 extract from PC12 cells. The products synthesized from either firefly luciferase mRNA or PC12 cell poly(A) RNAs in the PC12-S10 extract were increased by the addition of the HSW from PC12 cells. Increases in the translation of luciferase mRNA by the addition of PC12-HSW were dose-dependent and also dependent on the time of incubation. The translation of human epidermal growth factor receptor (hEGFR) mRNA could also be detected in the PC12-S10 extract translation system by immunoprecipitation.N-linked glycosylation of the translation products also was observed. The efficiency of translation was altered by the addition of Mg²⁺ or K⁺, and optimization of the concentrations of these ions was necessary for each mRNA. The translation system made from PC12 cells, then, is capable of the synthesis of proteins of relatively high molecular weight and should be useful for analyzing mechanisms of translational control during proliferation and differentiation of cells from a neuronal lineage. Special issue dedicated to Dr. Hans Thoenen.     

Barley hordothionin accumulates in transgenic oat seeds and purified protein retains anti-fungal properties in vitro

Summary Transgenic anti-fungal gene expression in heterologous species provides a means to test resistance protein combinations across species barriers. This is the first report of transgenic anti-fungal seed storage protein accumulation in oat seed. An anti-fungal barley (Hordeum vulgare L.) hordothionin (Hthl) gene was genetically engineered into oat (Avena sativa L.) to determine the effect of hordothionin on pathogen resistance. The transgene was expressed in both leaf and seed tissue, with transgenic protein accumulation occurring only in the seed. Transgenic oat line HTH-Av5 expressed c. 94 μg HTH/g seed, 19% of native barley seed levels. The anti-fungal activities of HTH fractions from barley cv. Morex and oat (transgenic and control) were tested in an in vitro growth assay against an important small grain pathogen. Fusarium graminearum. The partially purified HTH fractions from control oat seeds did not inhibit fungal growth, while HPLC-purified HTH positive control, as well as partially purified barley and transgenic oat HTH inhibited growth similarly over a range of concentrations. These results indicate hordothionin can be expressed in a heterologous cereal species and still maintain its anti-fungal properties. Future studies with HTH targeted to additional tissues are planned to test for increased fungal resistance. The University of Wisconsin and the USDA neither guarantee nor warrant the standard of the products named herein, and the use of the name by University of Wisconsin or USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Production and characterization of arboreous and fertile <Emphasis Type="Italic">Solanum melongena</Emphasis> + <Emphasis Type="Italic">Solanum marginatum </Emphasis>somatic hybrid plants

In crop plants the shift from being annuals to perennials may allow future agricultural systems requiring less energy inputs. The practicability of this was tested for Solanum melongena. Leaf protoplasts of S. melongena (2n = 2x = 24) and one of the related arborescent species Solanum marginatum (2n = 2x = 24) were electrofused and fertile somatic hybrids with arborescent habit regenerated. The magnetic cell sorter (MACS) technique was used for the selection of heterokaryons. The hybrid nature of 18 regenerated plants was assessed on the banding patterns generated by inter-simple sequence repeat PCR. When taken to maturity in the greenhouse, hybrids grew more vigorously compared to the parental species. Their morphological traits were intermediate between those of S. melongena and S. marginatum. Hybrids flowered and produced an average of 85% stainable viable pollen and fertile fruits. The somatic hybrids were maintained in the greenhouse for more than 3 years and continued to produce flowers developing into two types of fruits with plentiful seeds. Fruits were either striated green containing non-germinable seeds or yellow with fully germinable seeds. Their S₁ progenies showed common features with S₀ hybrids, including fertility and arborescent habit. Cytologically, somatic hybrids exhibited the expected chromosome number of 2n = 4x = 48, while chromosome pairing during microsporogenesis was associated with a low frequency of intergenomic pairing. It is concluded that an arborescent perennial species has been obtained by somatic hybridization. The usefulness of this species per se or in eggplant breeding will depend not only on the transmission of the arborescent habit to cultivated eggplant varieties, but also on the variability that should be created from backcrossing the S. melongena + S. marginatum hybrids to S. melongena.     

Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro

Immature embryos of Brassica napus were cultured in vitro with and without various concentrations of germination inhibitors, and the progress of embryogeny was monitored by comparing accumulation of storage proteins in culture with the normal accumulation in seeds. The two major B. napus storage proteins (12S and 1.7S) were purified from seed extracts and analyzed by rocket immunoelectrophoresis (12S protein) or by sodium lauryl sulfate polyacrylamide gel electrophoresis (1.7S protein). During embryo development within seeds both the 12S and 1.7S proteins were first detected when the cotyledons were well developed (embryo dry weight, 0.4 mg), and each storage protein accumulated at an average rate of 26 g d^-1 during maximum deposition. Accumulation of the 1.7S protein stopped when the water content of the embryo began to decline (embryo DW, 2.7 mg), but accumulation of the 12S protein continued until seed maturity (embryo DW, 3.6 mg). At the end of embryo development the 12S and the 1.7S proteins comprised approx. 60 and 20% of the total salt-soluble protein, respectively. When embryos were removed from seeds at day 27, just as storage protein was starting to accumulate, and placed in culture on a basal medium, they precociously germinated within 3d, and incorporation of amino acids into the 12S storage protein dropped from 3% of total incorporation to less than 1%. If 10^-6 M abscisic acid (ABA) was included in the medium, amino-acid incorporation into the 12S protein increased from 3% of total incorporation when embryos were placed into culture to 18%, 5d later, and the accumulation rate (27.1±2.6 g embryo^-1 d^-1) matched the maximum rate observed in the seed. High osmotica, such as 0.29 M sucrose or mannitol, added to the basal medium, also inhibited precocious germination, but there was a lag period before 12S-protein synthesis rates equaled the rates on ABA media. These results indicate that some factor in the seed environment is necessary for storage-protein synthesis to proceed, and that ABA is a possible candidate.Abbreviations ABA abscisic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium lauryl sulfate