Homology among 3S and 7S Globulins from Cereals and Pea

The globulins from wheat caryopses were found to consist primarily of protein sedimenting at approximately 3S and 7S. These proteins displayed a molecular weight distribution similar to that of the purified vicilin-like fractions from oat and pea, with variations occurring in the isoelectric points and relative quantities of their major subunits. concanavalin A Sepharose chromatography suggested that the major polypeptides of the wheat (3S + 7S) fraction are glycosylated. Western blot analysis using antioat (3S + 7S) globulin immunoglobulin G revealed the vicilins from pea and the globulin fractions of oat, wheat, barley, rye, corn, and rice to contain immunologically homologous polypeptides. Major groups of polypeptides were shared by all the cereals and pea, including subunits of approximately 75, 50, 40 kilodaltons and 20 to 25 kilodaltons. These results indicate that legume-like 3S and 7S globulins have been conserved and are being expressed in cereals.     

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.     

Separation and characterization of oat globulin polypeptides

The storage globulin of oat seeds was separated into its acidic (α) and basic (β) polypeptides by ion-exchange chromatography in 6 m urea and further characterized by several electrophoretic techniques. Molecular weights of the α and β polypeptides were 32,500–37,500 and 22,000–24,000, respectively. The unreduced protein existed as disulfide-linked αβ species of molecular weight 53,000–58,000. Isoelectric points were approximately 5.9–7.2 (α) and 8.7–9.2 (β). Two-dimensional electrophoresis showed considerable heterogeneity within both groups of polypeptides. More complete amino acid analyses of the globulin and its polypeptides are presented along with a proposed structure of the native protein based on previous and present data. Similarities were noted between the oat globulin and the legumin (11 S) class of storage proteins in certain legumes.     

Characterization of tonoplast polypeptides isolated from corn seedling roots

Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg²⁺ from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross-contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from corn roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast-specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions.     

Reproduction of Pratylenchus penetrans on Potato and Crops Grown in Rotation with Potato

The relative suitability of potato and crops frequently grown in rotation with potato as hosts for Pratylenchus penetrans was evaluated. Suitability of rye, wheat, corn, oat, sorgho-sudangrass, and potato were compared in pot studies based on ratios of final population : initial population density and densities of nematodes in roots at harvest. Population densities increased more on potato, oat, and corn than on rye, wheat, and sorgho-sudangrass. There were no differences among the four rye cultivars or between the two oat cultivars in host suitability. Population increases were not related to root weight or consistently to nematode densities in roots. Although rye and wheat were equally suitable hosts in pot studies, P. penetrans increased more on wheat than on rye in a field study, indicating that reproduction was reduced or mortality was increased on rye under field conditions.     

Gas chromatographic determination of 2(3)-benzoxazolinones from cereal plants

2(3)-Benzoxazolinone (BOA), 6-methoxy-2(3)-benzoxazolinone (MBOA) and 6,7-dimethoxy-2(3)-benzoxazolinone (dimethoxy-BOA) could be separated by GC. The identity of these components was verified by combined GC-MS. BOA and MBOA were determined quantitatively in 0·1 g samples of corn seedling. The presence of analogs not previously reported was demonstrated in seedlings of wheat, rye and in leaves of Job's tears. Seedlings of rice, barley, oat and sorghum did not contain any detectable amount of benzoxazolinones.     

Isolation and Characterization of Mustard (Sinapis alba L.) Seed Storage Proteins

A total storage protein fraction was prepared from mustard (Sinapis alba L.) seeds via isolated protein bodies and characterized by sedimentation, immunological, and electrophoretic techniques. Mustard seed storage protein consists of three fractions (1) a “legumin-like” 13-S complex composed of two pairs of disulfide-linked polypeptides (16.5 + 28.5 kDa and 19.5 + 34 kDa, respectively) and two single polypeptides (18 kDa and 26 kDa), (2) a “vicilin-like” 9-S complex composed of two glycoproteins (64 kDa and 77 kDa), and (3) two small polypeptides (10 kDa and 11 kDa) which probably represent the 1.7-S complex found in other Cruciferae. In contrast to related species, no glycosylated polypeptide was found in the 13-S complex. Immunological relationships were found between the paired polypeptides of the 13-S complex but not between polypeptides of the 13-S complex and polypeptides of the 9-S complex. Pulse-chase labeling and in vitro translation of polysomal RNA from young embryos demonstrated that the polypeptides of the 13-S complex originate from high molecular mass precursors, except for the 18 kDa polypeptide which appears to be synthesized in its final size. The amino-acid composition of the major polypeptides of the mustard storage protein is given.     

The Localization of Oat (Avena sativa L.) Seed Globulins in Protein Bodies

The major storage proteins of the oat grain are the 12S and7S globulins. Using sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) and immuno-difTusion assays we havedemonstrated that the 7S globulin is localized predominantlyin the embryo and the 12S globulin chiefly in the endosperm.Protein bodies have been isolated using aqueous and non-aqueousmedia and sucrose density gradient ultracentrifugation. Assayingmarker enzymes gave results consistent with the presence ofvacuolar protein bodies in the preparations. SDS-PAGE of sequentialsalt and alcohol extractions demonstrated the presence of globulinsand prolamins respectively and their distribution within thegradient suggested that they may be localized in different proteinbodies. Key words: Avena sativa L., Seed globulins, Protein bodies, Localization.     

A survey of Fusarium toxins in Austrian feed stuffs

During the last decade observations have been made concerning the occurrence of the fusariotoxins zearalenone and deoxynivalenol in Austria. Feed samples were tested in a large-scaled examination. They usually consisted of cereals (barley, oat, wheat, rye and corn), mixed feed (meal or pellets) and corn or corn cob silage.     

Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis.

SDS-PAGE and quantitative densitometric analysis revealed alterations in the protein pattern of subcellular fractions (100,000 x g) isolated from Paramecium aurelia (299s axenic) cells suppressed in phagocytosis as compared with the control. Two different agents were used to block phagocytosis: the beta-adrenergic antagonist-1-propranolol (200 microM) and inhibitor of calmodulin-dependent processes--trifluoperazine (20 microM). More than 40 polypeptides were identified in the cytosolic (soluble) fractions S1 and S2. A considerable decrease in band intensity was found for three polypeptides: by 60% for 87 kDa band, 52% for 75 kDa and 37% for 42 kDa in comparison to the control, when S2 fractions from propranolol-treated cells of equal load were quantified. TFP treatment evoked only a small decrease in the intensity of the same bands: 9%, 10% and 6%, respectively. The 42 kDa band was identified by Western blot analysis and chemiluminiscent detection to be actin. This result suggests that actin may be a primary target of pharmacological agents used in this study to inhibit Paramecium phagocytic activity.     

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

Total globulins from embryos and endosperms of barley, wheat,rye, and oats were separated by SDS-PAGE under reducing andnon-reducing conditions. The preparations from embryos of allfour cereals contained major groups of bands with M_r's of 50-60000,which were not affected by reduction. These have been characterizedpreviously from oats and shown to correspond to subunits ofthe 7S storage globulin. Immunochemical relationships betweenthese bands (and others with M_r's between 40000 and 70000) weredemonstrated by immunodiffusion and ‘Western Blotting’using antiserum raised against the major subunits of the oat7S globulins. The 7S globulins were also prepared from hand-dissectedembryos of the four cereals using sucrose density ultracentrifugation.Their amino acid compositions were broadly similar, but differedfrom those of the 7S vicilins of legumes. It is concluded thatstructurally-related 7S globulins are present in the embryosof the four species of cereals. Key words: Homologous globulins, embryo, wheat, barley, rye, oats     

Characterisation of cDNAs for ribulose bisphosphate carboxylase small subunit genes in different species of Avena (Poaceae)

ABSTRACT

We report the cloning and sequencing of 14 rbcS cDNAs in six species of Avena (Poaceae) with different genome and ploidy levels. The nucleotide sequences 504 bp long were aligned with the published sequences of cultivated hexaploid oat, wheat, barley, rye, rice and corn and subjected to cladistic and phenetic analyses. The parsimonious analysis generated a tree with a topology very similar to the phenogram generated by the Neighbor-Joining analysis based on the Jukes and Cantor distances. Within the monophyletic assemblage of the tribe Aveneae, consistent clades composed of rbcS clones belonging to different species are recognized. It is suggested that they correspond to orthologous genes belonging to different subfamilies, and that the “within-species?d homogenisation may have occurred at a slow rate with respect to species evolution. In the monophyletic group of Pooideae, the topologies place barley rbcS sequences closer to wheat and rye than to oat sequences. This grouping agrees with most taxonomic and phylogenetic views.     

Tobacco embryogenesis: Storage-protein-accumulating cells of embryo, suspensor, and endosperm are able to undergo cytokinesis

Summary It is widely accepted that seed storage proteins accumulate only in cells which have entered the cell expansion phase and do not continue to divide. Here we present data indicating that the accumulation of storage globulins in tobacco begins already during early embryogenesis in a period of sustained mitotic activity. Western blot analysis revealed that polypeptides of the legumin-like 12S globulins (M_r 60000, 40000, 20000) appear at mid/late globular stage, whereas the vicilin-like 7S globulin (M_r 50000) follows during the transition from heart to torpedo stage. The accumulation of legumin-like polypeptides begins first in the endosperm during the mid globular stage followed in the embryo-suspensor complex during the heart-shaped stage. The vicilin-related fraction appears first in the endosperm and three days later in the embryo. Examination of individual cells from squash preparations revealed that protein bodies are not confined to intermitotic cells, but are also present in cells undergoing mitosis. Protein bodies of dividing cells situated outside the mitotic apparatus are not metabolized during cytokinesis. The only cell type which loses its protein bodies completely prior to the first mitotic division is the primary hypophysis cell. Our finding that storage proteins can occur in dividing cells independent of their origin and developmental capacity indicates that the cell expansion hypothesis of storage protein accumulation has to be revised.     

Sugar uptake by cotton tissues: leaf disc versus cultured roots

The synthesis, transport, and posttranslational processing of reserve globulin in Avena sativa L. seeds were studied by pulse-chase labeling. Developing oat seeds were labeled with radioactive sulfate and tissue homogenates were used for globulin extraction.

Two globulin precursors (58-62 kilodaltons) were labeled after 1 hour pulse. The α and β globulin subunits appeared between 2 and 10 hours later, while simultaneously the 58 to 62 kilodaltons polypeptides gradually disappeared. This confirmed a precursor-product relationship. In a second pulse-chase experiment, the tissue extracts were fractionated on a sucrose gradient. The major portion of radioactivity was initially (1 hour pulse) associated with the endoplasmic reticulum. However, a significant amount of radioactivity shifted from the endoplasmic reticulum to protein bodies after 20 hours chase, suggesting the transport of the newly synthesized proteins. Protein bodies isolated from pulse-chased seeds were analyzed for the arrival of the newly synthesized globulin. Labeled precursors were detected after 2 hours chase and gradually disappeared. The α and β subunits appeared during the same chase period and assembled into a 12S oligomer.

The data indicated that oat globulin was synthesized as two large precursors which were transported from endoplasmic reticulum into protein bodies where they were processed to the α and β subunits forming a 12S oligomer.

     

Purification and characterization of arabinofuranosidase from the corn endophyte Acremonium zeae

Acremonium zeae, one of the most prevalent fungal colonists of preharvest corn, possesses a suite of hemicellulolytic activities including xylanase, xylosidase, and arabinofuranosidase. Two enzymes with arabinofuranosidase activity were purified from cell-free culture supernatants of A. zeae grown on oat spelt xylan. A 47 kDa enzyme (AF47) was optimally active at 37 °C and pH 6.0, and had a specific activity for 4-nitrophenyl-α-L-arabinofuranoside (4NPA) of 6.2 U/mg. A 30 kDa enzyme (AF30) was optimally active at 50 °C and pH 4.5, and had a specific activity for 4NPA of 12.4 U/mg. AF47 hydrolyzed 4-nitrophenyl-β-D-xylopyranoside, 4-nitrophenyl-β-D-glucopyranoside, and 4-nitrophenyl-β-D-cellobioside, as well as producing reducing sugars from corn fiber, wheat, and oat spelt arabinoxylan. AF30 had little detectable activity on the 4-nitrophenyl substrates, except for 4NPA, but activity on arabinoxylans from corn fiber, wheat, and oat spelt was at least 7-fold higher than AF47, with specific activities of 109, 358, and 153 U/mg, respectively. A combination of the two enzymes released 61 and 88% of the total arabinose from corn fiber and wheat arabinoxylans. The arabinofuranosidases produced by A. zeae may have industrial application for the enzymatic hydrolysis of recalcitrant lignocellulosic feedstocks such as corn fiber and wheat straw.     

Identification of legumin-like proteins in wheat

We have obtained several amino acid sequences from purified polypeptides of a wheat endosperm storage globulin previously described as triplet protein. The amino acid sequence data supported by immunochemical analysis using anti-oat 12S globulin antibodies, provide definitive evidence that the triplet protein is homologous to pea legumin and related seed storage proteins of oats, rice and several dicotyledonous species. Thus, it is now proposed that the triplet protein of wheat be renamed triticin. The oat globulin antibodies also cross-reacted strongly with the high-molecular-weight (HMW) glutenin subunits which have been implicated in bread-making quality.     

Characterization of developing oat seed mRNA: evidence for many globulin mRNAs

Polyadenylated mRNA from developing oat (Avena sativa L.) seeds was isolated and analyzed. Prominent mRNA species of 18S, 15S and 12S were observed; the 18S mRNA was judged to be esentially free of ribosomal RNA by hybridization analysis. Size fractionation andin vitro translation of this mRNA was performed. SDS, IEF-SDS gel electrophoresis and immunoprecipitation were used to analyze the translation products. It is shown that globulin mRNA (18S) accounts for roughly 30% of the total mRNA in developing seeds, the 12S and 15S mRNAs accounting for the remainder. The 18S mRNA directs the synthesis of a series of distinct but related polypeptides, suggesting that some of the heterogeneity seen in the oat globulins is at the amino acid sequence level.     

Storage globulins in Gnetopsida. 1. Recognition of legumin-like proteins

Abstract

Salt soluble storage proteins were extracted from seeds of Ephedra distachya, Ephedra foeminea, Gnetum gnemon, Gnetum montanum and Welwitschia mirabilis and separated by chromatographic procedures. The molecular weight of the main storage globulin ranges from 300 to 350 kD. Denaturation by SDS resolved the holoprotein in monomers of M_r 40 to 60 kD. Oligomers up to 120 kD were observed in Ephedra. Reduction of disulphide bridges by DTE resolved the monomers in paris of polypeptides of M_r 10 to 35 kD. The characters above indicate that the main storage globulin of Gnetopsida is a legumin-like protein.     

Peroxygenase-Catalyzed Fatty Acid Epoxidation in Cereal Seeds (Sequential Oxidation of Linoleic Acid into 9(S),12(S),13(S)-Trihydroxy-10(E)-Octadecenoic Acid)

Peroxygenase-catalyzed epoxidation of oleic acid in preparations of cereal seeds was investigated. The 105,000g particle fraction of oat (Avena sativa) seed homogenate showed high peroxygenase activity, i.e. 3034 [plus or minus] 288 and 2441 [plus or minus] 168 nmol (10 min)-1 mg-1 protein in two cultivars, whereas the corresponding fraction obtained from barley (Hordeum vulgare and Hordeum distichum), rye (Secale cereale), and wheat (Triticum aestivum) showed only weak activity, i.e. 13 to 138 nmol (10 min)-1 mg-1 protein. In subcellular fractions of oat seed homogenate, peroxygenase specific activity was highest in the 105,000g particle fraction, whereas lipoxygenase activity was more evenly distributed and highest in the 105,000g supernatant fraction. Incubation of [1-14C]linoleic acid with the 105,000g supernatant of oat seed homogenate led to the formation of several metabolites, i.e. in order of decreasing abundance, 9(S)-hydroxy-10(E),12(Z)-octadecadienoic acid, 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, cis-9,10-epoxy-12(Z)-octadecenoic acid [mainly the 9(R),10(S) enantiomer], cis-12,13-epoxy-9(Z)-octadecenoic acid [mainly the 12(R),13(S) enantiomer], threo-12,13-dihydroxy-9(Z)-octadecenoic acid, and 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid. Incubation of linoleic acid with the 105,000g particle fraction gave a similar, but not identical, pattern of metabolites. Conversion of linoleic acid into 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, a naturally occurring oxylipin with antifungal properties, took place by a pathway involving sequential catalysis by lipoxygenase, peroxygenase, and epoxide hydrolase.