Location of a Protease and its Inhibitor in the Barley Kernel

Tissues of barley caryopsis and seedling were examined for the protease, BAPAase, and an inhibitor. The enzyme was present in extracts of alevn-one but was absent from aleurone incubation media and extracts of: embryo with scutellum; seedling with scutellum and rootlets, and endosperm that was free of aleurone tissue. The enzyme was present in non-incubated aleurone and did not increase significantly during incubation under conditions where alpha-amylase increased in the medium and tissue. Addition of gibberellie acid produced no detectable increase in BAPAase. Extracts of endosperm had weak BAPAase-inhibitory activity; embryo or seedling extracts produced strong inhibition. The inhibitor present in these extracts was dialyzable.     

Isolation and characterization of oat aleurone and starchy endosperm protein bodies

To compare oat (Avena sativa L. cv Froker) aleurone protein bodies with those of the starchy endosperm, methods were developed to isolate these tissues from mature seeds. Aleurone protoplasts were prepared by enzymic digestion and filtration of groat (caryopsis) slices, and starchy endosperm tissue was separated from the aleurone layer by squeezing slices of imbibed groats followed by filtration. Protein bodies were isolated from each tissue by sucrose density gradient centrifugation. Ultrastructure of the isolated protein bodies was not identical to that of the intact organelles, suggesting modification during isolation or fixation. Both aleurone and starchy endosperm protein bodies contained globulin and prolamin storage protein, but minor differences in the protein-banding pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. The amino acid compositions of the protein body fractions were similar and resembled that of oat globulin. The aleurone protein bodies contained phytic acid and protease activity, which were absent in starchy endosperm protein bodies.     

Changes in Levels of alpha-Amylase Components in Barley Tissues during Germination and Early Seedling Growth

Kernels of Klages barley (Hordeum vulgare L.) were germinated for 1 to 4 days on moist sand at 18°C. Representative kernels from each time period were dissected to give the following fractions: scutellum, subscutellar endosperm, aleurone-scutellum interface, remaining aleurone, subaleurone endosperm, and core endosperm. These tissues were analyzed for α-amylase components by isoelectric focusing and rocket-line immunoelectrophoresis. Although aleurone and scutellar tissues appeared to synthesize the same α-amylase components, enzyme was detected first in the scutellum. A larger proportion of scutellar α-amylase was excreted into the endosperm compared to aleurone synthesized α-amylase. Aleurone cells appeared to synthesize appreciably more α-amylase than did scutellar tissue.     

大麦胚和胚乳发育的相关性及贮藏营养物质的积累

大麦(Hordeum vulgare L.)开花后1d,见合子及退化助细胞,游离核胚乳尚未形成;开花后2～3d,胚为5及10个细胞,胚乳为游离核期;开花后4及5、6d,胚为梨形及长梨形,胚乳达细胞化期;开花后8d,胚为胚芽鞘期,糊粉层原始细胞产生;开花后10d,胚具1叶,糊粉层1～2层;开花后13d胚为2叶胚,亚糊粉层发生;开花后17d,3叶胚形成,糊粉层多为3层并停止分裂,菱柱形及不规则胚乳细胞分化;开花后21～29d,胚为4叶胚,胚乳进一步分化;开花后33d,胚为5叶成熟胚,胚乳亦成熟。淀粉、蛋白质在胚中积累始于开花后13d。在盾片中由基向顶发生,在胚芽鞘及叶原基中,首先在顶端出现。成熟盾片顶端的淀粉消失。开花后6d,胚乳开始积累淀粉;开花后10d,糊粉层及胚乳细胞积累蛋白质。开花17d后胚乳的蛋白质体多聚集,29d后蛋白质体显著减少。开花后17d,在盾片及糊粉层细胞中检测到油脂。果长或果长与稃片长之比和盾片长可作为不同发育期胚和胚乳的形态指标。     

Localization of carboxypeptidase I in germinating barley grain

Activity measurements and Northern blot hybridizations were used to study the temporal and spatial expression of carboxypeptidase I in germinating grains of barley (Hordeum vulgare L. cv Himalaya). In the resting grain no carboxypeptidase I activity was found in the aleurone layer, scutellum, or starchy endosperm. During germination high levels of enzyme activity appeared in the scutellum and in the starchy endosperm but only low activity was found in the aleurone layer. No mRNA for carboxypeptidase I was observed in the resting grain. By day 1 of germination the mRNA appeared in the scutellum where its level remained high for several days. In contrast, little mRNA was observed in the aleurone layer. These results indicate that the scutellum plays an important role in the production of carboxypeptidase I in germinating barley grain.     

Fusion of oil bodies in endosperm of oat grains

Few microscopical studies have been made on lipid storage in oat grains, with variable results as to the extent of lipid accumulation in the starchy endosperm. Grains of medium- and high-lipid oat (Avena sativa L.) were studied at two developmental stages and at maturity, by light microscopy using different staining methods, and by scanning and transmission electron microscopy. Discrete oil bodies occurred in the aleurone layer, scutellum and embryo. In contrast, oil bodies in the starchy endosperm often had diffuse boundaries and fused with each other and with protein vacuoles during grain development, forming a continuous oil matrix between the protein and starch components. The different microscopical methods were confirmative to each other regarding the coalescence of oil bodies, a phenomenon probably correlated with the reduced amount of oil-body associated proteins in the endosperm. This was supported experimentally by SDS-PAGE separation of oil-body proteins and immunoblotting and immunolocalization with antibodies against a 16 kD oil-body protein. Much more oil-body proteins per amount of oil occurred in the embryo and scutellum than in the endosperm. Immunolocalization of 14 and 16 kD oil-body associated proteins on sectioned grains resulted in more heavy labeling of the embryo, scutellum and aleurone layer than the rest of the endosperm. Observations on the appearance of oil bodies at an early stage of development pertain to the prevailing hypotheses of oil-body biogenesis.     

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.     

Sugars as repressors of gibberellin-induced synthesis of α-amylase in wheat

In germinating cereal caryopses, α-amylase is synthesized in the aleurone layer and scutellum epithelium. Produced enzyme is released into the endosperm, where starch is hydrolyzed. We investigated the effect of sugars on gibberellic acid (GA)-induced synthesis of this enzyme in both tissues of wheat (Triticum aestivum L.) seeds. α-Amylase synthesis in the embryo was much more sensitive to sugars, and their inhibitory effect was observed at the lower concentrations (10–20 mM), whereas in the aleurone layer the enzyme was only inhibited at a relatively high (above 100 mM) concentration of sugars in the medium. These results point to a specific (repressive) influence of sugars on embryonic α-amylase and probably to its nonspecific (osmotic) effect on the cells of the aleurone layer. It was found that phosphorylated sugars were more effective repressors of α-amylase than nonphosphorylated sugars.     

Spatial X-ray fluorescence micro-imaging of minerals in grain tissues of wheat and related genotypes

Main conclusion

Wheat and its related genotypes show distinct distribution patterns for mineral nutrients in maternal and filial tissues in grains. X-ray-based imaging techniques are very informative to identify genotypes with contrasting tissue-specific localization of different elements. This can help in the selection of suitable genotypes for nutritional improvement of food grain crops.

Abstract

Understanding mineral localization in cereal grains is important for their nutritional improvement. Spatial distribution of mineral nutrients (Mg, P, S, K, Ca, Fe, Zn, Mn and Cu) was investigated between and within the maternal and filial tissues in grains of two wheat cultivars (Triticum aestivum Cv. WH291 and WL711), a landrace (T. aestivum L. IITR26) and a related wild species Aegilops kotschyi, using micro-proton-induced X-ray emission (µ-PIXE) and micro-X-ray fluorescence (µ-XRF). Aleurone and scutellum were major storage tissues for macro (P, K, Ca and Mg) as well as micro (Fe, Zn, Cu and Mn) nutrients. Distinct elemental distribution patterns were observed in each of the four genotypes. A. kotschyi, the wild relative of wheat and the landrace, T. aestivum L. IITR26, accumulated more Zn and Fe in scutellum and aleurone than the cultivated wheat varieties, WH291 and WL711. The landrace IITR26, accumulated far more S in grains, Mn in scutellum, aleurone and embryo region, Ca and Cu in aleurone and scutellum, and Mg, K and P in scutellum than the other genotypes. Unlike wheat, lower Mn and higher Fe, Cu and Zn concentrations were noticed in the pigment strand of A. kotschyi. Multivariate statistical analysis, performed on mineral distribution in major grain tissues (aleurone, scutellum, endosperm and embryo region) resolved the four genotypes into distinct clusters.     

ANATOMY OF THE CARYOPSIS OF BRIZA MAXIMA (POACEAE)

Briza maxima (quaking grass) is a cosmopolitan grass common to Europe and North and South America. It grows in disturbed soils and on roadsides. The hemispherical caryopsis is embedded between a leaflike lemma and flattened palea. The embryo is of the festucoid type. The scutellum shows two surrounding ridges at the edge of the scutellum/endosperm boundary, and has lateral lobes. A broad epiblast extends toward the embryo apex and is continuous with the dorsal surface of the coleorhiza. The single-layered aleurone surrounds the starchy endosperm and is discontinuous around the embryo. The caryopsis coat is thin, except at the placental pad where it is thickened by the pigment strand and the nucellar projection.     

Localization of major endopeptidase activities in maize endosperms

Using a tissue print method, major endopeptidase activitieswere observed in the aleurone layer and along parts of scutellumsurface 1 d after imbibition. By day 2 the zone of activityhad spread into the subaleurone and starchy parenchyma cellsof the endosperm. Three days later, activity was detected throughoutthe endosperm tissue, but not in the embryo. Endosperm tissues,aleurone layers and scu-tella were dissected from the seedlingsat different stages after imbibition and endopeptidase activitywas analysed by an activity stain after native PAGE. At leastten different endopeptidase activities were detected in theendosperm tissues during the initial 5 d. Activities similarto these ten enzymes were also detected in aleurone layers.These results suggest that the main source of these endopeptidasesin the endosperm is the aleurone layer. The scutellum had adifferent spectrum of endopeptidases. One of these alternativeendopeptidases, which was detected on the first day after theaddition of water, was a metallo-enzyme with electrophoreticproperties similar to an activity found in endosperm tissueshortly after imbibition. Key words: Zea mays, endopeptidase localization, seed germination     

Immunohistochemical localization of β-amylase in resting barley seeds

The cellular localization of β-amylase (EC 3.2.1.2) in resting barley seeds was investigated by immunohistochemistry. The monospecificity of the antibodies used was shown by immunoelectrophoresis and western blotting. An adaptation of the immunofluorescence technique allowed the localization of β-amylase. free of autofluorescence, in the different parts of the seed. In endosperm, there was β-amylase protein in aleurone layers, only in the starchy endosperm, where the distribution of the enzyme was not uniform. The β-amylase of starchy endosperm. which can be in a free or a hound form, was mainly localized around starch granules of different sizes. In the embryo. β-amylase was present only in the part of the scutellum in front of the first leaf. By immunoquantitation after separation of the seed parts, its was shown that the ratio between the amounts of enzyme in embryo and endosperm was less than 1/3000.     

Nucleotide pyrophosphatase activity in dry and germinated seeds of Triticum,and its relationship to general acid phosphatase activity

A cytochemical investigation has been made of nucleotide pyrophosphatase activity in dry and germinated seeds of Triticum, and its distribution compared to that of general acid phosphatase reactions seen with naphthol AS-BI phosphate and p-nitrophenylphosphate as substrates. Acid phosphatase activity was present in the cytoplasm and in channels through the walls of the aleurone cells in both dry and germinated seeds. The cytoplasmic activity was even more marked with nucleotide pyrophosphatase which was almost entirely absent from the cell walls. Nucleotide pyrophosphatase was active in all endosperm cells but particularly in some cells adjacent to the aleurone layer. In addition, all cells of the scutellum and embryo were positive for nucleotide pyrophosphatase activity, especially the developing fibres and xylem elements of leaves and coleoptiles, mature leaf xylem and phloem elements, scutellar provascular and vascular tissues and the epidermis of dark grown coleoptiles.Abbreviation GA₃ gibberellic acid     

X-ray Analysis Studies of Elements Stored in Protein Body Globoid Crystals of Triticum Grains

Energy-dispersive x-ray analysis was used to investigate the elemental storage within protein bodies, specifically the globoid crystals, in grains of wheat. Areas of the grain investigated included various parts of the embryo, the aleurone layer plus starchy endosperm near the embryo and the aleurone layer plus starchy endosperm farthest from the embryo. Variations did occur grain-to-grain, cell-to-cell and, in certain regions, intracellularly. No protein bodies with electron-dense globoid crystals were found in the starchy endosperm. Generally globoid crystals contained P, K, and Mg in all areas investigated. Globoid crystals from the aleurone layer farthest from the embryo on occasion contained Ca, whereas aleurone globoid crystals near the embryo sometimes contained Fe. In most of the embryo regions examined, a few globoid crystals contained Ca along with P, K, and Mg. No specific pattern to the Ca distribution could be found. Welldefined elemental distribution occurred with Mn. Manganese was found only in globoid crystals located in the base and midregions of the stele in the radicle. Thus, in wheat there is some specific distribution of minerals dependent upon cell type and/or position in the grain.     

The structure and function of the scutellum of the Grarnineae

NEGBI, M., 1984. The structure and function of the scutellum of the Gramineae. Four kinds of scutella, of which only the first is universally known, can be distinguished in the Gramineae. (1) The scutellum sew stricto , the kind most commonly described in textbooks. In this scutellum the only growth activity during germination is the development of every epithelial cell into a separate elongated papilla. These papillae are involved in secretion of hydrolases, gibberellins and other hormonal factors which in their turn activate the aleurone layer; and in absorption of the mobilized endosperm reserves. (2) The kind characteristic of Auma is found in several genera. In this the scutellar tip elongates during germination, reaches the distal end of the endosperm sac and develops papillae over its whole surface. (3) The kind found in Cizuniu in which the scutellar tip elongates and extends to the far end of the caryopsis during embryo development, but not during germination. In this scutellum only the abaxial surface faces the bulk of the storage endosperm and probably only this surface becomes papillate. Several bamboo genera have the kind of scutellum characterized by Melocannu . This scutellum has evolved as a storage organ and in mature caryopses the endosperm is reduced. This kind is associated with vivipary and with the presence of storage tissue in the pericarp.
The vascularization and the structure of the scutellar epithelium, as studied mainly in a limited number of species belonging to the first kind, are related to the functions of the scutellum. The scutellum has a prime role in controlling the mobilization of endosperm reserves.     

Expression of the 30 kD cysteine endoprotease B in germinating barley seeds

The expression of a 30 kD cysteine endoprotease (EP-B) was studied by in situ hybridization and immunomicroscopy to clarify its role in germinating barley grains. At the beginning of germination, EP-B mRNA was expressed in the scutellar epithelium and aleurone cells next to the embryo. Later, mRNA levels were highest in the aleurone layer proceeding to the distal end of the grain. During the first day of germination, EP-B protein was strongly localized to the germ aleurone and scutellar epithelium from where the secretion into the starchy endosperm began. Secretion was also observed to proceed along the aleurone layer to the distal end. These results show that EP-B is differentially localized during germination, and both scutellum and aleurone layer are able to synthesize and secrete EP-B protein.     

Differential gene expression during seed germination in barley (Hordeum vulgare L.)

A barley cDNA macroarray comprising 1,440 unique genes was used to analyze the spatial and temporal patterns of gene expression in embryo, scutellum and endosperm tissue during different stages of germination. Among the set of expressed genes, 69 displayed the highest mRNA level in endosperm tissue, 58 were up-regulated in both embryo and scutellum, 11 were specifically expressed in the embryo and 16 in scutellum tissue. Based on Blast X analyses, 70% of the differentially expressed genes could be assigned a putative function. One set of genes, expressed in both embryo and scutellum tissue, included functions in cell division, protein translation, nucleotide metabolism, carbohydrate metabolism and some transporters. The other set of genes expressed in endosperm encodes several metabolic pathways including carbohydrate and amino acid metabolism as well as protease inhibitors and storage proteins. As shown for a storage protein and a trypsin inhibitor, the endosperm of the germinating barley grain contains a considerable amount of residual mRNA which was produced during seed development and which is degraded during early stages of germination. Based on similar expression patterns in the endosperm tissue, we identified 29 genes which may undergo the same degradation process. Electronic Publication     

Hormonal Regulation of Organic and Phosphoric Acid Release by Barley Aleurone Layers and Scutella

The release of acid from the aleurone layer and scutellum of barley (Hordeum vulgare L. cv Himalaya) was investigated. Aleurone layers isolated from mature barley grains acidify the external medium by releasing organic and phosphoric acids. Gibberellic acid and abscisic acid stimulate acid release 2-fold over control tissue incubated in 10 mM CACl2. Gibberellic acid causes medium acidification by stimulating the release of phosphoric and citric acids, whereas abscisic acid stimulates the release of malic acid. The accumulation of these acids in the incubation medium buffers the medium against changes in pH, particularly between pH 4 and 5. The amounts of amino acids that accumulate in the medium are low (2-12 nmol/layer) compared to other organic and phosphoric acids (100-500 nmol/layer). The scutellum does not play a major role in medium acidification but participates in the uptake of organic acids. The organic acid composition of the starchy endosperm changes after 3 d of imbibition; malic, succinic, and lactic acids decrease, whereas citric and phosphoric acids remain unchanged or increase. These results indicate that during postgerminative growth, the acidity of the starchy endosperm is maintained by acid production by the aleurone layer.