Publisher Summary This chapter discusses that the knowledge of phytic acid had its beginning in the discovery by Hartig, who isolated small particles or grains (which were not starch grains) from the seeds of various plants. Phytic acid has been generally regarded as the primary storage form of both phosphate and inositol in almost all seeds. The amount of phytic acid varies from 0.50% to 1.89% in cereals (except polished rice), from 0.40% to 2.06% in legumes, from 2.00% to 5.20% in oil seeds except soybeans and peanuts (grouped under legumes), and from 0.40% to 7.50% in protein products. Many foods and seeds contain myo-inositol hexaphosphate as an important source of phosphorus, and accurate methods for its determination are needed. Phytase (meso-inositol hexaphosphate phosphohydrolase, EC 3.1.3.8) is widely distributed in plants, animals, and fungi. In mature cereal grains, legumes, and oil seeds, the major portion of the total phosphorus is present in the form of phytic acid (phytate). Experiments with animals have suggested that phytic acid in plant foods complexes with dietary essential minerals such as calcium, zinc, iron, and magnesium and makes them biologically unavailable for absorption. The correlation of phytate with the cooking quality of peas was first suggested by Mattson. Phytic acid, myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate serves as the main phosphorus store in mature seeds and grains. In cereals and legumes, phytic acid content ranges from 0.14% to 2.05%, which accounts for 18 to 88% of the total phosphorus.

Phytates in Legumes and Cereals

▪ Abstract The endosperm develops from the central cell of the megagametophyte after introduction of the second male gamete into the diploid central cell. Of the three forms of endosperm in angiosperms, the nuclear type is prevalent in economically important species, including the cereals. Landmarks in nuclear endosperm development are the coenocytic, cellularization, differentiation, and maturation stages. The differentiated endosperm contains four major cell types: starchy endosperm, aleurone, transfer cells, and the cells of the embryo surrounding region. Recent research has demonstrated that the first two phases of endosperm occur via mechanisms that are conserved among all groups of angiosperms, involving directed nuclear migration during the coenocytic stage and anticlinal cell wall deposition by cytoplasmic phragmoplasts formed in interzones between radial microtubular systems emanating from nuclear membranes. Complete cellularization of the endosperm coenocyte is achieved through centripetal growt...

ENDOSPERM DEVELOPMENT: Cellularization and Cell Fate Specification

Reserve polysaccharides are formed by plant cells at certain physiological stages of development, usually during periods of intense photosynthetic activity, and are later “digested” to deliver carbohydrate monomers which re-enter the cells’ metabolism. They are stored — that is temporarily withdrawn from cellular metabolism — either in the solid state or, less frequently, in a dissolved or highly hydrated colloidal state. Their deposition takes place on the outside of the plasmalemma in the cell wall region, in plastids or in cell vacuoles. As far as we know, starch is the only polysaccharide formed in plastids and the plastids constitute the only cellular compartment which forms starch in higher plants.

Reserve Polysaccharides Other Than Starch in Higher Plants

Methylation analysis and hydrolysis with specific enzymes indicates that aleurone cell wall preparations from wheat (Triticum aestivum cv. Insignia) and barley (Hordeum vulgare cv. Clipper) are composed of two main types of polysaccharides, heteroxylans and 1,3;1,4-β-glucans. Small amounts of glucomannan and cellulose are also present. Approximately 1% of a 1,3-β-glucan was also detected in the wall preparations using a specific 1,3-β-glucan exohydrolase. This material probably corresponds to the aniline blue-fluorescent deposits seen at the aleurone-endosperm interface. As isolated, the aleurone wall preparations are associated with protein, 10.5% in wheat and 16.0% in barley. Electron microscopic examination and amino acid analyses indicated that a major part of this protein arises from cytoplasmic protein deposited on the walls during isolation in organic solvents. Fractionation of the walls by conventional procedures showed that the heteroxylan and 1,3 ;1,4-β-glucan components were extracted by water, 8 M urea and alkaline solvents. Their differential solubility is discussed in terms of their structural organization and possible covalent interactions between polymers. Transmission electron microscopy of the walls at each stage of fractionation showed that the bilayered organization was retained after water and 8 M urea extraction but was lost following extraction in alkaline media.

Chemistry and Organization of Aleurone Cell Wall Components From Wheat and Barley

Publisher Summary This chapter discusses the regulation of synthesis and transport of secreted proteins in cereal aleurone. It is one of the few digestive tissues found in plants, and the synthesis and secretion of hydrolytic enzymes by this tissue are controlled primarily by the plant hormones gibberellic acid (GA) and abscisic acid (ABA), and by the calcium ion. The aleurone cells from various cereals have the same basic structure and function and play the same role in germination-related processes. The most extensively studied aleurone enzyme is α-amylase. α-Amylase plays a role in the endosperm during germination; it is made in very large amounts in aleurone; it is easily assayed, purified, and detected on electrophoretic gels; it is very stable; and it consists of a single polypeptide chain. The mechanisms that regulate the intracellular transport and secretion of hydrolytic enzymes from the barley aleurone cell as well as the mechanisms involved in the targeting of proteins to subcellular compartments and the identity of these compartments are reviewed in the chapter.

Regulation of synthesis and transport of secreted proteins in cereal aleurone.

Cytochemical methods have been used in conjunction with light and electron microscopy to determine the nature of the inclusions in aleurone grains of barley aleurone layers. Two kinds of inclusions were found: (1) Globoids within globoid cavities which were not enclosed by a membrane: the globoids stained red with toluidin blue due to the presence of phytin, and with lipid stains; (2) Protein-carbohydrate bodies which stained green with toluidin blue. The characteristics of globoids and protein-carbohydrate bodies as seen in the electron microscope are described in detail using both glutaraldehyde- and permanganatefixed tissues. The protein-carbohydrate body was identified by silver-hexaminestaining; this was not caused by carbohydrate but by some component which stained green in toluidin blue and which also occurred in cell walls in a thin band adjacent to the cytoplasm. The characteristics of both bodies are discussed in relation to apparent confusion in their identities in previous electron-microscope studies.

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The structure and composition of aleurone grains in the barley aleurone layer.

Acid phosphatase has been localized by cytochemical techniques in aleurone layers of dry barley (Hordeum vulgare L.) grains, in imbibed half-grains and in isolated layers treated with and without gibberellic acid (GA3). A major fraction of the enzyme activity is located in the cell walls. During imbibition and incubation of layers without GA3 a steady increase of enzyme activity in the inner wall region indicates a continued release of enzyme into the walls, but there is no essential change in the distribution of wall-enzyme sites. On the other hand, when GA3 is present enzyme activity is found for the first time in regions of the wall that become digested during GA3 treatment. These results indicate that the digested wall channels act as preferential routes through which acid phosphatase is released from the aleurone layer. No digested wall channels are formed in the absence of GA3 and, there being no route for release of the enzyme, it accumulates in the inner regions of the wall around aleurone cells. Assays of enzyme activity in vitro support the conclusions based on the histochemical data. They indicate that release of acid phosphatase from aleurone layers is under strict GA3 control, but that some of the increase in acid phosphatase activity in the isolated during incubation is not GA3 dependent.Acid phosphatase is present in the protein matrix of aleurone grains in all stages except the dry grain. Enzyme activity persists in aleurone grains throughout GA3 treatment when enlargement of the grains and mobilization of reserves takes place. It is suggested that this phosphatase hydrolyses phosphate reserves within the aleurone grains.

Cytochemical localization of phosphatase in barley aleurone cells: The pathway of gibberellic-acid-induced enzyme release

Gibberellic-acid(GA3)-induced α-amylase has been localised in barley aleurone layers using cytochemical methods and light microscopy. Evidence obtained from the use of a starch substrate film method as well as immunofluorescence indicated that the first amylase to appear in the cell was associated with aleurone grains, apparently with the outer membrane, and also with the peripheral cytoplasm. In GA3-treated tissue, the amylase distribution was much more diffuse, although patchy, throughout the cytoplasm and it tended to accumulate in the endosperm side of the cell. The possibility that the aleurone grain membrane is the site of gibberellin-induced enzyme synthesis and that it proliferates to become rough endoplasmic reticulum is considered. Immunological information was obtained which supports earlier indications that induced α-amylase consists of two different proteins, each with molecular heterogeneity.

Cytochemical localization and antigenicity of α-amylase in barley aleurone tissue.

Isolated barley (Hordeum vulgare L.) aleurone layers released a number of proteins into an aqueous medium in the absence of gibberellic acid (GA3). Evidence from molecular weight determinations and a number of immunological tests indicated that these proteins were water-soluble endosperm proteins which apparently arose from endosperm cells which adhered to the layers during isolation. They were not aleurone-cell proteins. By means of immunofluorescence, the water-soluble endosperm proteins were found to be concentrated around starch grains in the starchy endosperm. These proteins were resistant to hydrolysis by GA3-induced hydrolases released from aleurone tissue. Isolated aleurone layers could be washed free of soluble endosperm proteins. After treatment with GA3, such layers released another group of proteins which were shown by immunological and electrophoretic methods to be uncontaminated by soluble endosperm proteins. The pure GA3-induced proteins were separated, using SDS-acrylamide gel disc electrophoresis, into 12 components which had molecular weights (monomer) from 15500 to 81000. Ten of these protein bands became radioactive if GA3-treatment of layers was carried out in the presence of radioactive amino acids, and therefore probably contained de novo synthesized proteins. The two protein bands which were not labelled contributed about 40% of the protein released by washed aleurone layers after GA3 treatment, and their production appeared to be dependent on proteolysis.

J. V. Jacobsen

Papers

The structure and composition of aleurone grains in the barley aleurone layer.

Cytochemical localization of phosphatase in barley aleurone cells: The pathway of gibberellic-acid-induced enzyme release

Cytochemical localization and antigenicity of α-amylase in barley aleurone tissue.

The proteins released by isolated barley aleurone layers before and after gibberellic-acid treatment.