Aleurone Layer Cells Research Articles

Two cis-acting elements necessary and sufficient for gibberellin-upregulated proteinase expression in rice seeds.

In germinating rice seeds, a cysteine proteinase (REP-1), synthesized in aleurone-layer cells, is a key enzyme in the degradation of the major storage protein, glutelin. The expression of the gene for REP-1 (Rep1) is induced by gibberellins (GAs) and repressed by abscisic acid (ABA). To identify GA-responsive elements in the Rep1 promoter, we developed a transient expression system in rice aleurone cells. Deletion and point-mutation analyses indicated that the GA-response complex was composed of TAACAGA, TAACGTA, and two copies of CAACTC. The two former sequences were identical to GAREs conserved in the promoter of genes for alpha-amylase and proteinases in cereals. The latter, termed as CAACTC regulatory elements (CAREs), were novel GAREs. Gain-of-function experiments revealed that two pairs of GARE and CARE were necessary and sufficient to confer GA inducibility. The sequences were also required for effective transactivation by the transcription factor OsGAMyb. Four copies of either GARE or CARE showed transactivation neither by OsGAMyb nor by GA induction. CARE and GARE were also found in the promoters of a rice alpha-amylase gene, RAmy1A, and a barley proteinase gene, EPB1, which are expressed in germinating seeds. Mutations of CARE in their promoters caused a loss of GA inducibility and GAMyb transactivation, suggesting that CARE is the regulatory element for GA-inducible expression of hydrolase genes in the germinating seeds.

An Electron Paramagnetic Resonance Spin-Probe Study of Membrane-Permeability Changes with Seed Aging.

We developed an electron paramagnetic resonance spin-probe technique to study changes in the barrier properties of plasma membranes in wheat (Triticum aestivum L.) seeds during aging under dry storage. The estimation of these barrier properties was based on the differential permeability of membranes for the stable free radical 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy and the broadening agent ferricyanide. The line-height ratio between the water and lipid components in the electron paramagnetic resonance spectra of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (R value) allowed for the quantitative assessment of the plasma membrane permeability in small samples, enabling separate studies of the axis, scutellum, aleurone layer, and starchy endosperm tissue. High R values corresponded to low permeability and vice versa. Starchy endosperm cells had completely permeable plasma membranes even in mature, viable seeds. The loss of germinability with aging coincided with a considerably increased plasma membrane permeability of the embryo axis cells, but not of the scutellum and aleurone layer cells. The threshold R value for the individual axes associated with viability loss was established at 5 to 6, with the total ranging from 0 to more than 12. We suggest that the R value of an individual axis is the result of contributions from all individual cells, each of them characterized by a different permeability. The loss of viability, therefore, corresponds to the accumulation of cells having permeability above a critical level.

Premature Drying Increases the GA-Responsiveness of Developing Aleurone Layers of Barley (Hordeum vulgare L.) Grain

The effect of premature drying on the sensitivity of aleurone layer cells of developing barley (Hordeum vulgare L.) grain to gibberellic acid (GA3) was investigated. The capacity of barley aleurone layer cells to respond to GA3, as indicated by a-amylase synthesis and secretion by de-embryonated grain, increased during the later stages of development. Aleurone layers of immature grain (younger than 30 d after anthesis; DAA) were not capable of producing aamylase in response to GA3; however, premature drying at this stage promoted GA-responsiveness resulting in the induction of mRNA for a-amylase and increased a-amylase synthesis and secretion. Preincubation of the immature grain or its maintenance at 100% relative humidity prior to exposure of the de-embryonated grain to GA 3 also led to an enhanced capacity of the aleurone layer to produce aamylase and its mRNA as compared to the fresh, untreated grain. However, the amount of mRNA and enzyme produced was much lower than that induced by premature drying. Moreover, following these nondrying treatments, the aleurone layer cells remained unresponsive to exogenous GA3; the same amount of enzyme was produced in the absence of applied GA3. Transient expression of chimeric gene constructs in aleurone layer cells of de-embryonate d grain suggest that drying up-regulates the a-amylase gene promoter in response to GA3. We conclude that desiccation is required for barley aleurone layer cells to become responsive to GA 3 and hence express their full potential for aamylase synthesis and secretion.

Tissue-specific localization of aspartic proteinase in developing and germinating barley grains

Resting seeds of several plant species, including barley grains, have been reported to contain aspartic proteinase (EC 3.4.23) activity. Here, the expression of the Hordeum vulgare L. aspartic proteinase (HvAP) was studied in developing and germinating grains by activity measurements as well as by immunocytochemical and in-situ hybridization techniques. Southern blotting suggests the presence of one to two HvAP-encoding genes in the barley genome, while Northern analysis reveals a single 2.1-kb mRNA in grains and vegetative tissues. Western blotting with antibodies to HvAP shows the same subunit structure in different grain parts. In developing grains, HvAP is produced in the embryo, aleurone layer, testa and pericarp, but in the starchy endosperm HvAP is present only in the crushed and depleted area adjacent to the scutellum. During seed maturation, HvAP-encoding mRNA remains in the aleurone layer and in the embryo, but the enzyme disappears from the aleurone cells. The enzyme, however, remains in the degenerating tissues of the testa and pericarp as well as in resting embryo and scutellum. During the first three days of germination, the enzyme reappears in the aleurone layer cells but is not secreted into the starchy endosperm. The HvAP is also expressed in the flowers, stem, leaves, and roots of barley. The wide localization of HvAP in diverse tissues suggests that it may have several functions appropriate to the needs of different tissues.

Tissue- and cell-specific expression of the two sucrose synthase isoenzymes in developing maize kernels

The localization of the two known sucrose synthase isoenzymes of Zea mays L., sucrose synthase 1 and sucrose synthase 2, was studied during kernel development by indirect immunohistochemistry. These enzymes are encoded by the Sh and Sus genes, respectively. Since the antiserum used cross-reacts with both enzymes, tissue sections of Sh and sh kernels were compared. In the latter tissue no sucrose synthase 1 is expressed and thus the signal obtained was ascribed to sucrose synthase 2. We found that the isoenzymes are differentially expressed. While sucrose synthase 1 is expressed only in the endosperm, sucrose synthase 2 is found in almost all tissues of the kernel with cxpression levels specific for cell type and developmental stage. Sucrose synthase 2 is expressed strongly in the aleurone and subaleurone cell layers, where the signal detected is as strong as or even stronger than the sucrose synthase 1 signal in the inner endosperm. The distribution of the enzymes changes characteristically during development.

Mycotoxins in cereal grain. Part X. Invasion of fungi mycelium into wheat and barley kernels.

Histochemical examination of wheat kernels invaded by Aspergilli and Penicillia proved penetration of fungal mycelium into subaleurone and endosperm cells through aleurone layer cells. Walls of aleurone layer cells in invaded kernels were found to be thicker and split. Formation of spores in cleistothecia's in endosperm cells was found. The observation of kernels under a stereoscopic microscope was found to be a sensitive way to detect fungi invasion in cereal kernels.

Responses of Enzymically Isolated Aleurone Cells of Oat to Gibberellin A3

Oat (Avena sativa L.) aleurone layer cells (spheroplasts) were isolated by maceration of the aleurone layer with a mixture of commercially available cellulase and pectinase. About 20% of the cells present in intact layers were released as spheroplasts and 79 +/- 9% of the spheroplast population was viable as judged by methylene blue staining. The spheroplasts became disorganized in solutions containing less than 0.4 md-mannitol. When the spheroplasts were incubated for 48 hours, total activities of acid p-nitrophenyl phosphatase and acid proteinase increased and protein levels decreased. These changes were not effected by gibberellin A(3). Isolated aleurone layers incubated under the same conditions as the spheroplasts showed reduced responses to gibberellin A(3). It is concluded that the necessary presence of an osmoticum limits the value of spheroplasts as a system for studying the mechanism of action of gibberellin A(3) in the aleurone cell.

Maize with Multilayer Aleurone of High Protein Content1

Coroico corn (Zea mays L.), a South American race of floury maize contained two to six layers of aleurone cells instead of the customary single aleurone cell layer found in ordinary yellow dent corn. In Coroico with an average of 3.7 aleurone cell layers, the aleurone made up 4.3% of the endosperm as against 2.1% in yellow dent corn. Total protein in Coroico aleurone is 35 to 38% compared with 22% in yellow dent corn. Lysine levels in aleurone proteins of all corns examined, both Coroico and yellow dent were comparable ranging from 4.0 to 4.4 g/100 g of protein. Because of the additive effect of the multiple aleurone and the high protein content of the aleurone, lysine in Coroico endosperm was higher than in that of yellow dent corn. Multiple aleurone is transmitted as a partial dominant character over the normal single aleurone condition. Improvement of protein nutritional quality using Coroico germ plasm appears worthwhile.

Chapter 4 Hormonal Control of A Secretory Tissue

The aleurone layer of the seeds of gramineae is a secretory tissue surrounding the endosperm reserves of the seed. During germination, gibberellins from the embryo induce the aleurone layer cells to synthesize and secrete several hydrolases, including amylases and proteases that hydrolyze the reserve starch to maltose and glucose and the reserve proteins to amino acids. These end products of hydrolysis are transported to the growing regions of the embryo. In the intact germinating seed, the embryo, both the scutellum and the nodal region, synthesizes the gibberellins, probably GA, and GA that control the aleurone response. This is appropriate and logical, because it is the embryo that gains from an ordered mobilization of the endosperm reserves. Thus, the cereal grain provides a convenient system for the study of the plant's use of a hormone to integrate the various functions of the different tissues and organs.

Aleurone Nuclei and 2C Meristematic Nuclei inTriticum Durum. a Comparative Cytophotometric Study

Abstract In unripe caryopses of Triticum durum Desf., DNA content per nucleus, histone content per nucleus and DNA/histone ratio were detected both in the meristem cell population and in the Aleurone cell layer. The collected data evidentiate that Aleurone nuclei have not only a DNA content 3C (G1) and 6C (G2) but reach 12C and higher values. This fact indicates that at least an additional endoreduplication occurred (endopolyploidy). Histone content per nucleus is almost constant in meristematic nuclei while it is etherogeneus in Aleurone nuclei. As to DNA/histone ratio, it is seen that 3C nuclei show an higher value than meristematic nuclei, while the ratio decreases in 6C nuclei and reaches the lowest values in 12C nuclei. The significance of these observations and the possible relation between metabolic differentiation and genetic inactivation by histones are discussed.

Ultrastructure of the Developing Aleurone Cells of Wheat Grain

The developing aleurone layer cells of the wheat kernel have been investigated by electron microscopy and the results compared with those of light microscopy. Two weeks after flowering vacuoles appear in the cells and deposits accumulate in these until maturity when the cells are filled 'with the resulting "vacuolar units" 2-3p. in diameter, corresponding to the aleurone grains of light microscopy. The wheat aleurone grain consists of a bounding membrane (of vacuole origin) enclosing a matrix in which are embedded spherical deposits. Some of these deposits are translucent and others opaque to electrons after potassium permanganate and osmium tetroxide fixation. At all stages examined the cytoplasm of aleurone cells contained large numbers of small unidentified bodies with irregular outline and dense contents. At first they are dispersed, but towards maturity are organized as a monolayer over the surface of each aleurone grain and the inner surface of the cell walls. The apparent specificity of these structures to aleurone cells is discussed in relation to future chemical and physiological studies of the tissue.