Endosperm-specific demethylation and activation of specific alleles of α-tubulin genes of Zea mays L.

We have investigated the methylation status of the α-tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the α-tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tubα2 and tubα4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two α-tubulin genes, tubα3 and tubα4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.     

Maize α-tubulin genes are expressed according to specific patterns of cell differentiation

In the past few years many - and -tubulin genes of different organisms have been cloned and studied, and in most systems studied they constitute multigene families. In plants, most studies have been done in Arabidopsis thaliana and Zea mays. In this paper, the study of mRNA accumulation by in situ hybridization and the activity of three maize -tubulin gene promoters (tua1, tua2 and tua3) in transgenic tobacco plants are described. In maize, the expression of these three tubulin isotypes differ in the root and shoot apex and is associated with different groups of cells throughout the distinct stages of cell differentiation. In transgenic tobacco plants the promoters of the genes, fused to the uidA reporter gene (GUS), direct expression to the same tissues observed by in situ hybridization experiments. The tua1 promoter is mainly active in cortex-producing meristematic cells and in pollen, whereas tua3 is active in cells which are differentiating to form vascular bundles in the root and shoot apices. The accumulation of tua2 mRNA is detected by RNA blot in a similar form as tua1, but at a very much low level. In situ hybridization indicates that the tua2 mRNA specifically accumulates in the maize root epidermis. No GUS staining was detected in transgenic tobacco plants with the tua2 promoter. The difference in expression of the specific genes may be linked to processes where microtubules have different functions, suggesting that in plants, as in animals, there are differences in the function of the tubulin isotypes.     

Monensin inhibits the secretion of α-amylase but not polysaccharide slime from seedling tissues ofZea mays

Summary The effect of monensin on polysaccharide slime secretion by root tips of corn (Zea mays) was studied. Various treatment times and ionophore concentrations were tested: none resulted in inhibition of slime secretion. Because monensin changes the pH of the medium, its effect was also monitored in strongly buffered media and at different pH's. Even in such media, monensin did not inhibit slime secretion. We also measured the effect of the drug after a pulse with [³H]fucose or a pulse followed by a chase. The amount of labeled slimed secreted was not altered by the ionophore. However, 10M monensin affected the development of root tips and drastically reduced their growth. We showed that monensin inhibits the secretion of -amylase by the scutellum of the same plantlet. The importance of the nature of the secretory compound in relation to monensin inhibition of its secretion is discussed.Abbrevations Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sul-fonic acid - Mes 2-(N-morpholino)ethane-sulfonic acid     

Distribution of acetylated α-tubulin in brain

Summary We studied the solubility properties of brain acetylated -tubulin, as well as the localization of this tubulin in brain tissue. Endogenous unpolymerized tubulin and cytoskeletal tubulin were fractionated after brain Triton-solubilization. Using the immunoblotting technique, we found that acetylated -tubulin was recovered in the cytoskeletal fraction, and that most (92%) of the acetylated microtubules of this fraction were depolymerized by cold/Ca²⁺ treatment. In another set of experiments, axonal and soma-dendritic preparations were found to have equivalent amounts of acetylated -tubulin. By immunogold electron microscopy, we established that acetylated microtubules are widely distributed in dendrites of the central nervous system.     

A highly conserved α-tubulin sequence from Prunus amygdalus

The sequence of an -tubulin from Prunus amygdalus has been obtained by cDNA cloning. When this sequence is compared to that of the Tub1 gene from maize it shows a very high degree of similarity, much higher than any of the -tubulin sequences reported so far from plants. The expression of this gene is high in the stages of seed development where a high divisional activity is present. It is preferentially expressed in the radicular tissues as it is gene Tub1 in maize. Southern analysis indicates that this gene may from a subfamily of -tubulin genes having similar sequence and tissue specificity and existing at least in maize and in Prunus.     

A truncated form of α-tubulin detected in purified proteasome complexes

The 26S proteasome is a multisubunit protein complex responsible for selective protein degradation in the cell. A number of proteins with known and unknown functions were shown to be permanently or temporarily associated with 26S proteasomes. Identification of proteins that interact with proteasomes is an important step in the understanding of the proteasome functions in the cell and the mechanisms of their regulation. Using MALDI–ICR mass spectrometry, we have shown that some proteins of the cytoskeleton, such as actin, α-actinin 4, and α- and β-tubulins are associated with proteasomes obtained by affinity purification from the human myelogenous leukemia cell line K562. Western blot analysis showed that a truncated form of α-tubulin was associated with the purified proteasomes. The presence of the α-tubulin isoform in complex with affinity purified proteasomes was also observed in the human embryonic kidney cell line 293.     

Electron-microscopic demonstration of α-tubulin immunoreactivity in astroglia

Summary Vibratome sections of the cerebral cortex, hippocampus and cerebellum were immunostained for -tubulin using the TU-Ol monoclonal antibody. In all three regions, electron microscopy of the immunostained preparations revealed — in addition to the previously described reaction of pyramidal apical dendritic microtubules — consistent staining of the ribosomal apparatus of astrocytes.     

Uneven distribution of α-naphthyl acetate in tissues

Summary In the mouse ear in which esterases had been inactivated with warm formol the distribution of -naphthyl acetate was studied by an UV absorption technique. The substrate was found to concentrate in structures containing lipids. The physicochemical properties of substrates used in enzyme chemistry seem to be such that it might prove difficult to find any substrate that would distribute evenly in tissues. So far it does not seem to have been ruled out that this influences enzyme reactions by some mass law effect.

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Zusammenfassung Die Verteilung von -Naphthylazetat wurde mit Hilfe einer UV Absorptionstechnik in einem Mausohr untersucht, in dem die Esterasen mit heißem Formol inaktiviert worden waren. Das Substrat konzentrierte sich in lipoid-haltigen Strukturen. Die physikalisch-chemischen Eigenschaften histochemischer Enzym substratescheinen derartig zu sein, daß es schwer fallen dürfte, ein Substrat zu finden, das sich in den Geweben gleichmäßig verteilen würde. Bis aufs weitere scheint es durchaus nicht ausgeschlossen zu sein, daß dies Enzymreaktionen durch irgendein Massenwirkungsgesetz beeinflußt.

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Aided by a grant from Sigrid Jusélius Foundation.     

Embryo Rescue and Induction of Somatic Embryogenesis as a Method to Overcome Seed Inviability in Zea mays ssp. mays × ; Zea mays ssp. parviglumis Crosses

Zea mays ssp. mays (2n=40) and Z. mays ssp. parviglumis (2n=20) were crossed to obtain hybrid plants by embryo rescue. Hybrid embryos were isolated and cultured on García et al. (1992) basic medium supplemented with 2,4-dichlorophenoxyacetic acid and/or kinetin in different concentrations. Caryopses harvested 23 d after pollination (DAP) were turgid, with 0.3 to 0.5 mm long embryos, while those harvested 30 DAP were shrunken, with 1 to 1.5 mm long embryos. Twenty days after plating, 100 % of the younger embryos gave rise to white, compact embryogenic calli. Subsequently, coleoptiles, leaf-like structures, shoots and roots originated from them and 35 hybrid plants were regenerated from 60 embryos. Embryogenic or organogenic calli frequencies did not differ among hormonal treatments, but they decreased, on average, from 90.5 to 44.3 %, comparing 50 and 120-d-old cultures. The older embryos regenerated plants only by germination, although they gave rise to organogenic callus with low frequencies. Regenerated plants showed a somatic chromosome number of 2n=30, pollen fertility of 40 to 80 % and 15 % viable naked caryopses.