Histone acetylation in Zea mays.I. Activities of histone acetyltransferases and histone deacetylases

DEAE-Sepharose chromatography of extracts from Zea mays meristematic cells revealed multiple histone acetyltransferase and histone deacetylase enzyme forms. An improved method for nuclear isolation allowed us to discriminate nuclear and cytoplasmic enzymes. Two nuclear histone acetyltransferases, A1 and A2, a cytoplasmic B-enzyme and two nuclear histone deacetylases, HD1 and HD2, have been identified. The histone specificity of the different enzyme forms has been studied in an in vitro system, using chicken erythrocyte histones as substrate. The cytoplasmic histone acetyltransferase B is the predominant enzyme, which acetylates mainly histone H4 and to a lesser extent H2A. The nuclear histone acetyltransferase A1 preferentially acetylates H3 and also H4, whereas enzyme A2 is specific for H3. This substrate specificity was confirmed with homologous Z. mays histones. The two histone deacetylases differ from each other with respect to ionic strength dependence, inhibition by acetate and butyrate, and substrate specificity. The strong inhibitory effect of acetate on histone deacetylases was exploited to distinguish different histone acetyltransferase forms.     

Cytological study on water stress during germination of Zea mays

Summary Kernels of Zea mays were subjected to dehydration treatment at various times during germination. Embryos from kernels dehydrated during the first 36 h of germination are resistant to dehydration and subsequently germinate earlier than controls. Dehydration of kernels germinated during 72h leads to an irreversible arrest of growth of the embryos. However, autoradiographic observations showed that these embryos are still able to incorporate [³H] uridine and probably [4-5-³H] lysine. Incorporation of [³H] thymidine does not occur. The effect of dehydration on root ultrastructure was studied. In embryos dehydrated after 24 h and 72 h of germination, condensation of chromatin is seen and association of elements of rough endoplasmic reticulum with vacuoles and glyoxysomes can be noted. These changes are reversible in drought-resistant embryos and irreversible in drought-sensitive embryos. However, more notable changes than those seen after 24 h can be observed in embryos dehydrated after 72 h of germination: mitochondria and proplastids can not be distinguished with certainty, glyoxysomes fuse and preferably dispose at the periphery of the cell. Rehydration of drought-sensitive embryos causes breakdown in plasma and nuclear membranes, which leads to the loss of cellular compartimentalization. Moreover, the chromatin remains definitively condensed and has lost its function of genetic regulation.     

Induction of acid phosphatase activity during germination of maize (Zea mays) seeds.

Acid phosphatase activity (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) increased during the first 24 h of maize (Zea mays) seed germination. The enzyme displayed a pH optimum of 4.5-5.5. Catalytic activity in vitro displayed a linear time course (60 min) and reached its half maximum value at 0.47 mM p-nitrophenyl phosphate (pNPP). Phosphatase activity towards phosphoamino acids was greatest for phosphotyrosine. The phosphatase activity was strongly inhibited by ammonium molybdate, vanadate and NaF and did not require divalent cations for the catalysis. The temperature optimum for pNPP hydrolysis was 37 degrees C. Under the same conditions, no enzyme activity was detected with phytic acid as substrate. Western blotting of total homogenates during seed germination revealed proteins/polypeptides that were phosphorylated on tyrosine residues; a protein of approximately 14 kDa is potentially a major biological substrate for the phosphatase activity. The results presented in this study suggest that the acid phosphatase characterized under the tested conditions is a member of the phosphotyrosine phosphatase family.     

Histone acetylation and hormone action. Early effects of aldosterone on histone acetylation in rat kidney

Histone acetylation in the kidney of the adrenalectomized rat was increased 5 min after a physiological dose of aldosterone. The increase was exclusively in the histone F2a1. The increase in histone acetylation was given by 9α-fluorocortisol, deoxycorticosterone and cortisol, but not by progesterone. The increase in histone acetylation was abolished by prior treatment of the animals with the anti-mineralocorticoid agent, SC14266 [potassium 3-(3-oxo-17β-hydroxy-4,6-androstadien-17-yl) propionate]; the basal degree of histone acetylation was unaffected. Pretreatment of the animals with actinomycin D or cyclo-heximide did not affect the stimulation, showing the stimulation was not due to increased synthesis of enzyme.     

Specificity of Zea mays histone deacetylase is regulated by phosphorylation.

Mono Q ion exchange high performance liquid chromatography (HPLC) reveals that the main histone deacetylase activity (HD1) of germinating Zea mays embryos consists of multiple enzyme forms. Chromatography of HD1 after treatment with alkaline phosphatase yields two distinct histone deacetylase forms (HD1-A, HD1-B). The same is true for chromatography after phosphatase treatment of a total cell extract. One of these enzyme forms (HD1-A) is subject to phosphorylation, which causes a change in the substrate specificity of the enzyme, as shown with HPLC-purified individual core histone species; the substrate specificity for H2A increases more than 2-fold after phosphorylation, whereas the specificity for H3 decreases to about 60%. The total histone deacetylase activity is quantitatively released from isolated nuclei after extraction with moderate ionic strength buffers; no significant residual enzyme activity could be detected in the nuclear matrix.     

Histone acetylation and histone synthesis in mouse fibroblasts during quiescence and restimulation into S-phase

Mouse NIH 3T3 fibroblasts were starved by serum depletion and subsequently restimulated by addition of serum. Histone acetylation and histone synthesis were studied from the beginning of starvation to the point where most of the cells were in S-phase utilizing electrophoretic and fluorographic techniques. We found that the major part of histone acetylation and histone synthesis occurs during S-phase but that also in the absence of DNA synthesis there are significant changes in the acetylation and synthesis rates of the core histones which occur during the first 6 hours of serum stimulation of quiescent cells, and between 24 and 48 hours of serum starvation.     

Actin expression is induced and three isoforms are differentially expressed during germination in Zea mays

Previous analysis of actin in a dicotyledonous plant, Phaseolus vulgaris (or common bean), showed very low actin levels in cotyledons but they were concentrated in the embryo axis. Upon imbibition, actin expression increased 5-fold and a maximum of four actin isoforms were observed, two of them transient and two major ones were steadily expressed. In this work, analysis of the actin expression in a monocotyledonous plant, Zea mays (or maize), and over a longer period of germination/growth, showed that striking similarities exist. Actin is present in all the seed components, but it is mainly concentrated in the embryo axis. The expression of maize actin was induced during post-imbibition at both the protein and mRNA levels. Sharp increases in actin appeared from 24-48 h and again from 72-96 h. A more modest and steady actin mRNA increase in expression was observed; however, it did not appear as dramatic as in the case of common bean due to the presence of readily detectable amounts of message in the dry maize seed. The isoform distribution in the dry seed showed a pattern of at least three isovariants of pIs approximately 5.0, 5.1, and 5.2, which were differentially expressed at the various post-imbibition times analysed. Two of the actin isoforms at 48 h post-imbibition cross-reacted with a phosphotyrosine-specific antibody and they are the product of three expressed genes as shown by in vitro translation assays. These data indicate that maize actin protein and mRNA expression is induced upon the trigger of germination, and the isoform expression kinetics and patterns resemble those from bean, suggesting that, in both species, actin expression at these early germination/growth stages is a highly regulated event.     

Absence of storage protein synthesis in the embryo of Zea mays

Polypeptides were identified in the total protein extract from isolated maize embryos having a similar behaviour of endosperm zeins and glutelins by means of two-dimensional gel electrophoresis. They reacted with antisera ellicited against the same proteins as seen by immunoblotting. In contrast, mRNAs coding for these proteins were not identified in the poly(A +) or poly(A −) RNA fractions prepared from embryo tissues by Northern hybridization experiments or immunoprecipitation of the in vitro translated products. ‘In situ’ hybridization experiments with zein cDNA clearly show that the corresponding mRNAs are confined to the endosperm and they are not present in the embryo tissues. Possible hypotheses to explain these results are discussed.     

Nonadditive protein accumulation patterns in Maize (Zea mays L.) hybrids during embryo development

Heterosis describes the superior performance of heterozygous F(1)-hybrid plants compared to their homozygous parental inbred lines. In the present study, heterosis was detected for length, weight, and the time point of seminal root primordia initiation in maize (Zea mays L.) embryos of the reciprocal F(1)-hybrids UH005xUH250 and UH250xUH005. A two-dimensional gel electrophoresis (2-DE) proteome survey of the most abundant proteins of the reciprocal hybrids and their parental inbred lines 25 and 35 days after pollination revealed that 141 of 597 detected proteins (24%) exhibited nonadditive accumulation in at least one hybrid. Approximately 44% of all nonadditively accumulated proteins displayed an expression pattern that was not distinguishable from the low parent value. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analyses and subsequent functional classification of the 141 proteins revealed that development, protein metabolism, redox-regulation, glycolysis, and amino acid metabolism were the most prominent functional classes among nonadditively accumulated proteins. In 35-day-old embryos of the hybrid UH250xUH005, a significant up-regulation of enzymes related to glucose metabolism which often exceeded the best parent values was observed. A comparison of nonadditive protein accumulation between rice and maize embryo data sets revealed a significant overlap of nonadditively accumulated proteins suggesting conserved organ- or tissue-specific regulatory mechanisms in monocots related to heterosis.     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1'in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.     

Histone acetylation in gene regulation.

Genetic information is packaged in the highly dynamic nucleoprotein structure called chromatin. Many biological processes are regulated via post-translational modifications of key proteins. Acetylation of lysine residues at the N-terminal histone tails is one of the most studied covalent modifications influencing gene regulation in eukaryotic cells. This review focuses on the role of enzymes involved in controlling both histone and non-histone proteins acetylation levels in the cell, with particular emphasis on their effects on cancer.     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1' in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1'. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.     

Interchromosomal recombination in Zea mays.

A new allele of the 27-kD zein locus in maize has been generated by interchromosomal recombination between chromosomes of two different inbred lines. A continuous patch of at least 11,817 bp of inbred W64A, containing the previously characterized Ra allele of the 27-kD zein gene, has been inserted into the genome of A188 by a single crossover. While both junction sequences are conserved, sequences of the two homologs between these junctions differ considerably. W64A contains the 7313-bp-long retrotransposon, Zeon-1. A188 contains a second copy of the 27-kD zein gene and a 2-kb repetitive element. Therefore, recombination results in a 7.3-kb insertion and a 14-kb deletion compared to the original S+A188 allele. If nonpairing sequences are looped out, 206 single base changes, frequently clustered, are present. The structure of this allele may explain how a recently discovered example of somatic recombination occurred in an A188/W64A hybrid. This would indicate that despite these sequence differences, pairing between these alleles could occur early during plant development. Therefore, such a somatically derived chimeric chromosome can also be heritable and give rise to new alleles.