Functions and mechanisms of plant histone deacetylases

Lysine acetylation, one of the major types of post-translational modifications, plays critical roles in regulating gene expression and protein function. Histone deacetylases(HDACs) are responsible for removing acetyl groups from lysines of both histone and non-histone proteins. While tremendous progress has been made in understanding the function and mechanism of HDACs in animals in the past two decades, nearly half of the HDAC studies in plants were reported within the past five years. In this review,we summarize the major findings on plant HDACs, with a focus on the model plant Arabidopsis thaliana, and highlight the components, regulatory mechanisms, and biological functions of HDAC complexes.     

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology,chromosomal localization and evolutionary aspects

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.     

Oryzalin,a dinitroaniline herbicide,binds to plant tubulin and inhibits microtubule polymerization in vitro

The effects of oryzalin, a dinitroaniline herbicide, on chromosome behavior and on cellular microtubules (MTs) were examined by light microscopy and immunogold staining, respectively, in endosperm cells from Haemanthus katherinae Bak. Brief treatments with 1.0·10^-8 M oryzalin reduced markedly the migration rate of anaphase chromosomes and 1.0·10^-7 M oryzalin stopped migration abruptly. Oryzalin (1.0·10^-7 M) depolymerized MTs and prevented the polymerization of new MTs at all stages of the mitotic cycle. The chromosome condensation cycle was unaffected by oryzalin. Endothelial cells from the heart of Xenopus leavis showed no chromosomal or microtubular rearrangements after oryzalin treatment. The inhibition by oryzalin of the polymerization of tubulin isolated from cultured cells of Rosa sp. cv. Paul's scarlet was examined in vitro by turbidimetry, electron microscopy and polymer sedimentation analysis. Oryzalin inhibited the rapid phase of taxol-induced polymerization of rose MTs at 24°C with an apparent inhibition constant (K _i ) of 2.59·10⁶ M. Shorter and fewer MTs were formed with increasing oryzalin concentrations, and maximum inhibition of taxol-induced polymerization occurred at approx. 1:1 molar ratios of oryzalin and tubulin. Oryzalin partially depolymerized taxol-stabilized rose MTs. Ligand-binding experiments with [¹⁴C]oryzalin demonstrated the formation of a tubulin-oryzalin complex that was time- and pH-dependent. The tubulin-oryzalin interaction (24°C, pH 7.1) had an apparent affinity constant (K _app) of 1.19·10⁵ M^-1. Oryzalin did not inhibit taxol-induced polymerization of bovinebrain MTs and no appreciable binding of oryzalin to brain tubulin or other proteins was detected. The results demonstrate pharmacological differences between plant and animal tubulins and indicate that the most sensitive mode of action of the dinitroaniline herbicides is the direct poisoning of MT dynamics in cells of higher plants.Abbreviations MT microtubule - SIB sucrose isolation buffer - TO tubulin-oryzalin complex     

The chalcone synthase multigene family of Petunia hybrida (V30): differential,light-regulated expression during flower development and UV light induction

We have analysed the expression of the 8–10 members of the gene family encoding the flavonoid biosynthetic enzyme chalcone synthase (CHS) from Petunia hybrida. During normal plant development only two members of the gene family (CHS-A and CHS-J) are expressed. Their expression is restricted to floral tissues mainly. About 90% of the total CHS mRNA pool is transcribed from CHS-A, wheares CHS-J delivers about 10% in flower corolla, tube and anthers. Expression of CHS-A and CHS-J during flower development is coordinated and (red) light-dependent. In young seedlings and cell suspension cultures expression of CHS-A and CHS-J can be induced with UV light. In addition to CHS-A and CHS-J, expression of another two CHS genes (CHS-B and CHS-G) is induced in young seedlings by UV light, albeit at a low level. In contrast to CHS genes from Leguminoseae, Petunia CHS genes are not inducible by phytopathogen-derived elicitors. Expression of CHS-A and CHS-J is reduced to a similar extent in a regulatory CHS mutant, Petunia hybrida Red Star, suggesting that both genes are regulated by the same trans-acting factors. Comparison of the promoter sequences of CHS-A and CHS-J reveals some striking homologies, which might represent cis-acting regulatory sequences.     

Antisense genes in plants: an overview

Plants are the first multicellular higher eukaryotic organisms in which artificial antisense genes have been shown to down-regulate target gene expression. Manipulations with an antisense gene can serve as a tool to study the effect of a particular plant gene inactivation, the interaction of gene products whose genes are coordinately expressed, or the functional analysis of cryptic genes. Transgenic plants harbouring an antisense gene already gave rise to patentable new characteristics, showing that the technique has great scientific and economic value.     

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology,chromosomal localization and evolutionary aspects

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Crystallization of immunoglobulin Fab fragments specific for DNA

The purification and crystallization of Fab fragments of two mouse monoclonal immunoglobulins specific for different DNA structures are described. In each case, papain digestion of the immunoglobulins produced a mixture of Fab species differing in their isoelectric points. Purification of one of these species was required to obtain suitable crystals. One of these antibodies, Jel 72, is specific for right-handed duplex poly(dG).poly(dC). An Fab fragment of Jel 72 with a pI of 8.8 was purified by anion-exchange chromatography and used to obtain crystals from 56% saturated ammonium sulfate and 50 mM sodium acetate, pH 4.2, that diffract to 2.6-A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 94.6, b = 102.6, c = 92.4 A. The other antibody, Jel 318, binds triple-stranded DNA poly[d(Tm5C)].poly[d(GA)].poly[d(m5C + T)]. Jel 318 Fab fragments with isoelectric points of 7.6 and 7.8 were also purified by anion-exchange chromatography, and crystals were obtained from 12% polyethylene glycol 8000, 50 mM NaCl, and 10 mM Tris.HCl, pH 7.8. These crystals diffract to about 2.4-A resolution and also belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 82.4, b = 139.5, and c = 42.0 A. For both Fab fragments, crystal size and quality improved dramatically upon purification of an individual isoelectric species.