Revised and Improved Procedure for Immunolocalization of Male Meiotic Chromosomal Proteins and Spindle in Plants without the Use of Enzymes.

Immunolocalization studies to visualize the distribution of proteins on meiotic chromosomes have become an integral part of studies on meiosis in the model organism . These techniques have been used to visualize a wide range of meiotic proteins involved in different aspects of meiosis, including sister chromatid cohesion, recombination, synapsis, and chromosome segregation. However, the analysis of meiotic spindle structure by immunofluorescence is of outstanding importance in plant reproductive biology and is very challenging.

In Favor of Establishment: Regulation of Chromatid Cohesion in Plants.

In eukaryotic organisms, the correct regulation of sister chromatid cohesion, whereby sister chromatids are paired and held together, is essential for accurate segregation of the sister chromatids and homologous chromosomes into daughter cells during mitosis and meiosis, respectively. Sister chromatid cohesion requires a cohesin complex comprised of structural maintenance of chromosome adenosine triphosphatases and accessory proteins that regulate the association of the complex with chromosomes or that are involved in the establishment or release of cohesion. The cohesin complex also plays important roles in the repair of DNA double-strand breaks, regulation of gene expression and chromosome condensation.

The Opposing Actions of Arabidopsis CHROMOSOME TRANSMISSION FIDELITY7 and WINGS APART-LIKE1 and 2 Differ in Mitotic and Meiotic Cells.

Sister chromatid cohesion, which is mediated by the cohesin complex, is essential for the proper segregation of chromosomes during mitosis and meiosis. Stable binding of cohesin with chromosomes is regulated in part by the opposing actions of CTF7 (CHROMOSOME TRANSMISSION FIDELITY7) and WAPL (WINGS APART-LIKE). In this study, we characterized the interaction between Arabidopsis thaliana CTF7 and WAPL by conducting a detailed analysis of wapl1-1 wapl2 ctf7 plants.

Overexpression of a truncated CTF7 construct leads to pleiotropic defects in reproduction and vegetative growth in Arabidopsis.

Background: Eco1/Ctf7 is essential for the establishment of sister chromatid cohesion during S phase of the cell cycle. Inactivation of Ctf7/Eco1 leads to a lethal phenotype in most organisms. Altering Eco1/Ctf7 levels or point mutations in the gene can lead to alterations in nuclear division as well as a wide range of developmental defects.

Arabidopsis thaliana WAPL is essential for the prophase removal of cohesin during meiosis.

Sister chromatid cohesion, which is mediated by the cohesin complex, is essential for the proper segregation of chromosomes in mitosis and meiosis. The establishment of stable sister chromatid cohesion occurs during DNA replication and involves acetylation of the complex by the acetyltransferase CTF7. In higher eukaryotes, the majority of cohesin complexes are removed from chromosomes during prophase.

Arabidopsis thaliana glyoxalase 2-1 is required during abiotic stress but is not essential under normal plant growth.

The glyoxalase pathway, which consists of the two enzymes, GLYOXALASE 1 (GLX 1) (E.C.: 4.

Expression of epitope-tagged SYN3 cohesin proteins can disrupt meiosis in Arabidopsis.

α-kleisins are core components of meiotic and mitotic cohesin complexes. Arabidopsis contains genes encoding four α-kleisins. SYN1, a REC8 ortholog, is essential for meiosis, while SYN2 and SYN4 appear to be SCC1 orthologs and function in mitosis.

Arabidopsis CHROMOSOME TRANSMISSION FIDELITY 7 (AtCTF7/ECO1) is required for DNA repair, mitosis and meiosis.

The proper transmission of DNA in dividing cells is crucial for the survival of eukaryotic organisms. During cell division, faithful segregation of replicated chromosomes requires their tight attachment, known as sister chromatid cohesion, until anaphase. Sister chromatid cohesion is established during S-phase in a process requiring an acetyltransferase that in yeast is known as Establishment of cohesion 1 (Eco1).

Immunolocalization protocols for visualizing meiotic proteins in Arabidopsis thaliana: method 3.

Immunolocalization studies to visualize the distribution of proteins on meiotic chromosomes have become an integral part of studies on meiosis in the model organism Arabidopsis thaliana. This chapter describes fixation, sample preparation, and immunolocalization techniques used in our laboratory. These techniques have been used to visualize a wide range of meiotic proteins involved in different aspects of meiosis, including sister chromatid cohesion, recombination, synapsis, and chromosome segregation.

Arabidopsis ETHE1 encodes a sulfur dioxygenase that is essential for embryo and endosperm development.

Mutations in human (Homo sapiens) ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) result in the complex metabolic disease ethylmalonic encephalopathy, which is characterized in part by brain lesions, lactic acidemia, excretion of ethylmalonic acid, and ultimately death. ETHE1-like genes are found in a wide range of organisms; however, the biochemical and physiological role(s) of ETHE1 have not been examined outside the context of ethylmalonic encephalopathy. In this study we characterized Arabidopsis (Arabidopsis thaliana) ETHE1 and determined the effect of an ETHE1 loss-of-function mutation to investigate the role(s) of ETHE1 in plants.

The Arabidopsis SYN3 cohesin protein is important for early meiotic events.

α-Kleisins are core components of meiotic and mitotic cohesin complexes. Arabidopsis contains four genes that encode α-kleisin proteins: SYN1, SYN2, SYN3 and SYN4. SYN1, a REC8 ortholog, is essential for meiosis, while SYN2 and SYN4 appear to be SCC1 orthologs and function in mitosis.

The radially swollen 4 separase mutation of Arabidopsis thaliana blocks chromosome disjunction and disrupts the radial microtubule system in meiocytes.

The caspase-family protease, separase, is required at the onset of anaphase to cleave the cohesin complex that joins replicated sister chromatids. However, in various eukaryotes, separase has acquired additional and distinct functions. A single amino-acid substitution in separase is responsible for phenotypes of the Arabidopsis thaliana mutant, radially swollen 4 (rsw4).

Plant cohesins, common themes and unique roles.

Cohesin complexes are critical for holding sister chromatids together during nuclear division. They also play important roles in the compaction of chromosomes and their bipolar attachment to the spindle, DNA double strand break repair, and the regulation of gene expression. Studies on sister chromatid cohesion in a wide range of organisms have shown that the proteins involved, and the general events of this important process are conserved between yeast, plants and animals.

Converting GLX2-1 into an active glyoxalase II.

Arabidopsis thaliana glyoxalase 2-1 (GLX2-1) exhibits extensive sequence similarity with GLX2 enzymes but is catalytically inactive with SLG, the GLX2 substrate. In an effort to identify residues essential for GLX2 activity, amino acid residues were altered at positions 219, 246, 248, 325, and 328 in GLX2-1 to be the same as those in catalytically active human GLX2. The resulting enzymes were overexpressed, purified, and characterized using metal analyses, fluorescence spectroscopy, and steady-state kinetics to evaluate how these residues affect metal binding, structure, and catalysis.

Proper levels of the Arabidopsis cohesion establishment factor CTF7 are essential for embryo and megagametophyte, but not endosperm, development.

CTF7 is an essential gene in yeast that is required for the formation of sister chromatid cohesion. While recent studies have provided insights into how sister chromatid cohesion is established, less is known about how specifically CTF7 facilitates the formation of cohesion, and essentially nothing is known about how sister chromatid cohesion is established in plants. In this report, we describe the isolation and characterization of CTF7 from Arabidopsis (Arabidopsis thaliana).

The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity.

In an effort to better understand the structure, metal content, the nature of the metal centers, and enzyme activity of Arabidopsis thaliana Glx2-2, the enzyme was overexpressed, purified, and characterized using metal analyses, kinetics, and UV-vis, EPR, and (1)H NMR spectroscopies. Glx2-2-containing fractions that were purple, yellow, or colorless were separated during purification, and the differently colored fractions were found to contain different amounts of Fe and Zn(II). Spectroscopic analyses of the discrete fractions provided evidence for Fe(II), Fe(III), Fe(III)-Zn(II), and antiferromagnetically coupled Fe(II)-Fe(III) centers distributed among the discrete Glx2-2-containing fractions.

Arabidopsis thaliana mitochondrial glyoxalase 2-1 exhibits beta-lactamase activity.

In an effort to determine the physiological role of Arabidopsis thaliana Glx2-1, we attempted to uncover a substrate for the enzyme. Glx2-1 did not effectively process 192 diverse substrates found in a commercial screen used for microorganism identification or exhibit arylsulfatase, lactonase, or phosphotriesterase activities. However, Glx2-1 does exhibit beta-lactamase activity with k(cat)/KM values from 10(3) to 10(5) M(-1) s(-1).

Arabidopsis separase functions beyond the removal of sister chromatid cohesion during meiosis.

Separase is a capase family protease that is required for the release of sister chromatid cohesion during meiosis and mitosis. Proteolytic cleavage of the alpha-kleisin subunit of the cohesin complex at the metaphase-to-anaphase transition is essential for the proper segregation of chromosomes. In addition to its highly conserved role in cleaving the alpha-kleisin subunit, separase appears to have acquired additional diverse activities in some organisms, including involvement in mitotic and meiotic anaphase spindle assembly and elongation, interphase spindle pole body positioning, and epithelial cell reorganization.

Human glyoxalase II contains an Fe(II)Zn(II) center but is active as a mononuclear Zn(II) enzyme.

Human glyoxalase II (Glx2) was overexpressed in rich medium and in minimal medium containing zinc, iron, or cobalt, and the resulting Glx2 analogues were characterized using metal analyses, steady-state and pre-steady-state kinetics, and NMR and EPR spectroscopies to determine the nature of the metal center in the enzyme. Recombinant human Glx2 tightly binds nearly 1 equiv each of Zn(II) and Fe. In contrast to previous reports, this study demonstrates that an analogue containing 2 equiv of Zn(II) cannot be prepared.

Arabidopsis thaliana GLX2-1 contains a dinuclear metal binding site, but is not a glyoxalase 2.

In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV-visible, EPR, and (1)H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV-visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein.

Spectroscopic studies on Arabidopsis ETHE1, a glyoxalase II-like protein.

ETHE1 (ethylmalonic encephalopathy protein 1) is a beta-lactamase fold-containing protein that is essential for the survival of a range of organisms. In spite of the apparent importance of this enzyme, very little is known about its function or biochemical properties. In this study Arabidopsis ETHE1 was over-expressed and purified and shown to bind tightly to 1.

SWI1 is required for meiotic chromosome remodeling events.

The Arabidopsis dsy10 mutant was previously identified as being defective in the synapsis of meiotic chromosomes resulting in male and female sterility. We report here the molecular analysis of the mutation and show that it represents a T-DNA insertion in the third exon of the SWI1 gene. Four mutations have now been identified in SWI1, several of which exhibit different phenotypes.

Side chain and backbone dynamics of phospholamban in phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy.

2H and 15N solid-state NMR spectroscopic techniques were used to investigate both the side chain and backbone dynamics of wild-type phospholamban (WT-PLB) and its phosphorylated form (P-PLB) incorporated into 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC) phospholipid bilayers. 2H NMR spectra of site-specific CD3-labeled WT-PLB (at Leu51, Ala24, and Ala15) in POPC bilayers were similar under frozen conditions (-25 degrees C). However, significant differences in the line shapes of the 2H NMR spectra were observed in the liquid crystalline phase at and above 0 degrees C.

The Arabidopsis cohesin protein SYN3 localizes to the nucleolus and is essential for gametogenesis.

Alpha-kleisins are core components of meiotic and mitotic cohesin complexes. Arabidopsis contains genes for four alpha-kleisin proteins encoded by SYN genes. SYN1, a REC8 ortholog, is essential for meiosis, while SYN2 and SYN4 appear to be SCC1 orthologs and function in mitosis.

The Arabidopsis SKP1 homolog ASK1 controls meiotic chromosome remodeling and release of chromatin from the nuclear membrane and nucleolus.

During early stages of meiotic prophase I the nucleus undergoes considerable reorganization, including the clustering of telomeres, the release of contacts between chromosomes and the nuclear membrane, the reorganization of the nucleolus, and chromatin remodeling. Using a light squashing technique for the analysis of meiotic chromosomes along with fluorescent in situ hybridization, transmission electron microscopy and immunolocalization studies with antibodies to modified histones, we demonstrate that ASK1 is essential for early nuclear reorganization events. A relatively large number of meiotic alterations have been identified in ask1-1 plants.