Methyl recycling activities are co-ordinately regulated during plant development.

A large number of compounds including lignin, phospholipids, pectin, DNA, mRNA, and proteins require methyl groups for their functionality. A detailed study of the expression and activities of two enzymes, adenosine kinase (ADK) and S-adenosylhomocysteine hydrolase (SAHH), which are both required for the maintenance and recycling of S-adenosylmethionine-dependent methylation in plants, was carried out. The abundance and tissue localization of ADK and SAHH transcripts and protein were monitored along with their enzyme activities in leaves, stems, buds, siliques, and roots of Arabidopsis. In all but roots and seed coats, the transcript abundance of ADK and SAHH fluctuated co-ordinately, matching changes in their protein and enzyme activities. To evaluate whether this expression pattern was associated with methyl recycling, the protein content and distribution of S-adenosylmethionine synthetase and phosphoethanolamine N-methyltransferase, a key methyltransferase involved in phospholipid synthesis, were investigated. These were found to accumulate in a pattern similar to ADK and SAHH. ADK and SAHH protein and transcript amounts were shown to fluctuate similarly in tissues accumulating lignin. Additionally, the amounts of ADK and SAHH mRNAs were also found at high levels in inflorescence meristems likely to support their higher rates of cell division. Thus, the results point to a co-ordinated and probably transcriptional regulation of these genes in most organs of Arabidopsis; SAHH abundance is distinctly higher in seeds and roots which suggests it may have a non-methyl-related role in these organs.

DEX1, a novel plant protein, is required for exine pattern formation during pollen development in Arabidopsis.

To identify factors that are required for proper pollen wall formation, we have characterized the T-DNA-tagged, dex1 mutation of Arabidopsis, which results in defective pollen wall pattern formation. This study reports the isolation and molecular characterization of DEX1 and morphological and ultrastructural analyses of dex1 plants. DEX1 encodes a novel plant protein that is predicted to be membrane associated and contains several potential calcium-binding domains. Pollen wall development in dex1 plants parallels that of wild-type plants until the early tetrad stage. In dex1 plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. Sporopollenin is produced and randomly deposited on the plasma membrane in dex1 plants. However, it does not appear to be anchored to the microspore and forms large aggregates on the developing microspore and the locule walls. Based on the structure of DEX1 and the phenotype of dex1 plants, several potential roles for the protein are proposed.

Cloning and characterization of two Arabidopsis genes that belong to the RAD21/REC8 family of chromosome cohesin proteins.

Sister chromatid cohesion is required for proper chromosome segregation during cell division. One group of proteins that is essential for sister chromatid cohesion during mitosis and meiosis is the RAD21/REC8 family of cohesin proteins. Two cohesin proteins are found in yeast; one that functions mainly in mitosis while the other participates in meiosis. In contrast, only one cohesin gene appears to be present in Drosophila. In previous studies we identified an Arabidopsis cohesin protein that is required for meiosis. In this report we describe the isolation and characterization of two additional Arabidopsis cohesin genes. The structure of the genes suggests that they arose via a gene duplication event followed by extensive sequence evolution. Transcripts for the two genes are present throughout the plant and are highest in regions of active cell division, suggesting that the proteins may participate in chromosome cohesion during mitosis.

Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis.

Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.

Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis.

Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

Isolation and characterization of SYN1, a RAD21-like gene essential for meiosis in Arabidopsis.

The proper pairing, recombination, and segregation of chromosomes are central to meiosis and sexual reproduction. The syn1 mutation was previously identified as a synaptic mutant in a T-DNA-tagged population of plants. SYN1 has been isolated and found to exhibit similarity to Schizosaccharomyces pombe RAD21 and RAD21-like proteins, which are required for chromosome condensation and sister chromatid cohesion during mitosis. Plants homozygous for syn1 are male and female sterile and show defects in chromosome condensation and pairing beginning at leptonema of meiosis I. Fragmentation of the chromosomes was observed at metaphase I. Alternative promoters produced two SYN1 transcripts. One transcript was expressed at low levels in most tissues, whereas the other was expressed only in prebolting buds. DNA blot analyses suggest that Arabidopsis contains a small RAD21 gene family. Consistent with the DNA blot data, a second Arabidopsis RAD21-like gene has been identified. These results suggest that different RAD21-like proteins play essential roles in chromosome condensation and pairing during both meiosis and mitosis.

The radish (Raphanus sativus L.) mitochondrial cox2 gene contains an ACG at the predicted translation initiation site.

The mitochondrial cox2 gene has been sequenced from radish (Raphanus sativus L.). The gene is interrupted by a 1346-bp group-II intron and contains an ACG codon as the predicted translation initiation site. Analysis of cox2 cDNAs indicates that the ACG codon is not converted to an AUG codon in the mRNA, although 15 other RNA editing sites were identified. The cox2 gene from Raphanus raphanistrum, and other varieties of R. sativus, also contain an ACG as the predicted start codon; plants in the closely related genus, Brassica, do not. Western-blot analyses indicate that cox2 proteins in radish mitochondria are the same size as those found in Brassica mitochondria and different from cox2 proteins in plants where cox2 is nuclear-encoded. This finding, along with the observation that cox2 sequences are not present in the nuclear genome of radish, suggests that ACG is utilized as the radish cox2 initiation codon.

A defect in synapsis causes male sterility in a T-DNA-tagged Arabidopsis thaliana mutant.

Fluorescence microscopy was used to study meiosis in microsporocytes from wild-type Arabidopsis thaliana and a T-DNA-tagged meiotic mutant. Techniques for visualizing chromosomes and beta-tubulin in other plant species were evaluated and modified in order to develop a method for analyzing meiosis in A. thaliana anthers. Like most dicots, A. thaliana microsporocytes undergo simultaneous cytokinesis in which both meiotic divisions are completed prior to cytokinesis. However, two unique events were observed in wild-type A. thaliana that have not been reported in other angiosperms: (1) polarization of the microsporocyte cytoskeleton during prophase I prior to nuclear envelope breakdown, and (2) extensive depolymerization of microtubules just prior to metaphase II. The first observation could have implications regarding a previously uncharacterized mechanism for determining the axis of the metaphase I spindle during microsporogenesis. The second observation is peculiar since microtubules are known to be involved in chromosome alignment in other species; possible explanations will be discussed. A T-DNA-tagged meiotic mutant of A. thaliana (syn1), which had previously been shown to produce abnormal microspores with variable DNA content, was also cytologically characterized. The first observable defect occurs in microsporocytes at telophase I, where some chromosomes are scattered throughout the cytoplasm, usually attached to stray microtubules. Subsequent development stages are affected, leading to complete male sterility. Based on similarities to synaptic mutants that have been described in other species, it is suggested that this mutant is defective in synaptonemal complex formation and/or cohesion between sister chromatids.

Immunolocalization of integrin-like proteins in Arabidopsis and Chara.

Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

Molecular characterization of glyoxalase II from Arabidopsis thaliana.

Glyoxalase II is part of the glutathione-dependent glyoxalase detoxification system. In addition to its role in the detoxification of cytotoxic 2-oxo-aldehydes, specifically methylglyoxal, it has been suggested that the glyoxalase system may also play a role in controlling cell differentiation and proliferation. During the analysis of a T-DNA-tagged mutant of Arabidopsis we identified the gene for a glyoxalase II isozyme (GLY1) that appears to be mitochondrially localized. The cDNA encoding a glyoxalase II cytoplasmic isozyme (GLY2) was also isolated and characterized. Southern blot and sequence analyses indicate that glyoxalase II proteins are encoded by at least two multigene families in Arabidopsis. Escherichia coli cells expressing either GLY1 or GLY2 exhibit increased glyoxalase II activity, confirming that they do, in fact, encode glyoxalase II proteins. Northern analysis shows that the two genes are differentially expressed. Transcripts for the mitochondrial isozyme are most abundant in roots, while those for the cytoplasmic isozyme are highest in flower buds. The identification of glyoxalase II isozymes that are differentially expressed suggests that they may play different roles in the cell.

Glyoxalase II from A. thaliana requires Zn(II) for catalytic activity.

Cytosolic glyoxalase II from Arabidopsis thaliana, GLX2-2, was overexpressed and purified to homogeneity using Q-sepharose chromatography. MALDI-TOF mass spectrometry studies indicated a molecular weight of 28 767 Da. Using steady-state kinetics studies, the purified enzyme exhibited a Km of 660 +/- 100 microM and a kcat of 484 +/- 92 s(-1) at 37 degrees C. Metal analyses demonstrated that the enzyme binds 2.1 +/- 0.5 moles of Zn(II) per monomer; the binding of Zn(II) is essential for enzyme viability and activity. Sequence comparison of glyoxalase II enzymes from human, A. thaliana, and yeast and the metallo-beta-lactamases reveal that all metal binding ligands of the metallo-beta-lactamases are conserved in glyoxalase II enzymes, suggesting that all glyoxalase II enzymes are Zn(II) metalloenzymes. These results and their implications are discussed in light of previous studies on glyoxalase II, and an active site for the glyoxalase II enzymes is proposed.

Characterization of the radish mitochondrial nad3/rps12 locus: analysis of recombination repeats and RNA editing.

In order to further investigate sequences that are responsible for low-frequency recombination in plant mitochondrial DNAs and RNA editing in radish mitochondria, the nad3/rps12 locus has been isolated and characterized from a normal cultivar of radish and the male-sterile Ogura cytoplasm. A repeated sequence that has been implicated in other radish mitochondrial DNA rearrangements was identified at the breakpoint between the two loci indicating that it was also involved in the nad3/rps12 rearrangement. Similar to some other radish mitochondrial genes, nad3/rps12 genomic sequences already contain several, but not all, of the bases that are typically edited in plant mitochondrial nad3 and rps12 genes. Analysis of nad3/rps12 cDNAs indicated that the mRNAs are not edited. One partially edited transcript was identified out of the twenty two that were examined. This finding, along with the observation that nad3/rps12 RNAs are present at very low levels, raises the possibility that radish mitochondria may not encode functional copies of these genes. Consistent with this hypothesis, DNA-blot analysis detects nad3/rps12 sequences in the nucleus.

Organ-specific reduction in the abundance of a mitochondrial protein accompanies fertility restoration in cytoplasmic male-sterile radish.

The mitochondrial DNA of plants containing the male sterility-causing Ogura cytoplasm of radish contain a novel gene, orf138, that is transcribed as part of a bicistronic mRNA. Genetic studies have previously linked male sterility with the orf138 locus. To determine if orf138 is expressed at the protein level, and investigate the effect of fertility restoration on ORF138 levels, we have raised antibodies to an ORF138-glutathione S-transferase fusion protein. Anti-ORF138 antibodies detect a 20 kDa protein that is associated with the mitochondrial membrane of sterile Ogura radish plants. Nuclear restoration is accompanied by a dramatic reduction in the amount of this protein in mitochondria of flowers and leaves, but not roots of fertile Ogura radish plants. The presence or absence of fertility restoration genes has no detectable effect on the size, abundance, or RNA editing patterns of orf138 transcripts. These results support genetic studies that have implicated orf138 in Ogura cytoplasmic male sterility and suggest that the restorer genes may be affecting either the translation or stability of ORF138.

Intron loss from the NADH dehydrogenase subunit 4 gene of lettuce mitochondrial DNA: evidence for homologous recombination of a cDNA intermediate.

The mitochondrial gene coding for subunit 4 of the NADH dehydrogenase complex I (nad4) has been isolated and characterized from lettuce, Lactuca sativa. Analysis of nad4 genes in a number of plants by Southern hybridization had previously suggested that the intron content varied between species. Characterization of the lettuce gene confirms this observation. Lettuce nad4 contains two exons and one group IIA intron, whereas previously sequenced nad4 genes from turnip and wheat contain three group IIA introns. Northern analysis identified a transcript of 1600 nucleotides, which represents the mature nad4 mRNA and a primary transcript of 3200 nucleotides. Sequence analysis of lettuce and turnip nad4 cDNAs was used to confirm the intron/exon border sequences and to examine RNA editing patterns. Editing is observed at the 5' and 3' ends of the lettuce transcript, but is absent from sequences that correspond to exons two, three and the 5' end of exon four in turnip and wheat. In contrast, turnip transcripts are highly edited in this region, suggesting that homologous recombination of an edited and spliced cDNA intermediate was involved in the loss of introns two and three from an ancestral lettuce nad4 gene.

Subunit 6 of the Fo-ATP synthase complex from cytoplasmic male-sterile radish: RNA editing and NH2-terminal protein sequencing.

RNA editing and NH2-terminal processing of subunit 6 (atp6) of the mitochondrial Fo-ATPase complex has been investigated for the normal (fertile) and Ogura (male-sterile) radish cytoplasms to determine if previously identified differences between the Ogura atp6 locus and its normal radish counterpart are associated with cytoplasmic male sterility. Analysis of cDNA clones from five different sterile and fertile radish lines identified one C-to-U transition, which results in the replacement of a proline with a serine, in several of the lines. No editing of atp6 transcripts was observed in two lines, Scarlet Knight (normal radish) and sterile CrGC15 (Ogura radish). This is the first example of a naturally occurring plant mitochondrial gene that is not edited. The Ogura atp6 polypeptide is synthesized with a predicted NH2-terminal extension of 174 amino acids in contrast to the nine amino acid extension found in normal radish. In spite of the lack of similarity between the two extensions, NH2-terminal sequence analysis indicates that both polypeptides are processed to yield identical core proteins with a serine as the NH2-terminal residue. These results indicate that ATPase subunit 6 is synthesized normally in Ogura radish, and that it is unlikely that the atp6 locus is associated with Ogura cytoplasmic male sterility.

Characterization of the radish mitochondrial orfB locus: possible relationship with male sterility in Ogura radish.

The orfB locus of the normal (fertile) and Ogura (male-sterile) radish mitochondrial genomes has been characterized in order to determine if this region, which has previously been correlated with cytoplasmic male sterility (CMS) in Brassica napus cybrids (Bonhomme et al. 1991; Temple et al. 1992), could also be involved in radish CMS. In normal radish, orfB is expressed as a 600-nucleotide (nt) transcript. In Ogura radish, orfB is present as the second gene of a 1200-nt transcript that also contains a 138-codon open reading frame (orf138). Sequences showing similarity to orf138 are present in normal radish, but are not expressed.

Novel mitochondrial genomes in Brassica napus somatic hybrids.

The mitochondrial genomes of nine male-fertile and two Ogura cytoplasmic male-sterile (cms) Brassica napus somatic hybrids were probed with 46 mitochondrial DNA fragments. The distribution of information obtained from each fusion partner was not random. Several regions, including the coxI gene and a major recombination repeat sequence, were always derived from the Brassica campestris fusion partner, and some regions were always derived from the Ogura mitochondrial genome. Novel fragments occurred in seven distinct regions. Some of the rearrangement breakpoints were located near the evolutionary breakpoints relating the mitochondrial genomes of the Brassica species. The sizes of the mitochondrial genomes in the somatic hybrids ranged from 224.8 to 285.3 kb. A direct correlation between a specific gene and the cms phenotype was not observed; however, a possible cms-associated region was identified. It corresponds to a region that was identified through analysis of fertile revertants from a cms B. napus cybrid.

Variable intron content of the NADH dehydrogenase subunit 4 gene of plant mitochondria.

The gene nad4, encoding subunit four of the mitochondrial NADH dehydrogenase complex I, has been isolated and characterized from turnip, Brassica campestris. The 8 kb turnip nad4 gene contains four exons, which potentially encode a NAD4 polypeptide of 495 amino acids, and three large group II introns. Northern analysis identifies an abundant 2 kb transcript that most likely serves as the nad4 mRNA, while several larger transcripts (putative splicing intermediates) are also detected. Analysis of the nad4 locus in three distantly related dicotyledons indicates that introns 2 and 3 are optional. Mung bean has the same nad4 organization as turnip, whereas spinach nad4 contains introns 1 and 3, and lettuce nad4 has intron 1 only. We infer that all three group II introns were present in the nad4 gene of an angiosperm common ancestor and have persisted in certain lineages for over 200 million years, with two of the introns having been lost in other lineages.

The role of coxI-associated repeated sequences in plant mitochondrial DNA rearrangements and radish cytoplasmic male sterility.

The gene coxI, encoding subunit I of mitochondrial cytochrome c oxidase, has been characterized from the normal (fertile) and Ogura (male-sterile) cytoplasms of radish to determine if a previously identified mitochondrial DNA rearrangement, and its associated transcriptional differences, could play a role in Ogura cytoplasmic male sterility (CMS). The normal and Ogura loci are virtually identical for 2.8 kb, including a 527-codon open reading frame whose product is approximately 95% identical to other plant COXI polypeptides. A rearrangement 120 bp 5' to the coding region results in different 5' transcript termini for the two genes. A comparison of several crucifer mitochondrial DNAs indicates that this rearrangement also occurs in the normal radish cytoplasm and is, therefore, not involved in Ogura CMS. Sequences present at the coxI locus belong to at least two different dispersed repeat families, members of which are also associated with other rearranged genes in radish.

Atrazine-resistant cytoplasmic male-sterile-nigra broccoli obtained by protoplast fusion between cytoplasmic male-sterile Brassica oleracea and atrazine-resistant Brassica campestris.

Protoplast fusion was used to combine the cytoplasmic traits of atrazine resistance and male sterility in Brassica oleracea var. italica (broccoli). Leaf protoplasts from broccoli with the petaloid B. nigra type of cytoplasmic male sterility were fused with hypocotyl protoplasts from an atrazine-resistant biotype of B. campestris var. oleifera cv Candle (oilseed rape). A total of 19 colonies regenerated shoots, all of which were broccolilike in phenotype, i.e., lacked trichomes. Four shoots, all from one colony, were atrazine resistant, surviving and growing in the presence of 25 µM atrazine. A leaf piece assay also confirmed that they were atrazine resistant. Molecular analysis showed that they contain chloroplasts from the atrazine-resistant B. campestris parent and mitochondria from the B. nigra parent. No recombination or rearrangement of the mitochondrial genomes in the fusion products was detected. These four plants and their progeny all showed the petaloid B. nigra type of male sterility.