Arabidopsis thaliana GYRB3 does not encode a DNA gyrase subunit.

BACKGROUND: DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3. METHODOLOGY/PRINCIPAL FINDINGS: We found, contrary to previous data, that AtGyrB3 is not essential to the survival of A. thaliana. Bioinformatic analysis suggests AtGyrB3 is considerably shorter than other gyrase B subunits, lacking part of the ATPase domain and other key motifs found in all type II topoisomerases; but it does contain a putative DNA-binding domain. Partially purified AtGyrB3 cannot bind E. coli GyrA or support supercoiling. AtGyrB3 cannot complement an E. coli gyrB temperature-sensitive strain, whereas AtGyrB2 can. Yeast two-hybrid analysis suggests that AtGyrB3 cannot bind to AtGyrA or form a dimer. CONCLUSIONS/SIGNIFICANCE: These data strongly suggest that AtGyrB3 is not a gyrase subunit but has another unknown function. One possibility is that it is a nuclear protein with a role in meiosis in pollen.

BIN4, a novel component of the plant DNA topoisomerase VI complex, is required for endoreduplication in Arabidopsis.

How plant organs grow to reach their final size is an important but largely unanswered question. Here, we describe an Arabidopsis thaliana mutant, brassinosteroid-insensitive4 (bin4), in which the growth of various organs is dramatically reduced. Small organ size in bin4 is primarily caused by reduced cell expansion associated with defects in increasing ploidy by endoreduplication. Raising nuclear DNA content in bin4 by colchicine-induced polyploidization partially rescues the cell and organ size phenotype, indicating that BIN4 is directly and specifically required for endoreduplication rather than for subsequent cell expansion. BIN4 encodes a plant-specific, DNA binding protein that acts as a component of the plant DNA topoisomerase VI complex. Loss of BIN4 triggers an ATM- and ATR-dependent DNA damage response in postmitotic cells, and this response coincides with the upregulation of the cyclin B1;1 gene in the same cell types, suggesting a functional link between DNA damage response and endocycle control.

LITTLE NUCLEI genes affecting nuclear morphology in Arabidopsis thaliana.

Efforts to understand nuclear organization in plant cells have received little assistance from the better-studied animal nuclei, because plant proteomes do not contain recognizable counterparts to the key animal proteins involved in nuclear organization, such as lamin nuclear intermediate filament proteins. Previous studies identified a plant-specific insoluble nuclear protein in carrot (Daucus carota), called Nuclear Matrix Constituent Protein1 (NMCP1), which contains extensive coiled-coil domains and localizes to the nuclear periphery. Here, we describe a genetic characterization of two NMCP1-related nuclear proteins in Arabidopsis thaliana, LITTLE NUCLEI1 (LINC1) and LINC2. Disruption of either gene caused a reduction in nuclear size and altered nuclear morphology. Moreover, combining linc1 and linc2 mutations had an additive effect on nuclear size and morphology but a synergistic effect on chromocenter number (reduction) and whole-plant morphology (dwarfing). The reduction in nuclear size in the linc1 linc2 double mutant was not accompanied by a corresponding change in endopolyploidy. Rather, the density of DNA packaging at all endopolyploid levels in the linc1 linc2 mutants was increased significantly. Our results indicate that the LINC coiled-coil proteins are important determinants of plant nuclear structure.

Arabidopsis SPO11-2 functions with SPO11-1 in meiotic recombination.

The Spo11 protein is a eukaryotic homologue of the archaeal DNA topoisomerase VIA subunit (topo VIA). In archaea it is involved, together with its B subunit (topo VIB), in DNA replication. However, most eukaryotes, including yeasts, insects and vertebrates, instead have a single gene for Spo11/topo VIA and no homologues for topo VIB. In these organisms, Spo11 mediates DNA double-strand breaks that initiate meiotic recombination. Many plant species, in contrast to other eukaryotes, have three homologues for Spo11/topo VIA and one for topo VIB. The homologues in Arabidopsis, AtSPO11-1, AtSPO11-2 and AtSPO11-3, all share 20-30% sequence similarity with other Spo11/topo VIA proteins, but their functional relationship during meiosis or other processes is not well understood. Previous genetic evidence suggests that AtSPO11-1 is a true orthologue of Spo11 in other eukaryotes and is required for meiotic recombination, whereas AtSPO11-3 is involved in DNA endo-reduplication as a part of the topo VI complex. In this study, we show that plants homozygous for atspo11-2 exhibit a severe sterility phenotype. Both male and female meiosis are severely disrupted in the atspo11-2 mutant, and this is associated with severe defects in synapsis during the first meiotic division and reduced meiotic recombination. Further genetic analysis revealed that AtSPO11-1 and AtSPO11-2 genetically interact, i.e. plants heterozygous for both atspo11-1 and atspo11-2 are also sterile, suggesting that AtSPO11-1 and AtSPO11-2 have largely overlapping functions. Thus, the three Arabidopsis Spo11 homologues appear to function in two discrete processes, i.e. AtSPO11-1 and AtSPO11-2 in meiotic recombination and AtSPO11-3 in DNA replication.

Cell-wall structure and anisotropy in procuste, a cellulose synthase mutant of Arabidopsis thaliana.

In dark-grown hypocotyls of the Arabidopsis procuste mutant, a mutation in the CesA6 gene encoding a cellulose synthase reduces cellulose synthesis and severely inhibits elongation growth. Previous studies had left it uncertain why growth was inhibited, because cellulose synthesis was affected before, not during, the main phase of elongation. We characterised the quantity, structure and orientation of the cellulose remaining in the walls of affected cells. Solid-state NMR spectroscopy and infrared microscopy showed that the residual cellulose did not differ in structure from that of the wild type, but the cellulose content of the prc-1 cell walls was reduced by 28%. The total mass of cell-wall polymers per hypocotyl was reduced in prc-1 by about 20%. Therefore, the fourfold inhibition of elongation growth in prc-1 does not result from aberrant cellulose structure, nor from uniform reduction in the dimensions of the cell-wall network due to reduced cellulose or cell-wall mass. Cellulose orientation was quantified by two quantitative methods. First, the orientation of newly synthesised microfibrils was measured in field-emission scanning electron micrographs of the cytoplasmic face of the inner epidermal cell wall. The ordered transverse orientation of microfibrils at the inner face of the cell wall was severely disrupted in prc-1 hypocotyls, particularly in the early growth phase. Second, cellulose orientation distributions across the whole cell-wall thickness, measured by polarised infrared microscopy, were much broader. Analysis of the microfibril orientations according to the theory of composite materials showed that during the initial growth phase, their anisotropy at the plasma membrane was sufficient to explain the anisotropy of subsequent growth.

RHL1 is an essential component of the plant DNA topoisomerase VI complex and is required for ploidy-dependent cell growth.

How cells achieve their final sizes is a pervasive biological question. One strategy to increase cell size is for the cell to amplify its chromosomal DNA content through endoreduplication cycles. Although endoreduplication is widespread in eukaryotes, we know very little about its molecular mechanisms. Successful progression of the endoreduplication cycle in Arabidopsis requires a plant homologue of archaeal DNA topoisomerase (topo) VI. To further understand how DNA is endoreduplicated and how this process is regulated, we isolated a dwarf Arabidopsis mutant, hyp7 (hypocotyl 7), in which various large cell types that in the wild type normally endoreduplicate multiple times complete only the first two rounds of endoreduplication and stall at 8C. HYP7 encodes the RHL1 (ROOT HAIRLESS 1) protein, and sequence analysis reveals that RHL1 has similarity to the C-terminal domain of mammalian DNA topo IIalpha, another type II topo that shares little sequence homology with topo VI. RHL1 shows DNA binding activity in vitro, and we present both genetic and in vivo evidence that RHL1 forms a multiprotein complex with plant topo VI. We propose that RHL1 plays an essential role in the topo VI complex to modulate its function and that the two distantly related topos, topo II and topo VI, have evolved a common domain that extends their function. Our data suggest that plant topo II and topo VI play distinct but overlapping roles during the mitotic cell cycle and endoreduplication cycle.

The Arabidopsis WAVY GROWTH 2 protein modulates root bending in response to environmental stimuli.

To understand how the direction of root growth changes in response to obstacles, light, and gravity, we characterized an Arabidopsis thaliana mutant, wavy growth 2 (wav2), whose roots show a short-pitch pattern of wavy growth on inclined agar medium. The roots of the wav2 mutant bent with larger curvature than those of the wild-type seedlings in wavy growth and in gravitropic and phototropic responses. The cell file rotations of the root epidermis of wav2-1 in the wavy growth pattern were enhanced in both right-handed and left-handed rotations. WAV2 encodes a protein belonging to the BUD EMERGENCE 46 family with a transmembrane domain at the N terminus and an alpha/beta-hydrolase domain at the C terminus. Expression analyses showed that mRNA of WAV2 was expressed strongly in adult plant roots and seedlings, especially in the root tip, the cell elongation zone, and the stele. Our results suggest that WAV2 is not involved in sensing environmental stimuli but that it negatively regulates stimulus-induced root bending through inhibition of root tip rotation.

"Big it up": endoreduplication and cell-size control in plants.

Cells undergoing endoreduplication replicate chromosomal DNA without intervening mitoses. The resulting larger, higher-ploidy nucleus is often associated with an increase in cell size, but the molecular basis for this correlation remains poorly understood. Recent advances in characterising various mutants and transgenic plants are beginning to unravel how this unique type of cell cycling is regulated and how it contributes to cell-size control. Both cell growth (i.e. increase in cytoplasmic macromolecular mass) and cell expansion (i.e. increase in cell volume through vacuolation) contribute independently to increases in cell size in plants. A total organ-size checkpoint may also help to coordinate cell size and cell number within an organ, and can contribute to final cell-size determination in plants.

Tensile properties of Arabidopsis cell walls depend on both a xyloglucan cross-linked microfibrillar network and rhamnogalacturonan II-borate complexes.

The mechanical properties of plant organs depend upon anatomical structure, cell-cell adhesion, cell turgidity, and the mechanical properties of their cell walls. By testing the mechanical responses of Arabidopsis mutants, it is possible to deduce the contribution that polymers of the cell wall make to organ strength. We developed a method to measure the tensile parameters of the expanded regions of turgid or plasmolyzed dark-grown Arabidopsis hypocotyls and applied it to the fucose biosynthesis mutant mur1, the xyloglucan glycosyltransferase mutants mur2 and mur3, and the katanin mutant bot1. Hypocotyls from plants grown in the presence of increasing concentrations of dichlorobenzonitrile, an inhibitor of cellulose synthesis, were considerably weakened, indicating the validity of our approach. In order of decreasing strength, the hypocotyls of mur2 > bot1 and mur1 > mur3 were each found to have reduced strength and a proportionate reduction in modulus compared with wild type. The tensile properties of the hypocotyls and of the inflorescence stems of mur1 were rescued by growth in the presence of high concentrations of borate, which is known to cross-link the pectic component rhamnogalacturonan II. From comparison of the mechanical responses of mur2 and mur3, we deduce that galactose-containing side chains of xyloglucan make a major contribution to overall wall strength, whereas xyloglucan fucosylation plays a comparatively minor role. We conclude that borate-complexed rhamnogalacturonan II and galactosylated xyloglucan contribute to the tensile strength of cell walls.

DNA topoisomerase VI is essential for endoreduplication in Arabidopsis.

Endoreduplication is a common process in eukaryotes that involves DNA amplification without corresponding cell divisions. Cell size in various organisms has been linked to endoreduplication, but the molecular mechanisms are poorly understood. We have used a genetic strategy to identify molecules involved in endocycles in Arabidopsis. We isolated two extreme dwarf mutants, hypocotyl6 (hyp6) and root hairless2 (rhl2) [3], and cells of these mutants successfully complete only the first two rounds of endoreduplication and stall at 8C. In both mutants, large cell types, such as trichomes and some epidermal cells, that normally endoreduplicate their DNA are much reduced in size. We show that HYP6 encodes AtTOP6B, a plant homolog of the archaeal DNA topoisomerase VI subunit B, and that RHL2 encodes AtSPO11-3, one of the three Arabidopsis subunit A homologs. We propose that this topoisomerase VI complex is essential for the decatenation of replicated chromosomes during endocycles and that successive rounds of endoreduplication are required for the full growth of specific cell types.