Clare A. Hasenkampf

Mutations in Arabidopsis condensin genes disrupt embryogenesis, meristem organization and segregation of homologous chromosomes during meiosis.

Proper chromatin condensation and sister chromatid resolution are essential for the maintenance of chromosomal integrity during cell division, and is in part mediated by a conserved multisubunit apparatus termed the condensin complex. The core subunits of the complex are members of the SMC2 (Structural Maintenance of Chromosomes) and SMC4 gene families. We have cloned an Arabidopsis gene, AtCAP-E1, which is a functional ortholog of the yeast SMC2 gene. A second, highly homologous SMC2 gene, AtCAPE-2, was identified by the Arabidopsis genome project. SMC2 gene expression in Arabidopsis was correlated with the mitotic activity of tissues, with high level expression observed in meristematic cells. The two genes are differentially expressed with AtCAP-E1 accounting for more than 85% of the total SMC2 transcript pool. The titan3 mutant is the result of a T-DNA insertion into AtCAP-E1, but other than subtle endosperm defects, titan3 is viable and fecund. We identified a T-DNA insertion mutant of AtCAP-E2, which showed no obvious mutant phenotype, indicating that the two genes are functionally redundant. Genetic crosses were employed to examine the consequences of reduced SMC2 levels. Both male and female gametogenesis were compromised in double mutant spores. Embryo lethality was observed for both double homozygous and AtCAP-E1-/-, AtCAP-E2+/- plants; arrest occurred at or before the globular stage and was associated with altered planes of cell division in both the suspensor and the embryo. Down regulation of both genes by antisense technology, as well as in AtCAP-E1+/-, AtCAP-E2-/- plants results in meristem disorganization and fasciation. Our data are consistent with the interpretation that threshold levels of SMC2 proteins are required for normal development and that AtCAP-E2 may have a higher affinity for its target than AtCAP-E1.

Antibodies directed against a meiosis-specific, chromatin-associated protein identify conserved meiotic epitopes

The molecular mechanisms by which meiotic events are regulated are at present unknown. To approach this problem, we have exploited the natural synchrony of Lilium meiocytes to compare the nuclear protein profiles of a variety of stages of meiosis. This approach has facilitated the identification of a number of nuclear proteins that appear and disappear in a stagespecific fashion. Here we report the presence of an abundant nuclear protein that first appears during premeiotic interphase, a period during which the irreversible commitment to meiosis occurs. Antibodies directed against this protein demonstrate its meiosis specificity as well as conservation of the epitope(s) in both mono-and dicotyledonous plant species. Chromatin fractionation studies indicate that this protein, which we have termed meiotin-1, is associated with strings of nucleosomes. Implications for meiotic chromatin packaging and chromosome structure are discussed.

Disruption of the Arabidopsis SMC4 gene, AtCAP-C, compromises gametogenesis and embryogenesis

In higher eukaryotes, the condensin complex is a multisubunit apparatus that plays a pivotal role in the coordinated condensation of chromatin during mitosis. The catalytic subunits, CAP-E and CAP-C, members of the SMC family of ATPases, form a heterodimer, the activity of which is controlled by the non-SMC subunits CAP-D2, CAP-G and CAP-H. Here, we report the characterization of a T-DNA insertion mutant of the Arabidopsis CAP-C gene. Analysis of the progeny of selfed heterozygotes revealed that the homozygous null genotype is embryo lethal, with arrest occurring at or before the globular stage of development. Patterning defects associated with altered planes of cytokinesis were found in both the embryo and the suspensor. Crosses of heterozygotes with wild type plants revealed both male and female gametophytic defects. Stretched chromatin was observed between segregating mitotic chromosomes in pollen produced by selfed heterozygotes. Additionally, some plants heterozygous for the T-DNA insertion exhibited loss of apical dominance and mild fasciation, indicating a semi-dominant effect of the mutation. These results reveal a critical role for AtCAP-C during cell division and, unlike our previous studies on the AtCAP-E genes, suggest that no redundant factors for AtCAP-C exist in the Arabidopsis genome.

The synaptonemal complex — the chaperone of crossing over

During meiosis homologous chromosomes pair and exchange homologous chromosome segments. The synaptonemal complex (SC) forms between paired chromosomes. The role of the SC in the process of reciprocal exchange of flanking markers is a matter of debate. I propose a dual pathway for reciprocal exchange of flanking markers (REFM). In the first, SC-independent, path, two ‘half-nodules’ and an independent REFM protein combine to form a functional recombination nodule (RN). The RN binds to paired chromosomes and accomplishes reciprocal exchange of flanking markers. In the other, SC-dependent, pathway ‘half-nodules’ occur at pairing initiation sites. ‘Half-nodules’ move along the SC as it forms. Assisted by an SC-bound REFM protein, ‘half-nodules’ combine to form functional RNs. I propose that different organisms rely to different extents on the two pathways, and hence rely to different extents on the SC.

Ahp2 (Hop2) function in Arabidopsis thaliana (Ler) is required for stabilization of close alignment and synaptonemal complex formation except for the two short arms that contain nucleolus organizer regions.

A cytological comparative analysis of male meiocytes was performed for Arabidopsis wild type and the ahp2 (hop2) mutant with emphasis on ahp2’s largely uncharacterized prophase I. Leptotene progression appeared normal in ahp2 meiocytes; chromosomes exhibited regular axis formation and assumed a typical polarized nuclear organization. In contrast, 4′,6′-diamidino-2-phenylindole-stained ahp2 pachytene chromosome spreads demonstrated a severe reduction in stabilized pairing. However, transmission electron microscopy (TEM) analysis of sections from meiocytes revealed that ahp2 chromosome axes underwent significant amounts of close alignment (44% of total axis). This apparent paradox strongly suggests that the Ahp2 protein is involved in the stabilization of homologous chromosome close alignment. Fluorescent in situ hybridization in combination with Zyp1 immunostaining revealed that ahp2 mutants undergo homologous synapsis of the nucleolus-organizer-region-bearing short arms of chromosomes 2 and 4, despite the otherwise “nucleus-wide” lack of stabilized pairing. The duration of ahp2 zygotene was significantly prolonged and is most likely due to difficulties in chromosome alignment stabilization and subsequent synaptonemal complex formation. Ahp2 and Mnd1 proteins have previously been shown, “in vitro,” to form a heterodimer. Here we show, “in situ,” that the Ahp2 and Mnd1 proteins are synchronous in their appearance and disappearance from meiotic chromosomes. Both the Ahp2 and Mnd1 proteins localize along the chromosomal axis. However, localization of the Ahp2 protein was entirely foci-based whereas Mnd1 protein exhibited an immunostaining pattern with some foci along the axis and a diffuse staining for the rest of the chromosome.

Analysis of substage associations in prophase I of meiosis in floral buds of wild-type Arabidopsis thaliana (Brassicaceae).

We developed an improved cytological protocol for producing high quality, light microscope images of plant meiotic chromosomes. Because the technique works on species with small genomes and thick microsporocyte cell walls, it should be useful for studying the wild relatives of Arabidopsis and other eudicots with small genomes. Combining this improved fixation protocol with our new analysis of associated substages in floral buds, we can unambiguously assign individual meiotic cells to particular substages of prophase I in Arabidopsis thaliana, even for difficult distinctions such as that between late zygotene or early diplotene. In this report we provide the first estimate of the individual duration of the zygotene and pachytene substages (4.8 h and 10.0 h, respectively) in A. thaliana. We also have examined the diffuse substage of prophase I and report that during this post-pachytene substage, nuclei retain the association of homologous nucleolus organizer regions and homologous centromeres, despite the generally diffuse chromatin and generally unpaired chromosome regions. Additionally, we have observed that centromeric regions of the chromosomes of diffuse-stage nuclei are highly condensed, more so than those of any other substage of prophase I.

DNA, histone H1 and meiotin-1 immunostaining patterns along whole-mount preparations ofLilium longiflorum pachytene chromosomes

Meiotin-1 is a chromatin protein found in lily microsporocytes preceding and during meiosis. It reaches peak levels in the leptotene-pachytene interval. Here we report the pattern of immunostaining of meiotin-1 along pachytene chromosomes prepared using a whole-mount, synaptonemal complex spreading technique. Meiotin-1 immunostaining, like immunostaining for histone H1 and DNA, is found all along the lengths of the chromosomes. Unlike histone H1 and DNA immunostaining, there are several patches of particularly intense meiotin-1 immunostaining. The sites of intense staining may be clustered in thein vivo nucleus. The distribution of meiotin-1 along the width of the chromosomes was examined and compared with the pattern of histone H1 and DNA immunostaining. All three were distributed across the width of the chromosome, and did not appear to be preferentially associated with the synaptonemal complex. There was a slight suggestion that histone H1 and meiotin-1 may be located preferentially away from the synaptonemal complex. The temporal and spatial distribution of meiotin-1 in microsporocytes and tapetal cells, and the chemical and physical properties of meiotin-1, are discussed in the context of our hypothesis that meiotin-1 is a protein that functions to limit the degree of chromosome condensation during prophase I of meiosis.

Dmc1 fluorescent foci in prophase I nuclei of diploid, triploid and hybrid lilies

Abstract. We examined the distribution of meiotic epitopes for the Dmc1 protein of lilies in a normal diploid, a triploid, and in a diploid species-hybrid. The triploid has an extra chromosome set; all three sets align, but only two of the three axes intimately pair at a given location. Our findings with the triploid support the idea that retention of the foci until the pachytene stage requires a successful homology check and synaptonemal complex (SC) initiation; the number of foci in the triploid diminishes by approximately 30% from early zygotene to pachytene, and the triploid pachytene values are similar to the pachytene values of the diploid. The species-hybrid lacks chromosome homology, has reduced SC formation and few reciprocal genetic exchanges. In this species-hybrid the number of foci at early zygotene is similar to that in the normal diploid but is dramatically reduced by mid-zygotene. The extent to which the number of Dmc1 foci is reduced is similar to the extent that SC formation is reduced. In contrast the extent of the reduction in reciprocal genetic exchange in the species-hybrid is much greater than the reduction in the number of foci. We conclude that Dmc1 protein is involved in homology checking, but the impact of failure to find homology affects SC formation and reciprocal genetic exchange differentially.

In Situ Hybridization of Lilium Whole Mount Synaptonemal Complex Chromosomal Preparations

Whole mount meiotic preparations of the synaptonemal complex complement of Lilium have been used for in situ hybridization experiments. A probe of the maize ribosomal DNA gene cluster has been successfully hybridized to the lily preparations. Three strong signals, corresponding to the three known lily nucleolus organizer regions, have been seen in most of the chromosome preparations. In situ hybridization experiments using meiotic preparations should be useful for identifying specific chromosomes, and for investigating the role of particular DNA molecules important to meiotic function.

Chromosome fragile sites in Arabidopsis harbor matrix attachment regions that may be associated with ancestral chromosome rearrangement events.

Mutations in the BREVIPEDICELLUS (BP) gene of Arabidopsis thaliana condition a pleiotropic phenotype featuring defects in internode elongation, the homeotic conversion of internode to node tissue, and downward pointing flowers and pedicels. We have characterized five mutant alleles of BP, generated by EMS, fast neutrons, x-rays, and aberrant T–DNA insertion events. Curiously, all of these mutagens resulted in large deletions that range from 140 kbp to over 900 kbp just south of the centromere of chromosome 4. The breakpoints of these mutants were identified by employing inverse PCR and DNA sequencing. The south breakpoints of all alleles cluster in BAC T12G13, while the north breakpoint locations are scattered. With the exception of a microhomology at the bp-5 breakpoint, there is no homology in the junction regions, suggesting that double-stranded breaks are repaired via non-homologous end joining. Southwestern blotting demonstrated the presence of nuclear matrix binding sites in the south breakpoint cluster (SBC), which is A/T rich and possesses a variety of repeat sequences. In situ hybridization on pachytene chromosome spreads complemented the molecular analyses and revealed heretofore unrecognized structural variation between the Columbia and Landsberg erecta genomes. Data mining was employed to localize other large deletions around the HY4 locus to the SBC region and to show that chromatin modifications in the region shift from a heterochromatic to euchromatic profile. Comparisons between the BP/HY4 regions of A. lyrata and A. thaliana revealed that several chromosome rearrangement events have occurred during the evolution of these two genomes. Collectively, the features of the region are strikingly similar to the features of characterized metazoan chromosome fragile sites, some of which are associated with karyotype evolution.