Catastrophic chromosomal restructuring during genome elimination in plants.

Genome instability is associated with mitotic errors and cancer. This phenomenon can lead to deleterious rearrangements, but also genetic novelty, and many questions regarding its genesis, fate and evolutionary role remain unanswered. Here, we describe extreme chromosomal restructuring during genome elimination, a process resulting from hybridization of Arabidopsis plants expressing different centromere histones H3. Shattered chromosomes are formed from the genome of the haploid inducer, consistent with genomic catastrophes affecting a single, laggard chromosome compartmentalized within a micronucleus. Analysis of breakpoint junctions implicates breaks followed by repair through non-homologous end joining (NHEJ) or stalled fork repair. Furthermore, mutation of required NHEJ factor DNA Ligase 4 results in enhanced haploid recovery. Lastly, heritability and stability of a rearranged chromosome suggest a potential for enduring genomic novelty. These findings provide a tractable, natural system towards investigating the causes and mechanisms of complex genomic rearrangements similar to those associated with several human disorders

AtMND1 is required for homologous pairing during meiosis in Arabidopsis

BACKGROUND: Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1. RESULTS: We have analyzed the function of the Arabidopsis orthologue of the budding yeast MND1 gene (AtMND1). Loss of AtMND1 did not affect normal vegetative development but caused fragmentation and missegregation of chromosomes in male and female meiosis, formation of inviable gametes, and sterility. Analysis of the Atmnd1 Atspo11-1 double mutant indicated that chromosome fragmentation in Atmnd1 was suppressed by loss of Atspo11-1. Fluorescence in situ hybridization (FISH) analysis showed that homologous pairing failed to occur and homologues remained apart throughout meiosis. AtMND1 showed strong expression in meiocytes as revealed by RNA in situs. CONCLUSION: We conclude that AtMND1 is required for homologous pairing and is likely to play a role in the repair of DNA double strand breaks during meiosis in Arabidopsis, thus showing conservation of function with that of MND1 during meiosis in yeast

Meiosis-Specific Loading of the Centromere-Specific Histone CENH3 in Arabidopsis thaliana

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior

Gamete formation without meiosis in Arabidopsis

Apomixis, the formation of asexual seeds in plants, leads to populations that are genetically uniform maternal clones. The transfer of apomixis to crop plants holds great promise in plant breeding for fixation of heterozygosity and hybrid vigour because it would allow the propagation of hybrids over successive generations. Apomixis involves the production of unreduced (diploid) female gametes that retain the genotype of the parent plant (apomeiosis), followed by parthenogenetic development of the egg cell into an embryo and the formation of functional endosperm. The molecular mechanisms underlying apomixis are unknown. Here we show that mutation of the Arabidopsis gene DYAD/SWITCH1 (SWI1), a regulator of meiotic chromosome organization, leads to apomeiosis. We found that most fertile ovules in dyad plants form seeds that are triploid and that arise from the fertilization of an unreduced female gamete by a haploid male gamete. The unreduced female gametes fully retain parental heterozygosity across the genome, which is characteristic of apomeiosis. Our results show that the alteration of a single gene in a sexual plant can bring about functional apomeiosis, a major component of apomixis

The plant adherin AtSCC2 is required for embryogenesis and sister-chromatid cohesion during meiosis in Arabidopsis

Adherin plays an important role in loading the cohesin complex onto chromosomes, and is essential for the establishment of sister-chromatid cohesion. We have identified and analyzed the Arabidopsis adherin homolog AtSCC2. Interestingly, the sequence analysis of AtSCC2 and of other putative plant adherin homologs revealed the presence of a PHD finger, which is not found in their fungal and animal counterparts. AtSCC2 is identical to EMB2773, and mutants show early embryo lethality and formation of giant endosperm nuclei. A role for AtSCC2 in sister-chromatid cohesion was established by using conditional RNAi and examining meiotic chromosome organization. AtSCC2-RNAi lines showed sterility, arising from the following defects in meiotic chromosome organization: failure of homologous pairing, loss of sister-chromatid cohesion, mixed segregation of chromosomes and chromosome fragmentation. The mutant phenotype, which included defects in chromosome organization and cohesion in prophase I, is distinct from that of the Arabidopsis cohesin mutant Atrec8, which retains centromere cohesion up to anaphase I. Immunostaining experiments revealed the aberrant distribution of the cohesin subunit AtSCC3 on chromosomes, and defects in chromosomal axis formation, in the meiocytes of AtSCC2-RNAi lines. These results demonstrate a role for AtSCC2 in sister-chromatid cohesion and centromere organization, and show that the machinery responsible for the establishment of cohesion is conserved in plants

Establishment and inheritance of minichromosomes from Arabidopsis haploid induction.

Minichromosomes are small, sometimes circular, rearranged chromosomes consisting of one centromere and short chromosomal arms formed by treatments that break DNA, including plant transformation. Minichromosomes have the potential to serve as vectors to quickly move valuable genes across a wide range of germplasm, including into adapted crop varieties. To realize this potential, minichromosomes must be reliably generated, easily manipulated, and stably inherited. Here we show a reliable method for minichromosome formation in haploids resulting from CENH3-mediated genome elimination, a process that generates genome instability and karyotypic novelty specifically on one parental genome. First, we identified 2 out of 260 haploids, each containing a single-copy minichromosome originating from centromeric regions of chromosomes 1 and 3, respectively. The chromosome 1 minichromosome we characterized did not pair at meiosis but displayed consistent transmission over nine selfing generations. Next, we demonstrated that CENH3-based haploid induction can produce minichromosomes in a targeted manner. Haploid inducers carrying a selectable pericentromeric marker were used to isolate additional chromosome-specific minichromosomes, which occurred in 3 out of 163 haploids. Our findings document the formation of heritable, rearranged chromosomes, and we provide a method for convenient minichromosome production

Epigenetically mismatched parental centromeres trigger genome elimination in hybrids.

Wide crosses result in postzygotic elimination of one parental chromosome set, but the mechanisms that result in such differential fate are poorly understood. Here, we show that alterations of centromeric histone H3 (CENH3) lead to its selective removal from centromeres of mature Arabidopsis eggs and early zygotes, while wild-type CENH3 persists. In the hybrid zygotes and embryos, CENH3 and essential centromere proteins load preferentially on the CENH3-rich centromeres of the wild-type parent, while CENH3-depleted centromeres fail to reconstitute new CENH3-chromatin and the kinetochore and are frequently lost. Genome elimination is opposed by E3 ubiquitin ligase VIM1. We propose a model based on cooperative binding of CENH3 to chromatin to explain the differential CENH3 loading rates. Thus, parental CENH3 polymorphisms result in epigenetically distinct centromeres that instantiate a strong mating barrier and produce haploids