Synthetically induced Arabidopsis thaliana autotetraploids provide insights into the analysis of meiotic mutants with altered crossover frequency.

Mutations affecting crossover (CO) frequency and distribution lead to the presence of univalents during meiosis, giving rise to aneuploid gametes and sterility. These mutations may have a different effect after chromosome doubling. The combination of altered ploidy and mutations could be potentially useful to gain new insights into the mechanisms and regulation of meiotic recombination; however, studies using autopolyploid meiotic mutants are scarce. Here, we have analyzed the cytogenetic consequences in colchicine-induced autotetraploids (colchiploids) from different Arabidopsis mutants with an altered CO frequency. We have found that there are three types of mutants: mutants in which chiasma frequency is doubled after chromosome duplication (zip4, mus81), as in the control; mutants in which polyploidy leads to a higher-than-expected increase in chiasma frequency (asy1, mer3, hei10, and mlh3); and mutants in which the rise in chiasma frequency produced by the presence of two extrachromosomal sets is less than doubled (msh5, fancm). In addition, the proportion of class I/class II COs varies after chromosome duplication in the control. The results obtained reveal the potential of colchiploid meiotic mutants for better understanding of the function of key proteins during plant meiosis. This is especially relevant considering that most crops are polyploids.

Partial cytological diploidization of neoautotetraploid meiosis by induced cross-over rate reduction.

Polyploids, which arise from whole-genome duplication events, have contributed to genome evolution throughout eukaryotes. Among plants, novel features of neopolyploids include traits that can be evolutionarily or agriculturally beneficial, such as increased abiotic stress tolerance. Thus, in addition to being interesting from an evolutionary perspective, genome duplication is also increasingly recognized as a promising crop improvement tool. However, newly formed (neo)polyploids commonly suffer from fertility problems, which have been attributed to abnormal associations among the multiple homologous chromosome copies during meiosis (multivalents). Here, we test the long-standing hypothesis that reducing meiotic cross-over number may be sufficient to limit multivalent formation, favoring diploid-like bivalent associations (cytological diploidization). To do so, we developed Arabidopsis thaliana lines with low cross-over rates by combining mutations for HEI10 and TAF4b. Double mutants showed a reduction of ~33% in cross-over numbers in diploids without compromising meiotic stability. Neopolyploids derived from the double mutant show a cross-over rate reduction of about 40% relative to wild-type neotetraploids, and groups of four homologs indeed formed fewer multivalents and more bivalents. However, we also show that the reduction in multivalents comes with the cost of a slightly increased frequency of univalents and that it does not rescue neopolyploid fertility. Thus, while our results do show that reducing cross-over rates can reduce multivalent frequency in neopolyploids, they also emphasize that there are additional factors affecting both meiotic stability and neopolyploid fertility that will need to be considered in solving the neopolyploid fertility challenge.

The Role of DNA Topoisomerase Binding Protein 1 (TopBP1) in Genome Stability in Arabidopsis.

DNA topoisomerase II (TOPII) plays a very important role in DNA topology and in different biological processes such as DNA replication, transcription, repair, and chromosome condensation in higher eukaryotes. TOPII has been found to interact directly with a protein called topoisomerase II binding protein 1 (TopBP1) which also seems to have important roles in DNA replication and repair. In this study, we conducted different experiments to assess the roles of TopBP1 in DNA repair, mitosis, and meiosis, exploring the relationship between TOPII activity and TopBP1. We found that topbp1 mutant seedlings of Arabidopsis thaliana were hypersensitive to cisplatin treatment and the inhibition of TOPII with etoposide produced similar hypersensitivity levels. Furthermore, we recognised that there were no significant differences between the WT and topbp1 seedlings treated with cisplatin and etoposide together, suggesting that the hypersensitivity to cisplatin in the topbp1 mutant could be related to the functional interaction between TOPII and TopBP1. Somatic and meiotic anaphase bridges appeared in the topbp1 mutant at similar frequencies to those when TOPII was inhibited with merbarone, etoposide, or ICFR-187. The effects on meiosis of TOPII inhibition were produced at S phase/G2 stage, suggesting that catenanes could be produced at the onset of meiosis. Thus, if the processing of the catenanes is impaired, some anaphase bridges can be formed. Also, the appearance of anaphase bridges at first and second division is discussed.

How to Perform an Accurate Analysis of Metaphase I Chromosome Configurations in Autopolyploids of Arabidopsis thaliana.

During meiosis, accurate segregation of chromosomes requires the formation of bivalents at metaphase I. In autopolyploids, there are more than two copies of each chromosome with the same chance to form chiasmata at meiosis. This leads to the formation of multivalent configurations in which chiasma quantification is rather complicated. Here, we present an improved cytological protocol, including fluorescence in situ hybridization, to obtain high quality spreads of metaphase I chromosomes from Arabidopsis thaliana autotetraploids. This method allows an accurate analysis of the different meiotic configurations and enables the assessment of the number of chiasmata formed by each tetrasome (group of four homologs).

The Cdk1/Cdk2 homolog CDKA;1 controls the recombination landscape in Arabidopsis.

Little is known how patterns of cross-over (CO) numbers and distribution during meiosis are established. Here, we reveal that cyclin-dependent kinase A;1 (CDKA;1), the homolog of human Cdk1 and Cdk2, is a major regulator of meiotic recombination in ArabidopsisArabidopsis plants with reduced CDKA;1 activity experienced a decrease of class I COs, especially lowering recombination rates in centromere-proximal regions. Interestingly, this reduction of type I CO did not affect CO assurance, a mechanism by which each chromosome receives at least one CO, resulting in all chromosomes exhibiting similar genetic lengths in weak loss-of-function cdka;1 mutants. Conversely, an increase of CDKA;1 activity resulted in elevated recombination frequencies. Thus, modulation of CDKA;1 kinase activity affects the number and placement of COs along the chromosome axis in a dose-dependent manner.

Competition for Chiasma Formation Between Identical and Homologous (But Not Identical) Chromosomes in Synthetic Autotetraploids of Arabidopsis thaliana.

Polyploid organisms provide additional opportunities to study meiosis in a more complex context since more than two potential homologous chromosomes are available. When the chromosome complement of a diploid individual is duplicated, each chromosome is accompanied by one identical and two homologous chromosomes within the same nucleus. In this situation, a competition in pairing/synapsis/chiasma formation between identical and homologous (but not necessarily identical) chromosomes can occur. Several studies have been conducted in different species to address whether there are preferences in crossover formation between identical rather than homologous chromosomes. In this study, multivalent and chiasma frequencies were cytologically analyzed in synthetic autotetraploids of Arabidopsis thaliana including the accessions Col, Ler, and the Col/Ler hybrid. Fluorescence in situ hybridization was conducted to identify each chromosome at metaphase I. The new Col and Ler tetraploids showed high multivalent frequencies, exceeding the theoretical 66.66% expected on a simple random end-pairing model, thus indicating that there are more than two autonomous synaptic sites per chromosome despite their small size. However, a significant excess of bivalent pairs was found in the Col/Ler hybrid, mainly due to the contribution of chromosomes 2 and 3. The mean chiasma frequencies of the three artificial autotetraploids were about twofold the corresponding mean cell chiasma frequencies of their diploid counterparts. The relative contribution of each chromosome to the total chiasma frequency was similar in the three genotypes, with the exception of a lower contribution of chromosome 3 in the hybrid. Preferences for chiasma formation between identical and homologous chromosomes were analyzed in Col/Ler 4x, taking advantage of the cytological differences between the accessions: variations in the size of the 45S rDNA region on the short arm of chromosome 2 and changes in the size and localization of the 5S rDNA region in chromosome 3. We observed a different behavior of chromosomes 2 and 3, i.e., random chiasma formation between identical and homologous chromosomes 2, and preferences for chiasma formation between homologous chromosomes 3. Hence, our results reveal the existence of chromosome-specific mechanisms responsible for these preferences.