The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called "CENP-A") is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923-937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wildtype A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest. T he generation and use of haploids is one of the most powerful biotechnological means to accelerate the breeding process of cultivated plants. The advantage of haploid plants for breeders is that homozygosity can be achieved at all loci in a single generation via whole-genome duplication, without the need of selfing or backcrossing over many generations as conventionally required to obtain true-breeding lines. Haploids can be obtained in vitro or in vivo, although many species and genotypes are recalcitrant to these processes (reviewed in ref. 1). Alternatively, substantial changes to centromeric histone H3 (CENH3), such as replacing the hypervariable N-terminal tail of CENH3 with the tail of conventional histone H3 and fusing it to GFP (producing "tailswap-cenh3"), or complementing the cenh3.2-null mutant with homologs from the mustard family CENH3s creates haploid inducer lines in the model plant Arabidopsis thaliana (2-4). Haploidization occurred only when such a haploid inducer was crossed with a wild-type plant. The haploid inducer line proved to be stable upon selfing, suggesting that competition between modified and wild-type centromeres in the developing hybrid embryo results in the inactivation of the centromeres from the inducer parent. Consequently, chromosomes from the inducer parent are lost, and progeny can be recovered that retain only the haploid chromosome set of the wild-type parent.Because CENH3 is almost universal in eukaryotes, this method has the potential to produce haploids in any plant species. To elucidate whether, in addition to the severe conformational change using the CENH3-tailswap (2, 3), nontransgenic-induced minimal mutations in endogenous CENH3 also could affect the centromere function for haploid induction, we screened a population of barley (Hordeum vulgare) produced by ethyl methanesulfonate-induced targeting of local lesions in genomes (TILLING) (5); this diploid species has two functional variants...

SummaryElucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non‐coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long‐standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR–dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR–dCas9 can be combined with fluorescence‐labelled proteins to visualize DNA–protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.

Based on the analysis of 20 different monocot and eudicot species, we propose that the centromeric distribution of the phosphorylated histone H2AThr120 is evolutionary highly conserved across species with mono- and holocentric chromosomes. Therefore, antibodies recognizing the phosphorylated threonine 120 of the histone H2A can serve as a universal marker for the cytological detection of centromeres of mono- and holokinetic plant species. In addition, super resolution microscopy of signals specific to the centromere-specific histone H3 variant CENH3 and to H2AThr120ph revealed that these histone variants are incorporated into different nucleosomes, which form distinct, partly intermingled chromatin domains. This specific arrangement of both histone variants suggests different centromeric functions during the cell cycle.

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SUMMARYAurora is an evolutionary conserved protein kinase family involved in monitoring of chromosome segregation via phosphorylation of different substrates. In plants, however, the involvement of Aurora proteins in meiosis and in sensing microtubule attachment remains to be proven, although the downstream components leading to the targeting of spindle assembly checkpoint signals to anaphase-promoting complex have been described. To analyze the three members of Aurora family (AtAurora1, -2, and -3) of Arabidopsis we employed different combinations of T-DNA insertion mutants and/or RNAi transformants. Meiotic defects and the formation of unreduced pollen were revealed including plants with an increased ploidy level. The effect of reduced expression of Aurora was mimicked by application of the ATP-competitive Aurora inhibitor II. In addition, strong overexpression of any member of the AtAurora family is not possible. Only tagged or truncated forms of Aurora kinases can be overexpressed. Expression of truncated AtAurora1 resulted in a high number of aneuploids in Arabidopsis, while expression of AtAurora1-TAPi construct in tobacco resulted in 4C (possible tetraploid) progeny. In conclusion, our data demonstrate an essential role of Aurora kinases in the monitoring of meiosis in plants.