The SMC5/6 Complex Subunit NSE4A Is Involved in DNA Damage Repair and Seed Development.

The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.

The H3 histone chaperone NASPSIM3 escorts CenH3 in Arabidopsis.

Centromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non-nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASPSIM3 is co-expressed with Arabidopsis CenH3 in dividing cells and binds directly to both the N-terminal tail and the histone fold domain of non-nucleosomal CenH3. Reduced NASPSIM3 expression negatively affects CenH3 deposition, identifying NASPSIM3 as a CenH3 histone chaperone.

Proteomic analysis of barley cell nuclei purified by flow sorting.

Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.

Overexpressed TPX2 causes ectopic formation of microtubular arrays in the nuclei of acentrosomal plant cells.

TPX2 performs multiple roles in microtubule organization. Previously, it was shown that plant AtTPX2 binds AtAurora1 kinase and colocalizes with microtubules in a cell cycle-specific manner. To elucidate the function of TPX2 further, this work analysed Arabidopsis cells overexpressing AtTPX2-GFP. Distinct arrays of bundled microtubules, decorated with AtTPX2-GFP, were formed in the vicinity of the nuclear envelope and in the nuclei of overexpressing cells. The microtubular arrays showed reduced sensitivity to anti-microtubular drugs. TPX2-mediated formation of nuclear/perinuclear microtubular arrays was not specific for the transition to mitosis and occurred independently of Aurora kinase. The fibres were not observed in cells with detectable programmed cell death and, in this respect, they differed from TPX2-dependent microtubular assemblies functioning in mammalian apoptosis. Colocalization and co-purification data confirmed the interaction of importin with AtTPX2-GFP. In cells with nuclear foci of overexpressed AtTPX2-GFP, strong nuclear signals for Ran and importin diminished when microtubular arrays were assembled. This observation suggests that TPX2-mediated microtubule formation might be triggered by a Ran cycle. Collectively, the data suggest that in the acentrosomal plant cell, in conjunction with importin, overexpressed AtTPX2 reinforces microtubule formation in the vicinity of chromatin and the nuclear envelope.

Identification of Plant Nuclear Proteins Based on a Combination of Flow Sorting, SDS-PAGE, and LC-MS/MS Analysis.

In the plant nucleus, the majority of cellular DNA content is stored and maintained. This makes this highly specialized organelle the major coordinator of almost all essential processes in plant cells such as transcription, DNA replication, and repair. None of these biological pathways can be fully understood without a comprehensive characterization of nuclear proteins. Nevertheless, the interest of the proteomic community in the plant nuclear proteome has been very limited so far. This is probably due to the high integrity of plant cell, presence of many interfering metabolites, and considerable endogenous proteolytic activity which make the sample preparation problematic. Hereby, we describe a novel protocol for the high-throughput plant nuclear protein identification that combines a flow cytometric sorting of formaldehyde-fixed nuclei with protein and peptide separation and their subsequent LC-MS/MS analysis.

UNcleProt (Universal Nuclear Protein database of barley): The first nuclear protein database that distinguishes proteins from different phases of the cell cycle.

Proteins are the most abundant component of the cell nucleus, where they perform a plethora of functions, including the assembly of long DNA molecules into condensed chromatin, DNA replication and repair, regulation of gene expression, synthesis of RNA molecules and their modification. Proteins are important components of nuclear bodies and are involved in the maintenance of the nuclear architecture, transport across the nuclear envelope and cell division. Given their importance, the current poor knowledge of plant nuclear proteins and their dynamics during the cell's life and division is striking. Several factors hamper the analysis of the plant nuclear proteome, but the most critical seems to be the contamination of nuclei by cytosolic material during their isolation. With the availability of an efficient protocol for the purification of plant nuclei, based on flow cytometric sorting, contamination by cytoplasmic remnants can be minimized. Moreover, flow cytometry allows the separation of nuclei in different stages of the cell cycle (G1, S, and G2). This strategy has led to the identification of large number of nuclear proteins from barley (Hordeum vulgare), thus triggering the creation of a dedicated database called UNcleProt, http://barley.gambrinus.ueb.cas.cz/ .