The centromeric histone H3 (CENH3) substitutes histone H3 within the nucleosomes of active centromeres in all eukaryotes. CENH3 deposition at centromeres is needed to assemble the kinetochore, a complex of conserved proteins responsible for correct chromosome segregation during nuclear division. Histones of regular nucleosomes are loaded during replication in S phase, while CENH3 deposition deviates from this pattern in yeast, human, and Drosophila melanogaster cells. Little is known about when and how CENH3 targets centromeric loci. Therefore, we determined the location and quantity of recombinant enhanced yellow fluorescent protein (EYFP)-CENH3 in mitotic root and endopolyploid leaf nuclei of transgenic Arabidopsis thaliana cells. Our data indicate significant loading of A. thaliana CENH3 during G2 (before splitting into sister kinetochores) rather than during the S or M phase of the cell cycle. The histone fold domain of the C-terminal part of CENH3 is sufficient to target A. thaliana centromeres. A. thaliana EYFP-CENH3 can recognize and target three different centromeric repeats of Arabidopsis lyrata but not field bean (Vicia faba) centromeres.

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy. The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steadystate RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in E. coli. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.

The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a KINETOCHORE NULL2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4, and 9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.

SUMMARYThe histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated CENH3 knockdown in Arabidopsis thaliana caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error-free. CENH3 RNAi transformants had reduced fertility because of frequently disturbed meiotic chromosome segregation. N-terminally truncated EYFP-CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes, but cannot be loaded onto centromeres of meiotic nuclei. Thus the N-terminal part is apparently required for CENH3 loading during meiosis. EYFP-CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and lack of meiotic incorporation of EYFP-CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.

The ability to generate (doubled) haploid plants significantly accelerates the crop breeding process. Haploids have been induced mainly through the generation of plants from cultivated gametophic (haploid) cells and tissues, i.e., in vitro haploid technologies, or through the selective loss of a parental chromosome set upon inter- or intraspecific hybridization. Here, we focus our review on the mechanisms responsible for the in vivo formation of haploids in the context of inter- and intraspecific hybridization. The application of a modified CENH3 for uniparental genome elimination, the IG1 system used for paternal as well as the BBM-like and the patatin-like phospholipase essential for maternal haploidy induction are discussed in detail.

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The use of herbicides to control undesirable vegetation has become a universal practice. For the broad application of herbicides the risk of damage to crop plants has to be limited. We introduced a gene into the genome of tobacco (Nicotiana tabacum) plants encoding the plastid-located protoporphyrinogen oxidase of Arabidopsis, the last enzyme of the common tetrapyrrole biosynthetic pathway, under the control of the cauliflower mosaic virus 35S promoter. The transformants were screened for low protoporphyrin IX accumulation upon treatment with the diphenyl ether-type herbicide acifluorfen. Leaf disc incubation and foliar spraying with acifluorfen indicated the lower susceptibility of the transformants against the herbicide. The resistance to acifluorfen is conferred by overexpression of the plastidic isoform of protoporphyrinogen oxidase. The in vitro activity of this enzyme extracted from plastids of selected transgenic lines was at least five times higher than the control activity. Herbicide treatment that is normally inhibitory to protoporphyrinogen IX oxidase did not significantly impair the catalytic reaction in transgenic plants and, therefore, did not cause photodynamic damage in leaves. Therefore, overproduction of protoporphyrinogen oxidase neutralizes the herbicidal action, prevents the accumulation of the substrate protoporphyrinogen IX, and consequently abolishes the light-dependent phytotoxicity of acifluorfen.Protoporphyrinogen oxidase (EC 1.3.3.4) (PPOX) is the last enzyme in the common tetrapyrrole biosynthesis pathway before the pathway branches toward chlorophyll and heme synthesis. PPOX catalyzes the oxygen-dependent oxidation of non-fluorescent protoporphyrinogen IX to fluorescent protoporphyrin IX and is associated with the chloroplast envelope and the thylakoid membrane (Matringe et al., 1992a). Parallel to the plastidic tetrapyrrolic pathway, activities of the last two enzymes of the heme synthesizing pathway, PPOX and ferrochelatase, were found in mitochondria (Jacobs et al., 1991; Smith et al., 1993) Thus, protoporphyrinogen IX is distributed between the plastidic pathway and the mitochondrial heme synthesis pathway. The control mechanism of the intercompartmental allocation is still not known. We recently identified two different cDNA sequences for PPOX in tobacco (Nicotiana tabacum).The deduced protein sequences designated as PPOX I and II have molecular masses of 60 and 55 kD, respectively, and share only 30% similarity. Translocation studies and immunological analysis proved that the proteins are imported either exclusively into plastids (PPOX I) or into mitochondria (PPOX II) (Lermontova et al., 1997).PPOX I and PPOX II closely resemble known PPOX sequences, e.g.

In plants as in other eukaryotes, the structural maintenance of chromosome (SMC) protein complexes cohesin, condensin and SMC5/6 are essential for sister chromatid cohesion, chromosome condensation, DNA repair and recombination. The presence of paralogous genes for various components of the different SMC complexes suggests the diversification of their biological functions during the evolution of higher plants. In Arabidopsis thaliana, we identified two candidate genes (Cap-D2 and Cap-D3) which may express conserved proteins presumably associated with condensin. In silico analyses using public databases suggest that both genes are controlled by factors acting in a cell cycle-dependent manner. Cap-D2 is essential because homozygous T-DNA insertion mutants were not viable. The heterozygous mutant showed wild-type growth habit but reduced fertility. For Cap-D3, we selected two homozygous mutants expressing truncated transcripts which are obviously not fully functional. Both mutants show reduced pollen fertility and seed set (one of them also reduced plant vigour), a lower chromatin density and frequent (peri)centromere association in interphase nuclei. Sister chromatid cohesion was impaired compared to wild-type in the cap-D3 mutants but not in the heterozygous cap-D2 mutant. At superresolution (Structured Illumination Microscopy), we found no alteration of chromatin substructure for both cap-D mutants. Chromosome-associated polypeptide (CAP)-D3 and the cohesin subunit SMC3 form similar but positionally non-overlapping reticulate structures in 2C-16C nuclei, suggesting their importance for interphase chromatin architecture in differentiated nuclei. Thus, we presume that CAP-D proteins are required for fertility, growth, chromatin organisation, sister chromatid cohesion and in a process preventing the association of centromeric repeats.