Hieronim Golczyk

Horizontal genome transfer as an asexual path to the formation of new species

Allopolyploidization, the combination of the genomes from two different species, has been a major source of evolutionary innovation and a driver of speciation and environmental adaptation. In plants, it has also contributed greatly to crop domestication, as the superior properties of many modern crop plants were conferred by ancient allopolyploidization events. It is generally thought that allopolyploidization occurred through hybridization events between species, accompanied or followed by genome duplication. Although many allopolyploids arose from closely related species (congeners), there are also allopolyploid species that were formed from more distantly related progenitor species belonging to different genera or even different tribes. Here we have examined the possibility that allopolyploidization can also occur by asexual mechanisms. We show that upon grafting—a mechanism of plant–plant interaction that is widespread in nature—entire nuclear genomes can be transferred between plant cells. We provide direct evidence for this process resulting in speciation by creating a new allopolyploid plant species from a herbaceous species and a woody species in the nightshade family. The new species is fertile and produces fertile progeny. Our data highlight natural grafting as a potential asexual mechanism of speciation and also provide a method for the generation of novel allopolyploid crop species.

Molecular Marker Systems for Oenothera Genetics

The genus Oenothera has an outstanding scientific tradition. It has been a model for studying aspects of chromosome evolution and speciation, including the impact of plastid nuclear co-evolution. A large collection of strains analyzed during a century of experimental work and unique genetic possibilities allow the exchange of genetically definable plastids, individual or multiple chromosomes, and/or entire haploid genomes (Renner complexes) between species. However, molecular genetic approaches for the genus are largely lacking. In this study, we describe the development of efficient PCR-based marker systems for both the nuclear genome and the plastome. They allow distinguishing individual chromosomes, Renner complexes, plastomes, and subplastomes. We demonstrate their application by monitoring interspecific exchanges of genomes, chromosome pairs, and/or plastids during crossing programs, e.g., to produce plastome–genome incompatible hybrids. Using an appropriate partial permanent translocation heterozygous hybrid, linkage group 7 of the molecular map could be assigned to chromosome 9·8 of the classical Oenothera map. Finally, we provide the first direct molecular evidence that homologous recombination and free segregation of chromosomes in permanent translocation heterozygous strains is suppressed.

Chloroplast DNA in Mature and Senescing Leaves: A Reappraisal

The fate of plastid DNA (ptDNA) during leaf development has become a matter of contention. Reports on little change in ptDNA copy number per cell contrast with claims of complete or nearly complete DNA loss already in mature leaves. We employed high-resolution fluorescence microscopy, transmission electron microscopy, semithin sectioning of leaf tissue, and real-time quantitative PCR to study structural and quantitative aspects of ptDNA during leaf development in four higher plant species (Arabidopsis thaliana, sugar beet [Beta vulgaris], tobacco [Nicotiana tabacum], and maize [Zea mays]) for which controversial findings have been reported. Our data demonstrate the retention of substantial amounts of ptDNA in mesophyll cells until leaf necrosis. In ageing and senescent leaves of Arabidopsis, tobacco, and maize, ptDNA amounts remain largely unchanged and nucleoids visible, in spite of marked structural changes during chloroplast-to-gerontoplast transition. This excludes the possibility that ptDNA degradation triggers senescence. In senescent sugar beet leaves, reduction of ptDNA per cell to ∼30% was observed reflecting primarily a decrease in plastid number per cell rather than a decline in DNA per organelle, as reported previously. Our findings are at variance with reports claiming loss of ptDNA at or after leaf maturation.

Translocations of Chromosome End-Segments and Facultative Heterochromatin Promote Meiotic Ring Formation in Evening Primroses

This work reveals the arrangement of rRNA genes, euchromatin, and heterochromatin in cycling and noncycling cells of evening primroses. The results suggest a model of chromosomal evolution, with translocations of chromosome end-segments and selection against structurally heterozygous bivalents as mechanisms that promote permanent multichromosomal rings at meiosis. Due to reciprocal chromosomal translocations, many species of Oenothera (evening primrose) form permanent multichromosomal meiotic rings. However, regular bivalent pairing is also observed. Chiasmata are restricted to chromosomal ends, which makes homologous recombination virtually undetectable. Genetic diversity is achieved by changing linkage relations of chromosomes in rings and bivalents via hybridization and reciprocal translocations. Although the structural prerequisite for this system is enigmatic, whole-arm translocations are widely assumed to be the mechanistic driving force. We demonstrate that this prerequisite is genome compartmentation into two epigenetically defined chromatin fractions. The first one facultatively condenses in cycling cells into chromocenters negative both for histone H3 dimethylated at lysine 4 and for C-banding, and forms huge condensed middle chromosome regions on prophase chromosomes. Remarkably, it decondenses in differentiating cells. The second fraction is euchromatin confined to distal chromosome segments, positive for histone H3 lysine 4 dimethylation and for histone H3 lysine 27 trimethylation. The end-segments are deprived of canonical telomeres but capped with constitutive heterochromatin. This genomic organization promotes translocation breakpoints between the two chromatin fractions, thus facilitating exchanges of end-segments. We challenge the whole-arm translocation hypothesis by demonstrating why reciprocal translocations of chromosomal end-segments should strongly promote meiotic rings and evolution toward permanent translocation heterozygosity. Reshuffled end-segments, each possessing a major crossover hot spot, can furthermore explain meiotic compatibility between genomes with different translocation histories.

Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species

Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms—a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity.

Meiotic events in Oenothera - a non-standard pattern of chromosome behaviour.

The genus Oenothera shows an intriguing extent of permanent translocation heterozygosity. Reciprocal translocations of chromosome arms in species or populations result in various kinds of chromosome multivalents in diakinesis. Early meiotic events conditioning such chromosome behaviour are poorly understood. We found a surprising uniformity of the leptotene-diplotene period, regardless of the chromosome configuration at diakinesis (ring of 14, 7 bivalents, mixture of bivalents and multivalents). It appears that the earliest chromosome interactions at Oenothera meiosis are untypical, since they involve pericentromeric regions. During early leptotene, proximal chromosome parts cluster and form a highly polarized Rabl configuration. Telomeres associated in pairs were seen at zygotene. The high degree of polarization of meiotic nuclei continues for an exceptionally long period, i.e., during zygotene-pachytene into the diplotene contraction stage. The Rabl-polarized meiotic architecture and clustering of pericentromeres suggest a high complexity of karyotypes, not only in structural heterozygotes but also in bivalent-forming homozygous species.

The arrangement of pericentromeres during meiotic prophase I in the permanent translocation heterozygote Rhoeo spathacea

Abstract A study on the organization of prophase I chromatin in a PTH (permanent translocation heterozygosity) plant -Rhoeo spathacea was carried out. The sequential DAPI/AMD-FISH technique showed that nuclear polarization and centromere clustering during preleptotene-pachytene is a striking feature of all the studied heterozygotic clones. It was revealed that during pachytene pericentromeric heterochromatins assemble into a strikingly regular continuous ring with empty space inside. The topological constraints exerted by the heterochromatic pachytene “ring” exclude both random and multiple centromere interactions, and together with peripheral disposition of GC-rich pericentromeric regions and their reduced number, conform to a condition where one pericentromere interacts side-by-side with its two neighbors. The relation of the meiotic centromere associations in Rhoeo to the processes regulating chromosome arm juxtaposition (e.g. synapsis) and/or segregation was discussed.

Emodin, a natural inhibitor of protein kinase CK2, suppresses growth, hyphal development, and biofilm formation of Candida albicans

Emodin (1,3,8‐trihydroxy‐6‐methyl‐anthraquinone) is a natural secondary plant product, originally isolated from the rhizomes of Rheum palmatum. Many reports show its diuretic, vasorelaxant, antibacterial, antiviral, anti‐ulcerogenic, immunosuppressive, hepatoprotective, anti‐inflammatory and anticancer potential. Emodin is a pleiotropic molecule capable of interacting with several major molecular targets, e.g. NF‐κB, AKT/mTOR and STAT3. The compound can also act as an inhibitor of some protein kinases, with special affinity to protein kinase CK2. The aim of the presented report was to evaluate antifungal properties of emodin and its activity towards CK2 isolated from Candida cells. Our studies revealed that the compound suppressed growth of the cells of reference strains as well as clinical Candida strains, with minimal inhibitory concentration and minimal fungicidal concentration values between 12.5 and 200 μg/mL. Moreover, at a low concentration, the compound was able to effectively stop hyphal formation, thus showing a distinct antivirulent potential. Interestingly, we showed that emodin added to Candida culture inhibited the phosphorylation of many cellular proteins, presumably owing to the inhibition of protein kinase CK2. Notably, the enzyme isolated from the Candida cells was susceptible to emodin with IC50 of 2.8 μg/mL. Indeed, our computational modelling revealed that emodin was able to occupy the ATP‐binding pocket of CK2. Copyright

1,4-Naphthoquinone derivatives potently suppress Candida albicans growth, inhibit formation of hyphae and show no toxicity toward zebrafish embryos

Purpose. In this study, we applied various assays to find new activities of 1,4‐naphthoquinone derivatives for potential anti‐Candida albicans applications. Methodology. These assays determined (a) the antimicrobial effect on growth/cell multiplication in fungal cultures, (b) the effect on formation of hyphae and biofilm, (c) the influence on cell membrane integrity, (d) the effect on cell morphology using atomic force microscopy, and (e) toxicity against zebrafish embryos. We have demonstrated the activity of these compounds against different Candida species and clinical isolates of C. albicans. Key findings. 1,4‐Naphthoquinones significantly affected fungal strains at 8‐250 mg l−1 of MIC. Interestingly, at concentrations below MICs, the chemicals showed effectiveness in inhibition of hyphal formation and cell aggregation in Candida. Of note, atomic force microscopy (AFM) analysis revealed an influence of the compounds on cell morphological properties. However, at low concentrations (0.8‐31.2 mg l−1), it did not exert any evident toxic effects on zebrafish embryos. Conclusions. Our research has evidenced the effectiveness of 1,4‐naphthoquinones as potential anti‐Candida agents.

Biparental inheritance of chloroplasts is controlled by lipid biosynthesis in evening primroses

In most eukaryotes, organellar genomes are transmitted preferentially by the mother, but the reasons underlying this fundamental biological principle are far from being understood. It is believed that biparental inheritance promotes competition between the cytoplasmic organelles and allows the spread of selfish cytoplasmic elements. Those can be, for example, fast replicating chloroplasts (plastids) that are incompatible with the hybrid nuclear genome and therefore maladaptive. Here we show, that plastid competition is a metabolic phenotype determined by extremely rapidly evolving regions in the plastid genome of the evening primrose Oenothera. Polymorphisms in repeats of the regulatory region of accD (the only plastid-encoded subunit of the acetyl-CoA carboxylase, which catalyzes the first step of lipid biosynthesis), are responsible for the differences in competitive behavior of different plastid genotypes. These cytoplasmic drive loci change lipid synthesis and consequently lipid profiles of the plastid envelope membrane. This most likely determines plastid division and/or turn-over rates and hence competitiveness. Since plastid competition can result in uniparental inheritance (through elimination of the “weak” plastid) or biparental inheritance (when two similarly “strong” plastids are transmitted), this work uncovers for the first time a genetic determinant of organelle inheritance.In most eukaryotes, organellar genomes are transmitted preferentially by the mother, but molecular mechanisms and evolutionary forces underlying this fundamental biological principle are far from understood. It is believed that biparental inheritance promotes competition between the cytoplasmic organelles and allows the spread of so-called selfish cytoplasmic elements. Those can be, for example, fast replicating or aggressive chloroplasts (plastids) that are incompatible with the hybrid nuclear genome and therefore maladaptive. Here we show that the ability of plastids to compete against each other is a metabolic phenotype determined by extremely rapidly evolving genes in the plastid genome of the evening primrose Oenothera. Repeats in the regulatory region of accD (the plastid-encoded subunit of the acetyl-CoA carboxylase, which catalyzes the first and rate limiting step of lipid biosynthesis), as well as in ycf2 (a giant reading frame of still unknown function), are responsible for the differences in competitive behavior of plastid genotypes. Polymorphisms in these genes influence lipid synthesis and most likely profiles of the plastid envelope membrane. These in turn determine plastid division and/or turn-over rates and hence competitiveness. This work uncovers cytoplasmic drive loci controlling the outcome of biparental chloroplast transmission. Here, they define the mode of chloroplast inheritance, since plastid competitiveness can result in uniparental inheritance (through elimination of the 9weak9 plastid) or biparental inheritance (when two similarly 9strong9 plastids are transmitted).