3D cellular morphometrics of ovule primordium development in Zea mays reveal differential division and growth dynamics specifying megaspore mother cell singleness.

Introduction: Differentiation of spore mother cells marks the somatic-to-reproductive transition in higher plants. Spore mother cells are critical for fitness because they differentiate into gametes, leading to fertilization and seed formation. The female spore mother cell is called the megaspore mother cell (MMC) and is specified in the ovule primordium. The number of MMCs varies by species and genetic background, but in most cases, only a single mature MMC enters meiosis to form the embryo sac. Multiple candidate MMC precursor cells have been identified in both rice and Arabidopsis, so variability in MMC number is likely due to conserved early morphogenetic events. In Arabidopsis, the restriction of a single MMC per ovule, or MMC singleness, is determined by ovule geometry. To look for potential conservation of MMC ontogeny and specification mechanisms, we undertook a morphogenetic description of ovule primordium growth at cellular resolution in the model crop maize. Methods: We generated a collection of 48 three-dimensional (3D) ovule primordium images for five developmental stages, annotated for 11 cell types. Quantitative analysis of ovule and cell morphological descriptors allowed the reconstruction of a plausible developmental trajectory of the MMC and its neighbors. Results: The MMC is specified within a niche of enlarged, homogenous L2 cells, forming a pool of candidate archesporial (MMC progenitor) cells. A prevalent periclinal division of the uppermost central archesporial cell formed the apical MMC and the underlying cell, a presumptive stack cell. The MMC stopped dividing and expanded, acquiring an anisotropic, trapezoidal shape. By contrast, periclinal divisions continued in L2 neighbor cells, resulting in a single central MMC. Discussion: We propose a model where anisotropic ovule growth in maize drives L2 divisions and MMC elongation, coupling ovule geometry with MMC fate.

Environmental and Genetic Factors Affecting Apospory Expressivity in Diploid Paspalum rufum.

In angiosperms, gametophytic apomixis (clonal reproduction through seeds) is strongly associated with polyploidy and hybridization. The trait is facultative and its expressivity is highly variable between genotypes. Here, we used an F1 progeny derived from diploid apomictic (aposporic) genotypes of Paspalum rufum and two F2 families, derived from F1 hybrids with different apospory expressivity (%AES), to analyze the influence of the environment and the transgenerational transmission of the trait. In addition, AFLP markers were developed in the F1 population to identify genomic regions associated with the %AES. Cytoembryological analyses showed that the %AES was significantly influenced by different environments, but remained stable across the years. F1 and F2 progenies showed a wide range of %AES variation, but most hybrids were not significantly different from the parental genotypes. Maternal and paternal genetic linkage maps were built covering the ten expected linkage groups (LG). A single-marker analysis detected at least one region of 5.7 cM on LG3 that was significantly associated with apospory expressivity. Our results underline the importance of environmental influence in modulating apospory expressivity and identified a genomic region associated with apospory expressivity at the diploid level.

Differential Epigenetic Marks Are Associated with Apospory Expressivity in Diploid Hybrids of Paspalum rufum.

Apomixis seems to emerge from the deregulation of preexisting genes involved in sexuality by genetic and/or epigenetic mechanisms. The trait is associated with polyploidy, but diploid individuals of Paspalum rufum can form aposporous embryo sacs and develop clonal seeds. Moreover, diploid hybrid families presented a wide apospory expressivity variation. To locate methylation changes associated with apomixis expressivity, we compare relative DNA methylation levels, at CG, CHG, and CHH contexts, between full-sib P. rufum diploid genotypes presenting differential apospory expressivity. The survey was performed using a methylation content-sensitive enzyme ddRAD (MCSeEd) strategy on samples at premeiosis/meiosis and postmeiosis stages. Based on the relative methylation level, principal component analysis and heatmaps, clearly discriminate samples with contrasting apospory expressivity. Differential methylated contigs (DMCs) showed 14% of homology to known transcripts of Paspalum notatum reproductive transcriptome, and almost half of them were also differentially expressed between apomictic and sexual samples. DMCs showed homologies to genes involved in flower growth, development, and apomixis. Moreover, a high proportion of DMCs aligned on genomic regions associated with apomixis in Setaria italica. Several stage-specific differential methylated sequences were identified as associated with apospory expressivity, which could guide future functional gene characterization in relation to apomixis success at diploid and tetraploid levels.

Heterochronic reproductive developmental processes between diploid and tetraploid cytotypes of Paspalum rufum.

BACKGROUND AND AIMS: Apomixis is an asexual reproductive mode via seeds that generate maternal clonal progenies. Although apomixis in grasses is mainly expressed at the polyploid level, some natural diploid genotypes of Paspalum rufum produce aposporous embryo sacs in relatively high proportions and are even able to complete apomixis under specific conditions. However, despite the potential for apomixis, sexuality prevails in diploids, and apomixis expression is repressed for an as yet undetermind reason. Apomixis is thought to derive from a deregulation of one or a few components of the sexual pathway that could be triggered by polyploidy and/or hybridization. The objectives of this work were to characterize and compare the reproductive development and the timing of apospory initial (AI) emergence between diploid genotypes with potential for apomixis and facultative apomictic tetraploid cytotypes of P. rufum. METHODS: Reproductive characterization was performed by cytoembryological observations of cleared ovaries and anthers during all reproductive development steps and by quantitative evaluation of the ovule growth parameters. KEY RESULTS: Cytoembryological observations showed that in diploids, both female and male reproductive development is equally synchronized, but in tetraploids, megasporogenesis and early megagametogenesis are delayed with respect to microsporogenesis and early microgametogenesis. This delay was also seen when ovary growth was taken as a reference parameter. The analysis of the onset of AIs revealed that they emerge during different developmental periods depending on the ploidy level. In diploids, the AIs appeared along with the tetrad (or triad) of female meiocytes, but in tetraploids they appeared earlier, at the time of the megaspore mother cell. In both cytotypes, AIs can be seen even during megagametogenesis. CONCLUSIONS: Overall observations reveal that female sexual reproductive development is delayed in tetraploids as compared with diploid genotypes, mainly at meiosis. In tetraploids, AIs appear at earlier sexual developmental stages than in diploids, and they accumulate up to the end of megasporogenesis. The longer extension of megasporogenesis in tetraploids could favour AI emergence and also apomixis success.

A reference floral transcriptome of sexual and apomictic Paspalum notatum.

BACKGROUND: Paspalum notatum Flügge is a subtropical grass native to South America, which includes sexual diploid and apomictic polyploid biotypes. In the past decade, a number of apomixis-associated genes were discovered in this species through genetic mapping and differential expression surveys. However, the scarce information on Paspalum sequences available in public databanks limited annotations and functional predictions for these candidates. RESULTS: We used a long-read 454/Roche FLX+ sequencing strategy to produce robust reference transcriptome datasets from florets of sexual and apomictic Paspalum notatum genotypes and delivered a list of transcripts showing differential representation in both reproductive types. Raw data originated from floral samples collected from premeiosis to anthesis was assembled in three libraries: i) sexual (SEX), ii) apomictic (APO) and iii) global (SEX + APO). A group of physically-supported Paspalum mRNA and EST sequences matched with high level of confidence to both sexual and apomictic libraries. A preliminary trial allowed discovery of the whole set of putative alleles/paralogs corresponding to 23 previously identified apomixis-associated candidate genes. Moreover, a list of 3,732 transcripts and several co-expression and protein -protein interaction networks associated with apomixis were identified. CONCLUSIONS: The use of the 454/Roche FLX+ transcriptome database will allow the detailed characterization of floral alleles/paralogs of apomixis candidate genes identified in prior and future work. Moreover, it was used to reveal additional candidate genes differentially represented in apomictic and sexual flowers. Gene ontology (GO) analyses of this set of transcripts indicated that the main molecular pathways altered in the apomictic genotype correspond to specific biological processes, like biotic and abiotic stress responses, growth, development, cell death and senescence. This data collection will be of interest to the plant reproduction research community and, particularly, to Paspalum breeding projects.

Analysis of variation for apomictic reproduction in diploid Paspalum rufum.

BACKGROUND AND AIMS: The diploid cytotype of Paspalum rufum (Poaceae) reproduces sexually and is self-sterile; however, recurrent autopolyploidization through 2n + n fertilization and the ability for reproduction via apomixis have been documented in one genotype of the species. The objectives of this work were to analyse the variation in the functionality of apomixis components in diploid genotypes of P. rufum and to identify individuals with contrasting reproductive behaviours. METHODS: Samples of five individuals from each of three natural populations of P. rufum (designated R2, R5 and R6) were used. Seeds were obtained after open pollination, selfing, conspecific interploidy crosses and interspecific interploidy self-pollination induction. The reproductive behaviour of each plant was determined by using the flow cytometric seed screen (FCSS) method. Embryo sacs were cleared using a series of ethanol and methyl salicylate solutions and observed microscopically. KEY RESULTS: In open pollination, all genotypes formed seeds by sexual means and no evidence of apomeiotic reproduction was detected. However, in conspecific interploidy crosses and interspecific interploidy self-pollination induction, variations in the reproductive pathways were observed. While all plants from populations R2 and R6 formed seeds exclusively by sexual means, three genotypes from the R5 population developed seeds from both meiotic and aposporous embryo sacs, and one of them (R5#49) through the complete apomictic pathway (apospory + parthenogenesis + pseudogamy). Cytoembryological observations revealed the presence of both meiotic and aposporous embryo sacs in all the genotypes analysed, suggesting that parthenogenesis could be uncoupled from apospory in some genotypes. CONCLUSIONS: The results presented demonstrate the existence of variation in the functionality of apomixis components in natural diploid genotypes of P. rufum and have identified individuals with contrasting reproductive behaviours. Genotypes identified here can be crossed to generate segregating populations in order to study apomixis determinants at the diploid level. Moreover, analysis of their expression patterns, quantification of their transcript levels and an understanding of their regulation mechanisms could help to design new strategies for recreating apomixis in a diploid genome environment.

Characterization and expression analysis of SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes in sexual and apomictic Paspalum notatum.

The SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene plays a fundamental role in somatic embryogenesis of angiosperms, and is associated with apomixis in Poa pratensis. The objective of this work was to isolate, characterize and analyze the expression patterns of SERK genes in apomictic and sexual genotypes of Paspalum notatum. A conserved 200-bp gene fragment was amplified from genomic DNA with heterologous primers, and used to initiate a chromosomal walking strategy for cloning the complete sequence. This procedure allowed the isolation of two members of the P. notatum SERK family; PnSERK1, which is similar to PpSERK1, and PnSERK2, which is similar to ZmSERK2 and AtSERK1. Phylogenetic analyses indicated that PnSERK1 and PnSERK2 represent paralogous sequences. Southern-blot hybridization indicated the presence of at least three copies of SERK genes in the species. qRT-PCR analyses revealed that PnSERK2 was expressed at significantly higher levels than PnSERK1 in roots, leaves, reproductive tissues and embryogenic calli. Moreover, in situ hybridization experiments revealed that PnSERK2 displayed a spatially and chronologically altered expression pattern in reproductive organs of the apomictic genotype with respect to the sexual one. PnSERK2 is expressed in nucellar cells of the apomictic genotype at meiosis, but only in the megaspore mother cell in the sexual genotype. Therefore, apomixis onset in P. notatum seems to be correlated with the expression of PnSERK2 in nucellar tissue.

Perfil epidemiológico do adoecimento dos profissionais de enfermagem de um hospital universitário / The epidemiological profile of a university hospital workers´ health

Com o objetivo de identificar as causas que motivaram o afastamento do trabalho de profissionais de enfermagem do Hospital Universtário do Oeste do Paraná, realizou-se um levantamento bibliográfico para conhecer a estrutura hospitalar, o processo de trabalho da enfermagem e os riscos aos quais os trabalhadores de enfermagem estão submetidos, seguidos de pesquisa documental nos atestados médicos desses profissionais. Os dados foram analisados quantitativamente e os resultados evidenciaram que 68 por cento dos profissionais ausentaram-se do trabalho, sendo que os setores que obtiveram maior índice de ausência foram: UTI Geral e UTI Neonatal. Assim, acredita-se que a rotina estressante do ambiente hospitalar, bem como as condições de trabalho às quais esses trabalhadores estão expostos, contribuem para o adoecimento.