Cryptic Haploid Stages in the Life Cycle of Leathesia marina (Chordariaceae, Phaeophyceae) Under In Vitro Culture

We evaluated the life cycle of Leathesia marina through molecular analyses, culture studies, morphological observations, and ploidy measurements. Macroscopic sporophytes were collected from two localities in Atlantic Patagonia and were cultured under long-day (LD) and short-day (SD) conditions. Molecular identification of the microscopic and macroscopic phases was performed through the cox3 and rbcL genes and the phylogeny was assessed on the basis of single gene and concatenated datasets. Nuclear ploidy of each phase was estimated from the DNA contents of individual nuclei through epifluorescence microscopy and flow cytometry. Molecular results confirmed the identity of the Argentinian specimens as L. marina and revealed their conspecificity with L. marina from New Zealand, Germany, and Japan. The sporophytic macrothalli (2n) released mitospores from plurilocular sporangia, which developed into globular microthalli (2n), morphologically similar to the sporophytes but not in size, constituting a generation of small diploid thalli, with a mean fluorescent nuclei cross-sectional area of 3.21 ± 0.7 μm2. The unilocular sporangia released meiospores that developed two morphologically different types of microthalli: erect branched microthalli (n) with a nuclear area of 1.48 ± 0.07 µm2 that reproduces asexually, and prostrate branched microthalli (n) with a nuclear area of 1.24 ± 0.10 µm2 that reproduces sexually. The prostrate microthalli released gametes in LD conditions, which merged and produced macroscopic thalli with a nuclear cross-sectional area of 3.45 ± 0.09 µm2. Flow cytometry confirmed that the erect and prostrate microthalli were haploid and that the globular microthalli and macrothalli were diploid.Fil: Poza, Ailen Melisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; ArgentinaFil: Santiañez, Wilfred John E.. Hokkaido University; Japón. University of the Philippines Diliman; FilipinasFil: Croce, Maria Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Gauna, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Kogame, Kazuhiro. Hokkaido University; JapónFil: Parodi, Elisa Rosalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentin

Evolution of Life-Cycle-Related Developmental Processes in the Brown Algae

The molecular processes that regulate life cycle progression provide an essential foundation for the correct initiation of diverse biological processes, including multicellular development and sexual reproduction1. The life cycle of the model brown alga Ectocarpus2–4 involves an alternation between two independent multicellular organisms, the sporophyte and the gametophyte. We have shown that the identities of the two generations are not determined by ploidy, but rather are determined genetically. Mutations affecting either the OUROBOROS (ORO) or the SAMASARA (SAM) locus result in complete conversion of the sporophyte generation into a gametophyte5. Both ORO and SAM encode three amino acid loop extension homeodomain transcription factors (TALE HD TFs). Similarities between ORO/SAM and HD-TF-based life cycle regulation systems in other eukaryotic supergroups indicate not only that these systems have an extremely ancient origin but also that TALE HD TFs have been independently recruited to regulate sporophyte developmental programs in at least two different complex multicellular eukaryotic supergroups, Archaeplastida and Chromalveolata. The presentation will discuss genetic regulation of life cycle progression and will also look at some of the genetic and epigenetic events that occur downstream of the switch to either the sporophyte or gametophyte developmental programs

An Efficient Chromatin Immunoprecipitation Protocol for the Analysis of Histone Modification Distributions in the Brown Alga Ectocarpus

The brown algae are an important but understudied group of multicellular marine organisms. A number of genetic and genomic tools have been developed for the model brown alga Ectocarpus; this includes, most recently, chromatin immunoprecipitation methodology, which allows genome-wide detection and analysis of histone post-translational modifications. Post-translational modifications of histone molecules have been shown to play an important role in gene regulation in organisms from other major eukaryotic lineages, and this methodology will therefore be a very useful tool to investigate genome function in the brown algae. This article provides a detailed, step-by-step description of the Ectocarpus ChIP protocol, which effectively addresses the difficult problem of efficiently extracting chromatin from cells protected by a highly resistant cell wall. The protocol described here will be an essential tool for the future application of chromatin analysis methodologies in brown algal research

Histone modifications during the life cycle of the brown alga Ectocarpus

Background: Brown algae evolved complex multicellularity independently of the animal and land plant lineages and are the third most developmentally complex phylogenetic group on the planet. An understanding of developmental processes in this group is expected to provide important insights into the evolutionary events necessary for the emergence of complex multicellularity. Here, we focus on mechanisms of epigenetic regulation involving post-translational modifications of histone proteins. Results: A total of 47 histone post-translational modifications are identified, including a novel mark H2AZR38me1, but Ectocarpus lacks both H3K27me3 and the major polycomb complexes. ChIP-seq identifies modifications associated with transcription start sites and gene bodies of active genes and with transposons. H3K79me2 exhibits an unusual pattern, often marking large genomic regions spanning several genes. Transcription start sites of closely spaced, divergently transcribed gene pairs share a common nucleosome-depleted region and exhibit shared histone modification peaks. Overall, patterns of histone modifications are stable through the life cycle. Analysis of histone modifications at generation-biased genes identifies a correlation between the presence of specific chromatin marks and the level of gene expression. Conclusions: The overview of histone post-translational modifications in the brown alga presented here will provide a foundation for future studies aimed at understanding the role of chromatin modifications in the regulation of brown algal genomes

Chromatin dynamics associated with sexual differentiation in a UV sex determination system

In many eukaryotes, such as dioicous mosses and many algae, sex is determined by UV sex chromosomes and is expressed during the haploid phase of the life cycle. In these species, the male and female developmental programs are initiated by the presence of the U- or V-specific regions of the sex chromosomes but, as in XY and ZW systems, phenotypic differentiation is largely driven by autosomal sex-biased gene expression. The mechanisms underlying sex-biased transcription in XY, ZW or UV sexual systems currently remain elusive. Here, we set out to understand the extent and nature of epigenomic changes associated with sexual differentiation in the brown alga Ectocarpus, which has a well described UV system. Five histone modifications, H3K4me3, H3K27Ac, H3K9Ac, H3K36me3, H4K20me3, were quantified in near-isogenic male and female lines, leading to the identification of 13 different chromatin states across the Ectocarpus genome that showed different patterns of enrichment at transcribed, silent, housekeeping or narrowly-expressed genes. Chromatin states were strongly correlated with levels of gene expression indicating a relationship between the assayed marks and gene transcription. The relative proportion of each chromatin state across the genome remained stable in males and females, but a subset of genes exhibited different chromatin states in the two sexes. In particular, males and females displayed distinct patterns of histone modifications at sex-biased genes, indicating that chromatin state transitions occur preferentially at genes involved in sex-specific pathways. Finally, our results reveal a unique chromatin landscape of the U and V sex chromosomes compared to autosomes. Taken together, our observations reveal a role for histone modifications in sex determination and sexual differentiation in a UV sexual system, and suggest that the mechanisms of epigenetic regulation of genes on the UV sex chromosomes may differ from those operating on autosomal genes