Molecular Cross-Talk between Gravity- and Light-Sensing Mechanisms in Euglena gracilis.

Euglena gracilis is a photosynthetic flagellate. To acquire a suitable position in its surrounding aquatic environment, it exploits light and gravity primarily as environmental cues. Several physiological studies have indicated a fine-tuned relationship between gravity sensing (gravitaxis) and light sensing in E. gracilis. However, the underlying molecular mechanism is largely unknown. The photoreceptor photoactivated adenylyl cyclase (PAC) has been studied for over a decade. Nevertheless, no direct/indirect interaction partner (upstream/downstream) has been reported for PAC. It has been shown that a specific protein, kinase A (PKA), showed to be involved in phototaxis and gravitaxis. The current study reports the localization of the specific PKA and its relationship with PAC.

Transcriptome profiling of tobacco (Nicotiana tabacum) pollen and pollen tubes.

BACKGROUND: Pollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development. Cellular and regulatory mechanisms underlying pollen tube elongation are under intense investigation, which stands to greatly benefit from a comprehensive understanding of global gene expression profiles in pollen and pollen tubes. Here, RNA sequencing technology was applied to de novo assemble a Nicotiana tabacum male gametophytic transcriptome and to compare transcriptome profiles at two different stages of gametophyte development: mature pollen grains (MPG) and pollen tubes grown for six hours in vitro (PT6). RESULTS: De novo assembly of data obtained by 454 sequencing of a normalized cDNA library representing tobacco pollen and pollen tube mRNA (pooled mRNA isolated from mature pollen grains [MPG] and from pollen tubes grown in vitro for 3 [PT3] or 6 [PT6] hours) resulted in the identification of 78,364 unigenes. Among these unigenes, which mapped to 24,933 entries in the Sol Genomics Network (SGN) N. tabacum unigene database, 24,672 were predicted to represent full length cDNAs. In addition, quantitative analyses of data obtained by Illumina sequencing of two separate non-normalized MPG and PT6 cDNA libraries showed that 8979 unigenes were differentially expressed (differentially expressed unigenes: DEGs) between these two developmental stages at a FDR q-value of <0.0001. Interestingly, whereas most of these DEGs were downregulated in PT6, the minor fraction of DEGs upregulated in PT6 was enriched for GO (gene ontology) functions in pollen tube growth or fertilization. CONCLUSIONS: A major output of our study is the development of two different high-quality databases representing the tobacco male gametophytic transcriptome and containing encompassing information about global changes in gene expression after pollen germination. Quantitative analyses of these databases 1) indicated that roughly 30% of all tobacco genes are expressed in the male gametophyte, and 2) support previous observations suggesting a global reduction of transcription after pollen germination. Interestingly, a small number of genes, many of which predicted to function in pollen tube growth or fertilization, were found to be upregulated in elongating pollen tubes despite globally reduced transcription.

The Ectocarpus genome and the independent evolution of multicellularity in brown algae.

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae, closely related to the kelps (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic approaches to explore these and other aspects of brown algal biology further.

Computational prediction and experimental validation of microRNAs in the brown alga Ectocarpus siliculosus.

We used an in silico approach to predict microRNAs (miRNAs) genome-wide in the brown alga Ectocarpus siliculosus. As brown algae are phylogenetically distant from both animals and land plants, our approach relied on features shared by all known organisms, excluding sequence conservation, genome localization and pattern of base-pairing with the target. We predicted between 500 and 1500 miRNAs candidates, depending on the values of the energetic parameters used to filter the potential precursors. Using quantitative polymerase chain reaction assays, we confirmed the existence of 22 miRNAs among 72 candidates tested, and of 8 predicted precursors. In addition, we compared the expression of miRNAs and their precursors in two life cycle states (sporophyte, gametophyte) and under salt stress. Several miRNA precursors, Argonaute and DICER messenger RNAs were differentially expressed in these conditions. Finally, we analyzed the gene organization and the target functions of the predicted candidates. This showed that E. siliculosus miRNA genes are, like plant miRNA genes, rarely clustered and, like animal miRNA genes, often located in introns. Among the predicted targets, several widely conserved functional domains are significantly overrepresented, like kinesin, nucleotide-binding/APAF-1, R proteins and CED-4 (NB-ARC) and tetratricopeptide repeats. The combination of computational and experimental approaches thus emphasizes the originality of molecular and cellular processes in brown algae.

ETOILE regulates developmental patterning in the filamentous brown alga Ectocarpus siliculosus.

Brown algae are multicellular marine organisms evolutionarily distant from both metazoans and land plants. The molecular or cellular mechanisms that govern the developmental patterning in brown algae are poorly characterized. Here, we report the first morphogenetic mutant, étoile (etl), produced in the brown algal model Ectocarpus siliculosus. Genetic, cellular, and morphometric analyses showed that a single recessive locus, ETL, regulates cell differentiation: etl cells display thickening of the extracellular matrix (ECM), and the elongated, apical, and actively dividing E cells are underrepresented. As a result of this defect, the overrepresentation of round, branch-initiating R cells in the etl mutant leads to the rapid induction of the branching process at the expense of the uniaxial growth in the primary filament. Computational modeling allowed the simulation of the etl mutant phenotype by including a modified response to the neighborhood information in the division rules used to specify wild-type development. Microarray experiments supported the hypothesis of a defect in cell-cell communication, as primarily Lin-Notch-domain transmembrane proteins, which share similarities with metazoan Notch proteins involved in binary cell differentiation were repressed in etl. Thus, our study highlights the role of the ECM and of novel transmembrane proteins in cell-cell communication during the establishment of the developmental pattern in this brown alga.

Physcomitrium patens PpRIC, an ancestral CRIB-domain ROP effector, inhibits auxin-induced differentiation of apical initial cells.

RHO guanosine triphosphatases are important eukaryotic regulators of cell differentiation and behavior. Plant ROP (RHO of plant) family members activate specific, incompletely characterized downstream signaling. The structurally simple land plant Physcomitrium patens is missing homologs of key animal and flowering plant RHO effectors but contains a single CRIB (CDC42/RAC interactive binding)-domain-containing RIC (ROP-interacting CRIB-containing) protein (PpRIC). Protonemal P. patens filaments elongate based on regular division and PpROP-dependent tip growth of apical initial cells, which upon stimulation by the hormone auxin differentiate caulonemal characteristics. PpRIC interacts with active PpROP1, co-localizes with this protein at the plasma membrane at the tip of apical initial cells, and accumulates in the nucleus. Remarkably, PpRIC is not required for tip growth but is targeted to the nucleus to block caulonema differentiation downstream of auxin-controlled gene expression. These observations establish functions of PpRIC in mediating crosstalk between ROP and auxin signaling, which contributes to the maintenance of apical initial cell identity.

Auxin metabolism and function in the multicellular brown alga Ectocarpus siliculosus.

Ectocarpus siliculosus is a small brown alga that has recently been developed as a genetic model. Its thallus is filamentous, initially organized as a main primary filament composed of elongated cells and round cells, from which branches differentiate. Modeling of its early development suggests the involvement of very local positional information mediated by cell-cell recognition. However, this model also indicates that an additional mechanism is required to ensure proper organization of the branching pattern. In this paper, we show that auxin indole-3-acetic acid (IAA) is detectable in mature E. siliculosus organisms and that it is present mainly at the apices of the filaments in the early stages of development. An in silico survey of auxin biosynthesis, conjugation, response, and transport genes showed that mainly IAA biosynthesis genes from land plants have homologs in the E. siliculosus genome. In addition, application of exogenous auxins and 2,3,5-triiodobenzoic acid had different effects depending on the developmental stage of the organism, and we propose a model in which auxin is involved in the negative control of progression in the developmental program. Furthermore, we identified an auxin-inducible gene called EsGRP1 from a small-scale microarray experiment and showed that its expression in a series of morphogenetic mutants was positively correlated with both their elongated-to-round cell ratio and their progression in the developmental program. Altogether, these data suggest that IAA is used by the brown alga Ectocarpus to relay cell-cell positional information and induces a signaling pathway different from that known in land plants.

Analysis of the Localization of Fluorescent PpROP1 and PpROP-GEF4 Fusion Proteins in Moss Protonemata Based on Genomic "Knock-In" and Estradiol-Titratable Expression.

Tip growth of pollen tubes, root hairs, and apical cells of moss protonemata is controlled by ROP (Rho of plants) GTPases, which were shown to accumulate at the apical plasma membrane of these cells. However, most ROP localization patterns reported in the literature are based on fluorescent protein tagging and need to be interpreted with caution, as ROP fusion proteins were generally overexpressed at undefined levels, in many cases without assessing effects on tip growth. ROP-GEFs, important regulators of ROP activity, were also described to accumulate at the apical plasma membrane during tip growth. However, to date only the localization of fluorescent ROP-GEF fusion proteins strongly overexpressed using highly active promoters have been investigated. Here, the intracellular distributions of fluorescent PpROP1 and PpROP-GEF4 fusion proteins expressed at essentially endogenous levels in apical cells of Physcomitrella patens "knock-in" protonemata were analyzed. Whereas PpROP-GEF4 was found to associate with a small apical plasma membrane domain, PpROP1 expression was below the detection limit. Estradiol-titratable expression of a fluorescent PpROP1 fusion protein at the lowest detectable level, at which plant development was only marginally affected, was therefore employed to show that PpROP1 also accumulates at the apical plasma membrane, although within a substantially larger domain. Interestingly, RNA-Seq data indicated that the majority of all genes active in protonemata are expressed at lower levels than PpROP1, suggesting that estradiol-titratable expression may represent an important alternative to "knock-in" based analysis of the intracellular distribution of fluorescent fusion proteins in protonemal cells.

Alginates along the filament of the brown alga Ectocarpus help cells cope with stress.

Ectocarpus is a filamentous brown alga, which cell wall is composed mainly of alginates and fucans (80%), two non-crystalline polysaccharide classes. Alginates are linear chains of epimers of 1,4-linked uronic acids, ß-D-mannuronic acid (M) and α-L-guluronic acid (G). Previous physico-chemical studies showed that G-rich alginate gels are stiffer than M-rich alginate gels when prepared in vitro with calcium. In order to assess the possible role of alginates in Ectocarpus, we first immunolocalised M-rich or G-rich alginates using specific monoclonal antibodies along the filament. As a second step, we calculated the tensile stress experienced by the cell wall along the filament, and varied it with hypertonic or hypotonic solutions. As a third step, we measured the stiffness of the cell along the filament, using cell deformation measurements and atomic force microscopy. Overlapping of the three sets of data allowed to show that alginates co-localise with the stiffest and most stressed areas of the filament, namely the dome of the apical cell and the shanks of the central round cells. In addition, no major distinction between M-rich and G-rich alginate spatial patterns could be observed. Altogether, these results support that both M-rich and G-rich alginates play similar roles in stiffening the cell wall where the tensile stress is high and exposes cells to bursting, and that these roles are independent from cell growth and differentiation.

Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus.

BACKGROUND: Brown algae are plant multi-cellular organisms occupying most of the world coasts and are essential actors in the constitution of ecological niches at the shoreline. Ectocarpus siliculosus is an emerging model for brown algal research. Its genome has been sequenced, and several tools are being developed to perform analyses at different levels of cell organization, including transcriptomic expression analyses. Several topics, including physiological responses to osmotic stress and to exposure to contaminants and solvents are being studied in order to better understand the adaptive capacity of brown algae to pollution and environmental changes. A series of genes that can be used to normalise expression analyses is required for these studies. RESULTS: We monitored the expression of 13 genes under 21 different culture conditions. These included genes encoding proteins and factors involved in protein translation (ribosomal protein 26S, EF1alpha, IF2A, IF4E) and protein degradation (ubiquitin, ubiquitin conjugating enzyme) or folding (cyclophilin), and proteins involved in both the structure of the cytoskeleton (tubulin alpha, actin, actin-related proteins) and its trafficking function (dynein), as well as a protein implicated in carbon metabolism (glucose 6-phosphate dehydrogenase). The stability of their expression level was assessed using the Ct range, and by applying both the geNorm and the Normfinder principles of calculation. CONCLUSION: Comparisons of the data obtained with the three methods of calculation indicated that EF1alpha (EF1a) was the best reference gene for normalisation. The normalisation factor should be calculated with at least two genes, alpha tubulin, ubiquitin-conjugating enzyme or actin-related proteins being good partners of EF1a. Our results exclude actin as a good normalisation gene, and, in this, are in agreement with previous studies in other organisms.

Identification of a flagellar protein implicated in the gravitaxis in the flagellate Euglena gracilis.

Flagellated cells are of great evolutionary importance across animal and plant species. Unlike higher plants, flagellated cells are involved in reproduction of macro-algae as well as in early diverging land plants. Euglena gracilis is an emerging flagellated model organism. The current study reports that a specific calmodulin (CaM2) involved in gravitaxis of E. gracilis interacts with an evolutionary conserved flagellar protein, EgPCDUF4201. The subsequent molecular analysis showed clearly that EgPCDUF4201 is also involved in gravitaxis. We performed subcellular localization of CaM2 using immunoblotting and indirect immunofluorescence. By employing yeast two-hybrid screen, EgPCDUF4201 was identified as an interaction partner of CaM2. The C-terminus of EgPCDUF4201 is responsible for the interaction with CaM2. Silencing of N- and C-terminus of EgPCDUF4201 using RNAi resulted in an impaired gravitaxis. Moreover, indirect immunofluorescence assay showed that EgPCDUF4201 is a flagella associated protein. The current study specifically addressed some important questions regarding the signal transduction chain of gravitaxis in E. gracilis. Besides the fact that it improved the current understanding of gravity sensing mechanisms in E. gracilis, it also gave rise to several interesting research questions regarding the function of the domain of unknown function 4201 in flagellated cells.

Culture methods and mutant generation in the filamentous brown algae Ectocarpus siliculosus.

Ectocarpus siliculosus is a small filamentous alga that has recently emerged as the new model for fundamental research on brown algae. Here, we describe the basic culture protocols for propagating and collecting E. siliculosus material that can then be used in all types of molecular biology, biochemistry and cell biology techniques. In addition, procedures for carrying out genetic experiments (generation of mutants and genetic segregation analyses) on E. siliculosus are described.

Evaluation of reference genes for RT qPCR analyses of structure-specific and hormone regulated gene expression in Physcomitrella patens gametophytes.

The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions.

Plant Proteus: brown algal morphological plasticity and underlying developmental mechanisms.

Brown algae are multicellular photosynthetic marine organisms, ubiquitous on rocky intertidal shores at cold and temperate latitudes. Nevertheless, little is known about many aspects of their biology, particularly their development. Given their phylogenetic distance (1.6 billion years) from other plant organisms (land plants, and green and red algae), brown algae harbor a high, as-yet undiscovered diversity of biological mechanisms governing their development. They also show great morphological plasticity, responding to specific environmental constraints, such as sea currents, reduced light availability, grazer attacks, desiccation and UV exposure. Here, we show that brown algal morphogenesis is rather simple and flexible, and review recent genomic data on the cellular and molecular mechanisms known to date that can possibly account for this developmental strategy.

Space-time decoupling in the branching process in the mutant étoile of the filamentous brown alga Ectocarpus siliculosus.

Ectocarpus siliculosus is being developed as a model organism for brown algal genetics and genomics. Brown algae are phylogenetically distant from the other multicellular phyla (green lineage, red algae, fungi and metazoan) and therefore might offer the opportunity to study novel and alternative developmental processes that lead to the establishment of multicellularity. E. siliculosus develops as uniseriate filaments, thereby displaying one of the simplest architectures among multicellular organisms. The young sporophyte grows as a primary filament and then branching occurs, preferentially at the center of the filament. We recently described the first morphogenetic mutant étoile (etl) in a brown alga, produced by UVB mutagenesis in E. siliculosus. We showed that a single recessive mutation was responsible for a defect in both cell differentiation and the very early branching pattern (first and second branch emergences). Here, we supplement this study by reporting the branching defects observed subsequently, i.e. for the later stages corresponding to the emergence of up to the first six secondary filaments, and we show that the branching process is composed of at least two distinct components: time and position.

A stochastic 1D nearest-neighbour automaton models early development of the brown alga Ectocarpus siliculosus.

Early development of the filamentous brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye involves two cell types that are arranged in a polymorphic, but constrained, pattern. The present study aimed to decipher the cellular processes responsible for the establishment of this pattern. Thorough observations characterised five different events of division and differentiation that occurred during the early development. The hypothesis that a local control is responsible for these processes was tested. To do so, Ectomat, a stochastic automaton in which each cell only interacts with its closest neighbour(s), was created. The probabilities for the five events were adjusted to fit to the observations. Simulations with Ectomat reconstructed most of the essential properties of the sporophyte development, in terms of cell-type proportion, relative position and growth dynamics. The whole organism properties emerged by applying local transition rules. In conclusion, no global position information system was required at this development stage. Randomly occurring cell events, driven by simple contact interactions, are sufficient to account for the early filament development and establishment of the cell-type pattern of E. siliculosus.

EARLY DEVELOPMENT PATTERN OF THE BROWN ALGA ECTOCARPUS SILICULOSUS (ECTOCARPALES, PHAEOPHYCEAE) SPOROPHYTE(1).

The distant phylogenetic position of brown macroalgae from the other multicellular phyla offers the opportunity to study novel and alternative developmental processes involved in the establishment of multicellularity. At present, however, very little information is available about developmental patterning in this group. Ectocarpus siliculosus (Dillwyn) Lyngb. has uniseriate filaments and displays one of the simplest architectures in the Phaeophyceae. The aim of this study was to decipher the morphogenetic steps that lead to the development of the Ectocarpus sporophyte. We carried out a detailed morphometric study of the events that occurred between gamete germination and the 100-cell stage. This analysis was performed on two ecologically distant isolates to assess plasticity in developmental patterning within this species. Cell sizes were measured in both isolates, allowing the definition of two main cell types based on their shape (round and elongated). On average, the filament is composed of about 40% round cells, which are present in the central region of the filament, but different combinations of the two cell types within filaments were observed and quantified. Young sporophytes grew apically, with elongated cells progressively differentiating into round cells. Secondary filaments emerged preferentially on round cells, primarily from the older central cells. Statistical analyses showed that the pattern of branching was regulated to ensure a stereotyped architecture. This description of the developmental patterning during the growth of the E. siliculosus sporophyte will serve as a base for more detailed studies of development, in this species and in brown algae in general.

Development and physiology of the brown alga Ectocarpus siliculosus: two centuries of research.

Brown algae share several important features with land plants, such as their photoautotrophic nature and their cellulose-containing wall, but the two groups are distantly related from an evolutionary point of view. The heterokont phylum, to which the brown algae belong, is a eukaryotic crown group that is phylogenetically distinct not only from the green lineage, but also from the red algae and the opisthokont phylum (fungi and animals). As a result of this independent evolutionary history, the brown algae exhibit many novel features and, moreover, have evolved complex multicellular development independently of the other major groups already mentioned. In 2004, a consortium of laboratories, including the Station Biologique in Roscoff and Genoscope, initiated a project to sequence the genome of Ectocarpus siliculosus, a small filamentous brown alga that is found in temperate, coastal environments throughout the globe. The E. siliculosus genome, which is currently being annotated, is expected to be the first completely characterized genome of a multicellular alga. In this review we look back over two centuries of work on this brown alga and highlight the advances that have led to the choice of E. siliculosus as a genomic and genetic model organism for the brown algae.

[A Picasso among brown algae: the arduous conquest of symmetry by Ectocarpus]. / Un Picasso parmi les algues brunes: la difficile conquête de la symétrie chez Ectocarpus.

In response to environmental constraints, living organisms organise their body according to axes, rotation and translation plans, or asymmetries. Cellular and molecular processes are involved in the establishment of this architecture. Hence, this review aims at presenting the molecular mechanisms controlling the main symmetries and axes in plants. Several genes, coding for transcription factors, have been identified in land plants (mainly Arabidopsis thaliana), as controlling the establishment of apico-basal and adaxial-abaxial axes mainly. The establishment of these axes allows the development in other spatial directions of radial or bilateral symmetries. These processes seem in most cases to be under the control of the phytohormone auxin. In brown algae, which are all multicellular marine plants, polarity plans are less obvious than in land plants. The development of the model brown alga Ectocarpus siliculosus is currently being studied. E. siliculosus develops a filamentous architecture, and primary observations show that branching along the main axis occurs in a non-stereotyped and regular way, even though it is mainly centred. However, more detailed morphometrical studies, accompanied by probabilistic analyses, have shown that, among the overall population of individuals, organisms obey yet unidentified biological constraints, that aim at refining the radial symmetry as the organism grows. The role of this symmetry in the adaptation of E. siliculosus to its environment, as well as the molecular actors involved in this process, are currently under study in our laboratory.