Functional analysis and comparative genomics of expressed sequence tags from the lycophyte Selaginella moellendorffii

BACKGROUND: The lycophyte Selaginella moellendorffii is a member of one of the oldest lineages of vascular plants on Earth. Fossil records show that the lycophyte clade arose 400 million years ago, 150–200 million years earlier than angiosperms, a group of plants that includes the well-studied flowering plant Arabidopsis thaliana. S. moellendorffii has a genome size of approximately 100 Mbp, as small or smaller than that of A. thaliana. S. moellendorffii has the potential to provide significant comparative information to better understand the evolution of vascular plants. RESULTS: We sequenced 2181 Expressed Sequence Tags (ESTs) from a S. moellendorffii cDNA library. One thousand three hundred and one non-redundant sequences were assembled, containing 291 contigs and 1010 singletons. Approximately 75% of the ESTs matched proteins in the non-redundant protein database. Among 1301 clusters, 343 were categorized according to Gene Ontology (GO) hierarchy and were compared to the GO mapping of A. thaliana tentative consensus sequences. We compared S. moellendorffii ESTs to the A. thaliana and Physcomitrella patens EST databases, using the tBLASTX algorithm. Approximately 60% of the ESTs exhibited similarity with both A. thaliana and P. patens ESTs; whereas, 13% and 1% of the ESTs had exclusive similarity with A. thaliana and P. patens ESTs, respectively. A substantial proportion of the ESTs (26%) had no match with A. thaliana or P. patens ESTs. CONCLUSION: We discovered 1301 putative unigenes in S. moellendorffii. These results give an initial insight into its transcriptome that will aid in the study of the S. moellendorffii genome in the near future

A systemic gene silencing method suitable for high throughput, reverse genetic analyses of gene function in fern gametophytes

BACKGROUND: Ceratopteris richardii is a useful experimental system for studying gametophyte development and sexual reproduction in plants. However, few tools for cloning mutant genes or disrupting gene function exist for this species. The feasibility of systemic gene silencing as a reverse genetics tool was examined in this study. RESULTS: Several DNA constructs targeting a Ceratopteris protoporphyrin IX magnesium chelatase (CrChlI) gene that is required for chlorophyll biosynthesis were each introduced into young gametophytes by biolistic delivery. Their transient expression in individual cells resulted in a colorless cell phenotype that affected most cells of the mature gametophyte, including the meristem and gametangia. The colorless phenotype was associated with a 7-fold decrease in the abundance of the endogenous transcript. While a construct designed to promote the transient expression of a CrChlI double stranded, potentially hairpin-forming RNA was found to be the most efficient in systemically silencing the endogenous gene, a plasmid containing the CrChlI cDNA insert alone was sufficient to induce silencing. Bombarded, colorless hermaphroditic gametophytes produced colorless embryos following self-fertilization, demonstrating that the silencing signal could be transmitted through gametogenesis and fertilization. Bombardment of young gametophytes with constructs targeting the Ceratopteris filamentous temperature sensitive (CrFtsZ) and uroporphyrin dehydrogenase (CrUrod) genes also produced the expected mutant phenotypes. CONCLUSION: A method that induces the systemic silencing of target genes in the Ceratopteris gametophyte is described. It provides a simple, inexpensive and rapid means to test the functions of genes involved in gametophyte development, especially those involved in cellular processes common to all plants

Identification of molecular integrators shows that nitrogen activelycontrolsthephosphatestarvationresponseinplants

Nitrogen (N) and phosphorus (P) are key macronutrients sustaining plant growth and crop yield and ensuring food security worldwide. Understanding how plants perceive and interpret the combinatorial nature of these signals thus has important agricultural implications within the context of (1) increased food demand, (2) limited P supply, and (3) environmental pollution due to N fertilizer usage. Here, we report the discovery of an active control of P starvation response (PSR) by a combination of local and long-distance N signaling pathways in plants. We show that, in Arabidopsis (Arabidopsis thaliana), the nitrate transceptor CHLORINA1/NITRATE TRANSPORTER1.1 (CHL1/NRT1.1) is a component of this signaling crosstalk. We also demonstrate that this crosstalk is dependent on the control of the accumulation and turnover by N of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), a master regulator of P sensing and signaling. We further show an important role of PHOSPHATE2 (PHO2) as an integrator of the N availability into the PSR since the effect of N on PSR is strongly affected in pho2 mutants. We finally show that PHO2 and NRT1.1 influence each other’s transcript levels. These observations are summarized in a model representing a framework with several entry points where N signal influence PSR. Finally, we demonstrate that this phenomenon is conserved in rice (Oryza sativa) and wheat (Triticum aestivum), opening biotechnological perspectives in crop plants.This work was supported in the Honude group (Biochemistry & Plant Molecular Physiology) by Agence Nationale de la Recherche (IMANA ANR-14-CE19-0008 with a doctoral fellowship to A.S.), by the Centre National de la Recherche Scientifique (CNRS LIA-CoopNet to G.K.), and by the National Science Foundation (NSF IOS 1339362-NutriNet). Research in V.R.’s laboratory was funded by the Ministry of Economy and Competitiveness and AEI/FEDER/European (grants BIO2013-46539-R and BIO2016-80551-R)

Construction of a bacterial artificial chromosome library from the spikemoss Selaginella moellendorffii: a new resource for plant comparative genomics

BACKGROUND: The lycophytes are an ancient lineage of vascular plants that diverged from the seed plant lineage about 400 Myr ago. Although the lycophytes occupy an important phylogenetic position for understanding the evolution of plants and their genomes, no genomic resources exist for this group of plants. RESULTS: Here we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from the lycophyte Selaginella moellendorffii. Based on cell flow cytometry, this species has the smallest genome size among the different lycophytes tested, including Huperzia lucidula, Diphaiastrum digita, Isoetes engelmanii and S. kraussiana. The arrayed BAC library consists of 9126 clones; the average insert size is estimated to be 122 kb. Inserts of chloroplast origin account for 2.3% of the clones. The BAC library contains an estimated ten genome-equivalents based on DNA hybridizations using five single-copy and two duplicated S. moellendorffii genes as probes. CONCLUSION: The S. moellenforffii BAC library, the first to be constructed from a lycophyte, will be useful to the scientific community as a resource for comparative plant genomics and evolution

Epigenomic Consequences of Immortalized Plant Cell Suspension Culture

Plant cells grown in culture exhibit genetic and epigenetic instability. Using a combination of chromatin immunoprecipitation and DNA methylation profiling on tiling microarrays, we have mapped the location and abundance of histone and DNA modifications in a continuously proliferating, dedifferentiated cell suspension culture of Arabidopsis. We have found that euchromatin becomes hypermethylated in culture and that a small percentage of the hypermethylated genes become associated with heterochromatic marks. In contrast, the heterochromatin undergoes dramatic and very precise DNA hypomethylation with transcriptional activation of specific transposable elements (TEs) in culture. High throughput sequencing of small interfering RNA (siRNA) revealed that TEs activated in culture have increased levels of 21-nucleotide (nt) siRNA, sometimes at the expense of the 24-nt siRNA class. In contrast, TEs that remain silent, which match the predominant 24-nt siRNA class, do not change significantly in their siRNA profiles. These results implicate RNA interference and chromatin modification in epigenetic restructuring of the genome following the activation of TEs in immortalized cell culture

Planification urbaine: le cas de Belgrade

Il est devenu tout à fait ordinaire de poser des questions essentielles qui se rapprochent de cet ordre d'idées: «Qu'est-ce que la ville aujourd'hui? Qu'est-ce que l'on peut toujours nommer ville autour de nous? Les questions deviennent encore plus évasives: Ces entités dramatiquement défigurés et ces agglomérations «urbanomorphes» que l'on nomme encore villes ont-elles un réel sens et ce sens est-il du côté de l'homme ou lui est-il devenu si étranger jusqu'en advenir dangereux. L'étude qu'on propose à ceux intéressés par la problématique de la ville est seulement une des nombreuses pistes de recherche actuelles sur les questions d'urbanisme. Maintenant l'homme se pose des questions sur la ville et s'inquiète à propos de sa destinée. On devrait donc, en ce qui concerne les motifs et la matière qu'il traite, englober cet examen qu'on suggère dans le contexte de cette grande bibliographie moderne

Genetic and genomic studies of seed -free plants: Sex determination in ferns and whole genome analysis of lycopods

The fern Ceratopteris richardii is a model system for studying sex determination in plants. While many sex determining mutants have been identified in Ceratopteris, cloning these genes has proven difficult. A new reverse genetics approach to identify the sex determination genes is described. This technique was successfully used to show that the Ceratopteris SCL6-like gene, shared between ferns and flowering plants, is required for male sex differentiation. We also provide evidence that this gene may be regulated by a microRNA in a sex specific manner. We have recently discovered that the lycopside Selaginella moellendorffii has the smallest genome of any vascular plant reported (∼100 Mb). To initiate whole genome studies of this emerging model plant genome, a sample whole genome shotgun (SWGS) sequence dataset as well as 270 kb of two BAC clones sequences surrounding the S. moellendorffii Scarecrow and Coumaroyl-3′-shikimate hydroxylase genes were obtained. This information was used to determine the proportion of coding, non-coding and repetitive DNA, and determine the likely number of genes in this small genome. The S. moellendorffii SWGS dataset was also compared to a simulated SWGS from Arabidopsis thaliana, another small genome plant species with a whole genome sequence already available. The results show that the ∼100 Mb of S. moellendorffii genome is likely to encode 18000 to 22000 genes

Origins of novel phenotypic variation in polyploids

Polyploid species represent a special type of organism in nature, one that can survive and compete with three or more full sets of homologous chromosomes. While less common in the animal and fungal kingdoms, polyploid species are highly prevalent in the plant kingdom. Indeed, most agricultural crops are polyploids, typically because polyploidy confers greater robustness and therefore higher yields. Among many examples of novel phenotypic variation exhibited by polyploids are the production of larger fruits, reduced tillering, delays in the reproductive transition, and even the creation of visually stunning flower pigmentation patterns coveted by gardeners. The source of this novel variation in polyploids is still largely unclear. However, multiple cellular mechanisms have been proposed, with some supporting evidence, to explain novel variation. We review some of these mechanisms here