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Comprehensive assembly and analysis of the transcriptome of maritime pine developing embryos.

ABSTRACT: BACKGROUND:There are clear differences in embryo development between angiosperm and gymnosperm species. Most of the current knowledge on gene expression and regulation during plant embryo development has derived from studies on angiosperms species, in particular from the model plant Arabidopsis thaliana. The few published studies on transcript profiling of conifer embryogenesis show the existence of many putative embryo-specific transcripts without an assigned function. In order to extend the knowledge on the transcriptomic expression during conifer embryogenesis, we sequenced the transcriptome of zygotic embryos for several developmental stages that cover most of Pinus pinaster (maritime pine) embryogenesis. RESULTS:Total RNA samples collected from five zygotic embryo developmental stages were sequenced with Illumina technology. A de novo transcriptome was assembled as no genome sequence is yet published for Pinus pinaster. The transcriptome of reference for the period of zygotic embryogenesis in maritime pine contains 67,429 transcripts, which likely encode 58,527 proteins. The annotation shows a significant percentage, 31%, of predicted proteins exclusively present in pine embryogenesis. Functional categories and enrichment analysis of the differentially expressed transcripts evidenced carbohydrate transport and metabolism over-representation in early embryo stages, as highlighted by the identification of many putative glycoside hydrolases, possibly associated with cell wall modification, and carbohydrate transport transcripts. Moreover, the predominance of chromatin remodelling events was detected in early to middle embryogenesis, associated with an active synthesis of histones and their post-translational modifiers related to increased transcription, as well as silencing of transposons. CONCLUSIONS:Our results extend the understanding of gene expression and regulation during zygotic embryogenesis in conifers and are a valuable resource to support further improvements in somatic embryogenesis for vegetative propagation of conifer species. Specific transcripts associated with carbohydrate metabolism, monosaccharide transport and epigenetic regulation seem to play an important role in pine early embryogenesis and may be a source of reliable molecular markers for early embryogenesis.

SUBMITTER: Rodrigues AS

PROVIDER: S-EPMC6310951 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Pinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases. DESCRIPTION: EuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided. CONCLUSIONS: The EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.

Project description:BackgroundPredicting the adaptability of forest tree populations under future climates requires a better knowledge of both the adaptive significance and evolvability of measurable key traits. Phenotypic plasticity, standing genetic variation and degree of phenotypic integration shape the actual and future population genetic structure, but empirical estimations in forest tree species are still extremely scarce. We analysed 11 maritime pine populations covering the distribution range of the species (119 families and 8 trees/family, ca. 1300 trees) in a common garden experiment planted at two sites with contrasting productivity. We used plant height as a surrogate of fitness and measured five traits (mean and plasticity of carbon isotope discrimination, specific leaf area, needle biomass, Phenology growth index) related to four different strategies (acquisitive economics, photosynthetic organ size, growth allocation and avoidance of water stress).ResultsEstimated values of additive genetic variation would allow adaptation of the populations to future environmental conditions. Overall phenotypic integration and selection gradients were higher at the high productivity site, while phenotypic integration within populations was higher at the low productivity site. Response to selection was related mainly to photosynthetic organ size and drought-avoidance mechanisms rather than to water use efficiency. Phenotypic plasticity of water use efficiency could be maladaptive, resulting from selection for height growth.ConclusionsContrary to the expectations in a drought tolerant species, our study suggests that variation in traits related to photosynthetic organ size and acquisitive investment of resources drive phenotypic selection across and within maritime pine populations. Both genetic variation and evolvability of key adaptive traits were considerably high, including plasticity of water use efficiency. These characteristics would enable a relatively fast micro-evolution of populations in response to the ongoing climate changes. Moreover, differentiation among populations in the studied traits would increase under the expected more productive future Atlantic conditions.

Project description:Maritime pine (Pinus pinaster Aiton) is a coniferous native of the Mediterranean basin. Because of its adaptability to a wide range of environmental conditions, the species have become a model for studies in coniferous forest management and functional genomics. Somatic embryogenesis (SE) has been so far, the preferred biotechnological strategy for maritime pine breeding programs initiated at the middle-end of the 20th century. To overcome the limitations of the induction and maturation phases in maritime pine SE, we analyzed the possible maternal influence on the embryogenic capability of megagametophytes from controlled crosses, as well as the effect of the temperature and water availability during SE process on the production of plants. A strong maternal effect on the embryogenic potential of maritime pine megagametophytes was observed in our experiments using half-sib and full-sib progenies, while paternal effect was almost undetectable. Besides, it seems possible to improve somatic embryo production of maritime pine megagametophytes by adjusting optimal temperature throughout the process: 28°C during induction and proliferation, and 23°C during the maturation phase. Using induction and proliferation media with reduced water availability (6 g/L Gelrite) can also increase embryo production. Since other limitation of maritime pine SE is culture decline of embryogenic masses (EMs), that reduces embryo yield and germination, we assessed the profile of ABA and IAA and the expression of two embryogenesis-related genes (LEC1 and WOX2) during maturation of EMs of two morphotypes that differed in their maturation capability. Spiky morphotype (SK), with high maturation capability, had a steady increase in both hormones along the 12 weeks of the maturation, whereas ABA content in smooth morphotype picked at the 4th week and dropped. EMs with this morphotype also had a higher IAA content at the beginning of the maturation. A decrease of LEC1 and WOX2 gene expression over the course of embryo development was found to be characteristic of the SK with high maturation capability.

Project description:BackgroundPlants are subjected to continuous stimuli from the environment and have evolved an ability to respond through various growth and development processes. Phototropism and gravitropism responses enable the plant to reorient with regard to light and gravity.ResultsWe quantified the speed of maritime pine seedlings to reorient with regard to light and gravity over 22 days. Seedlings were inclined at 15, 30 and 45 degrees with vertical plants as controls. A lateral light source illuminated the plants and stem movement over time was recorded. Depending on the initial angle of stem lean, the apical response to the lateral light source differed. In control and 15° inclined plants, the apex turned directly towards the light source after only 2 h. In plants inclined at 30° and 45°, the apex first reoriented in the vertical plane after 2 h, then turned towards the light source after 24 h. Two-dimensional gel electrophoresis coupled with mass spectrometry was then used to describe the molecular response of stem bending involved in photo- and gravi-tropism after 22 hr and 8 days of treatment. A total of 486 spots were quantitatively analyzed using image analysis software. Significant changes were determined in the protein accumulation of 68 protein spots. Early response gravitropic associated proteins were identified, which are known to function in energy related and primary metabolism. A group of thirty eight proteins were found to be involved in primary metabolism and energy related metabolic pathways. Degradation of Rubisco was implicated in some protein shifts.ConclusionsOur study demonstrates a rapid gravitropic response in apices of maritime pine seedlings inclined >30°. Little or no response was observed at the stem bases of the same plants. The primary gravitropic response is concomitant with a modification of the proteome, consisting of an over accumulation of energy and metabolism associated proteins, which may allow the stem to reorient rapidly after bending.

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Comprehensive assembly and analysis of the transcriptome of maritime pine developing embryos.

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