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

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.

The pivotal role of small non-coding RNAs in the regulation of seed development.

Seeds represent a crucial stage of the seed plants life cycle. It is during seed development that the foundations of the future plant body, and the ability to give rise to a new plant capable of growing under sometimes adverse environmental conditions, are established. Small non-coding RNAs are major regulators of gene expression both at the post-transcriptional and transcriptional levels and, not surprisingly, these elements play major roles in seed development and germination. We review here the current knowledge about small RNA expression and functions in seed development, going from the morphogenesis phase comprehending embryo development and patterning, to the several steps of the maturation phase, ending in the transition to the germination. A special focus is given to the small RNAs for which functional studies have been conducted and their participation in regulatory networks operating in seeds. Many challenges remain ahead for dissecting the complex small RNA landscape in seeds, but this is a highly relevant issue in plant biology and advances in this area will most certainly impact plant breeding.

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster.

BACKGROUND: It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. RESULTS: Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in development, transcripts with homology to genes acting on modulation of auxin flow and determination of adaxial-abaxial polarity were up-regulated, as were putative orthologs of genes required for meristem formation and function as well as establishment of organ boundaries. Comparative analysis with A. thaliana embryogenesis also highlighted genes involved in auxin-mediated responses, as well as epigenetic regulation, indicating highly correlated transcript profiles between the two species. CONCLUSIONS: This is the first report of a time-course transcriptomic analysis of zygotic embryogenesis in a conifer. Taken together our results show that epigenetic regulation and transcriptional control related to auxin transport and response are critical during early to mid stages of pine embryogenesis and that important events during embryogenesis seem to be coordinated by putative orthologs of major developmental regulators in angiosperms.

Small RNA profiling in Pinus pinaster reveals the transcriptome of developing seeds and highlights differences between zygotic and somatic embryos.

Regulation of seed development by small non-coding RNAs (sRNAs) is an important mechanism controlling a crucial phase of the life cycle of seed plants. In this work, sRNAs from seed tissues (zygotic embryos and megagametophytes) and from somatic embryos of Pinus pinaster were analysed to identify putative regulators of seed/embryo development in conifers. In total, sixteen sRNA libraries covering several developmental stages were sequenced. We show that embryos and megagametophytes express a large population of 21-nt sRNAs and that substantial amounts of 24-nt sRNAs were also detected, especially in somatic embryos. A total of 215 conserved miRNAs, one third of which are conifer-specific, and 212 high-confidence novel miRNAs were annotated. MIR159, MIR171 and MIR394 families were found in embryos, but were greatly reduced in megagametophytes. Other families, like MIR397 and MIR408, predominated in somatic embryos and megagametophytes, suggesting their expression in somatic embryos is associated with in vitro conditions. Analysis of the predicted miRNA targets suggests that miRNA functions are relevant in several processes including transporter activity at the cotyledon-forming stage, and sulfur metabolism across several developmental stages. An important resource for studying conifer embryogenesis is made available here, which may also provide insightful clues for improving clonal propagation via somatic embryogenesis.

miRPursuit-a pipeline for automated analyses of small RNAs in model and nonmodel plants.

miRPursuit is a pipeline developed for running end-to-end analyses of high-throughput small RNA (sRNA) sequence data in model and nonmodel plants, from raw data to identified and annotated conserved and novel sequences. It consists of a series of UNIX shell scripts, which connect open-source sRNA analysis software. The involved parameters can be combined with convenient workflow management by users without advanced computational skills. miRPursuit presents several advantages when compared to other tools, including the possibility of processing several sRNA libraries in parallel, thus easily allowing a comparison of the differences in sRNA read accumulation among sRNA libraries. We validate miRPursuit by using datasets from a model plant and discuss its performance with the analysis of sRNAs from non-model species.