Transcript profiling for early stages during embryo development in Scots pine

Abstract

BackgroundCharacterization of the expression and function of genes regulating embryo development in conifers is interesting from an evolutionary point of view. However, our knowledge about the regulation of embryo development in conifers is limited. During early embryo development in Pinus species the proembyo goes through a cleavage process, named cleavage polyembryony, giving rise to four embryos. One of these embryos develops to a dominant embryo, which will develop further into a mature, cotyledonary embryo, while the other embryos, the subordinate embryos, are degraded. The main goal of this study has been to identify processes that might be important for regulating the cleavage process and for the development of a dominant embryo.ResultsRNA samples from embryos and megagametophytes at four early developmental stages during seed development in Pinus sylvestris were subjected to high-throughput sequencing. A total of 6.6 million raw reads was generated, resulting in 121,938 transcripts, out of which 36.106 contained ORFs. 18,638 transcripts were differentially expressed (DETs) in embryos and megagametophytes. GO enrichment analysis of transcripts up-regulated in embryos showed enrichment for different cellular processes, while those up-regulated in megagametophytes were enriched for accumulation of storage material and responses to stress. The highest number of DETs was detected during the initiation of the cleavage process. Transcripts related to embryogenic competence, cell wall modifications, cell division pattern, axis specification and response to hormones and stress were highly abundant and differentially expressed during early embryo development. The abundance of representative DETs was confirmed by qRT-PCR analyses.ConclusionBased on the processes identified in the GO enrichment analyses and the expression of the selected transcripts we suggest that (i) processes related to embryogenic competence and cell wall loosening are involved in activating the cleavage process; (ii) apical-basal polarization is strictly regulated in dominant embryos but not in the subordinate embryos; (iii) the transition from the morphogenic phase to the maturation phase is not completed in subordinate embryos. This is the first genome-wide transcript expression profiling of the earliest stages during embryo development in a Pinus species. Our results can serve as a framework for future studies to reveal the functions of identified genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0939-5) contains supplementary material, which is available to authorized users.

Identification of molecular processes that differ among Scots pine somatic embryogenic cell lines leading to the development of normal or abnormal cotyledonary embryos

“…In order to pinpoint molecular processes that might be disturbed during somatic embryogenesis in Scots pine, we have analyzed the expression pattern of selected transcripts during development of somatic embryos in a normal and an abnormal cell line of Scots pine. The selected transcripts have been previously shown to be differentially expressed during early zygotic embryogenesis in Scots pine (Merino et al 2016). Based on the transcripts that accumulated differentially between the two cell lines, we conclude that the apical-basal polarization in early somatic embryos and the transition from the morphogenic phase to the maturation phase are disturbed in the abnormal cell line.…”

“…We recently performed a transcriptome analysis during early zygotic embryo development in Scots pine (Merino et al 2016). A number of different transcripts were identified as potential regulators of the cleavage process and for the development of dominant embryos.…”

“…Three biological replicates, each with two technical replicates, were analyzed for each transcript. Transcript expression values were normalized against two reference genes, ELONGATION FACTOR 1 (EF1) and PHOSPHOGLUCOMUTASE (PHOS) (Merino et al 2016). The relative expression profile for each transcript was estimated by the Livak method (2 Table S1.…”

“…Traces of genomic DNA were removed using the DNA-free™ Kit (Ambion, Life Technologies), and the corresponding cDNA was synthesized using the PrimeScript™ RT reagent Kit (Takara), according to the manufacturer's instructions. To produce an entry clone, specific primers containing the cloning adapter CACC were designed by using the Scots pine zygotic transcriptome output sequences as templates (Merino et al 2016). The obtained PCR products, amplified with the Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific), were gel-purified with the GeneJET Gel Extraction Kit (Thermo Scientific) and introduced into the pENTR™/D-TOPO® vector by using the pENTR™ Directional TOPO® Cloning Kit (Invitrogen), following the manufacturer's instructions.…”

“…Since the transcript accumulation of TT7 was very low at stage 1, selection of 35S:TT7i sub-lines was based on the transcript abundance at stage 4. The primers used for analyzing transcript abundance in overexpressing sub-lines have been presented before (Merino et al 2016). The primers used to quantify CYP78A and TT7 in 35S:CYP78Ai and 35S:TT7i sublines are shown in Table S1.…”