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

Abstract

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.

Current Perspectives on the Auxin-Mediated Genetic Network that Controls the Induction of Somatic Embryogenesis in Plants

“…Moreover, the auxin-related processes including auxin biosynthesis and signalling was placed in centre of SE and ZE regulation. However, recent RNAseq data of an embryogenic culture of Arabidopsis and Pinus pinaster showed that the SE-transcriptome seems to be distinctly different from the transcriptome of a zygotic embryo [170,171]. This unexpected finding suggests that the molecular events that trigger SE induction might differ from the ones that operate during ZE and points to the epigenetic processes that orchestrate the transcriptomes of in vitro cultured somatic cells in response to plethora of endo-and exogenous factors, thus making them different from those found in ZE.…”

Research Tools for the Functional Genomics of Plant miRNAs During Zygotic and Somatic Embryogenesis

“…Nevertheless, comparisons of function of miRNAs in somatic versus zygotic embryos in the future may reveal an miRNA-based regulation network of the embryonic differentiation events, with common or specific miRNAs to both processes. In the ongoing debate about the ZE and SE similarities, recent analyses of an embryogenic culture of Arabidopsis and Pinus pinaster showed that the SE transcriptome seems to be distinctly different from the transcriptome of a zygotic embryo [ 38 , 39 ]. That finding highlighted how important the precise identification of the function of miRNAs in each of the different phases of both analysed processes is, and the miRNA-dedicated tools for miRNA functional analysis can make it possible.…”

“…miRNA profiling using NGS has revolutionised miRNA analysis and there are still new instruments, sequencing platforms, and new methods that appear, such as sRNA-seq using single-cell sequencing [ 81 ] which provide new opportunities for analyses also during embryogenesis. Until now, sRNA-seq analyses have been performed during embryogenesis in Picea sprus [ 82 ], P. pinaster [ 39 ], Picea glauca [ 30 ], Triticum aestivum [ 83 ], Arabidopsis [ 28 , 30 , 35 , 38 , 41 , 84 ], Dimocarpus longan [ 85 ], Zea mays [ 86 ], Citrus sinensis [ 87 ], Larix leptolepis [ 88 ], Gossypium hirsutum [ 89 , 90 ], Phyllostachys heterocycla [ 91 ], Oryza sativa [ 92 ], and Lilium pumilum [ 93 ] (for SE reviewed in [ 94 ]). Plant embryogenesis is challenging to analyse due to the small, early embryos that are deeply embedded in the maternal tissues, which often results in RNA contamination from maternal tissue, and due to difficult distinguishing of the cells that are undergoing SE within an explant tissue, protocols, which are specifically dedicated for ZE and SE, have been developed and successfully used for miRNA analysis [ 28 , 38 , 84 , 95 ].…”

“…RNA or dedicated kits/methods for isolating the sRNA fraction are available to analyse the miRNA expression in explants/tissues that are undergoing SE or ZE. The TRIzol ™ Reagent is primarily used in studies of D. longan [ 85 , 101 ], Hordeum vulgare [ 102 ], and Arabidopsis [ 62 , 63 , 79 ]; Quick-RNA MiniPrep is used in studies of Z. mays [ 86 , 103 ]; Plant/Fungi Total RNA purification is used in studies of P. pinaster [ 39 ]; C-TAB is used in studies of L. pumilum [ 93 ], and the mirVana TM miRNA Isolation Kit is used in studies of Arabidopsis [ 35 , 84 ], Acacia crassicorpe [ 104 ], and T. aestivum [ 83 ] to isolate RNA with an sRNA fraction to analyse miRNAs during embryogenesis.…”

Tissue-specific transposon-associated small RNAs in the gymnosperm tree, Norway spruce

“…Nevertheless, the factors involved in the RdDM pathway, such as RNA-DEPENDENT RNA POLY-MERASE 2 (RDR2), DICER-LIKE 3 (DCL3), ARGONAUTE 4 (AGO4) and most of Pol IV and Pol V components, are conserved between angiosperms and gymnosperms [17,21]. Indeed, gymnosperms have identifiable 24-nt sRNA populations in male cones and embryonic tissues [18,[21][22][23][24][25], revealing tissue-specific differences in sRNA production and possibly function in gymnosperms. More than half of the genomes in most gymnosperm species are occupied by TEs [18,26].…”