Abstract
Somatic embryogenesis (SE) is a method for producing embryos in vitro and is considered a highly promising approach for micropropagation and germplasm conservation. However, the application of SE for genetic breeding and ex situ conservation of certain species, such as Brazilian pine, faces several technical challenges, including the difficulty of inducing embryogenic cultures using tissues of mature trees, the loss of embryogenic competence of cell cultures and incomplete development of somatic embryos. In order to understand the genetic factors governing embryogenesis, a comparative transcriptome analysis was performed to elucidate differences between distinct cell cultures, early zygotic and somatic embryos and, unorthodox seed developmental stages. A total of 64 GB of sequence derived from high-throughput Illumina RNA-seq profiling was used for de novo transcriptome assembly. The reference transcriptome resulted in 112,772 predicted unigenes with an average length of 825 bp and an N50 of 1,638 bp. Sequence similarity searches using a public protein database revealed 19,947 unigenes that could be annotated with gene descriptions and gene ontology terms. Analysis of differential gene expression allowed pinpointing of genes whose products are predicted to be involved in cell line embryogenic potential, early somatic embryo formation and unorthodox seed development. The results expand our understanding of the complex molecular events that control embryogenesis suggesting that the regeneration impairment of Araucaria angustifolia cultures is consequence of the auxin signaling failure. The generated data lay the foundation for future functional genomic and evolutionary studies that will advance the understanding of conifer biology and unorthodox seed physiology.
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References
Armengaud J, Trapp J, Pible O, Geffard O, Chaumot A, Hartmann EM (2014) Non-model organisms, a species endangered by proteogenomics. J Proteomics. doi:10.1016/j.jprot.2014.01.007
Becwar MR, Noland TL, Wyckoff JL (1989) Maturation, germination, and conversion of Norway spruce (Picea abies L.) somatic embryos to plant. In Vitro Cell Dev Biol Plant 26:575–580
Birol I, Jackman SD, Nielsen CB, Qian JQ, Varhol R, Stazyk G, Morin RD, Zhao Y, Hirst M, Schein JE, Horsman DE, Connors JM, Gascoyne RD, Marra MA, Jones SJM (2009) De novo transcriptome assembly with ABySS. Bioinformatics 25:2872–2877
Burleigh JG, Barbazuk WB, Davis JM, Morse AM, Soltis PS (2012) Exploring diversification and genome size evolution in extant gymnosperms through phylogenetic synthesis. J Bot. ID 292857
Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, Schijlen EGWM, Hall RD, Bovy AG, Luo J, Martin C (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26:1301–1308
Cairney J, Pullman GS (2007) The cellular and molecular biology of conifer embryogenesis. New Phytol 176:511–536
Camacho C, Madden T, Ma N, Tao T, Agarwala R, Morgulis A (2013) BLAST command line applications user manual. National Center for Biotechnology Information (US). http://www.ncbi.nlm.nih.gov/books/NBK1763/. Accessed 09 Nov 2013
Canales J, Bautista R, Label P, Gómez MJ, Lesur I, Pozo NF, Rueda LM, Fernández D, Guerrero R, Vanessa C, Benzekri H, Cañas RA, Guevara MA, Andreia R (2013) De novo assembly of maritime pine transcriptome: implications for forest breeding and biotechnology. Plant Biotechnol J 1:1–14
Chénais B, Caruso A, Hiard S, Casse N (2012) The impact of transposable elements on eukaryotic genomes: from genome size increase to genetic adaptation to stressful environments. Gene 509:7–15
Christenhusz M, Zhang XC, Schneider H (2011) A linear sequence of extant families and genera of lycophytes and ferns. Phytotaxa 19:7–54
Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676
dos Santos ALW, Wiethölter N, El Gueddari NE, Moerschbacher BM (2006) Protein expression during seed development in Araucaria angustifolia: transient accumulation of class IV chitinases and arabinogalactan proteins. Physiol Plantarum 127:138–148
Filonova LH, Bozhkov PV, Brukhin VB, Daniel G, Zhivotovsky B, von Arnold S (2000) Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce. J Cell Sci 113:4399–4411
Francis WR, Christianson LM, Kiko R, Powers ML, Shaner NC, Haddock SHD (2013) A comparison across non-model animals suggests an optimal sequencing depth for de novo transcriptome assembly. BMC Genom 14:167
Futamura N, Totok Y, Toyoda A, Igasaki T, Nanjo T, Seki M, Sakaki Y, Mari A, Shinozaki K, Shinohara K (2008) Characterization of expressed sequence tags from a full-length enriched cDNA library of Cryptomeria japonica male strobili. BMC Genom 9:383
Gentleman R, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5:R80
Gliwicka M, Nowak K, Balazadeh S, Mueller-Roeber B, Gaj MD (2013) Extensive modulation of the transcription factor transcriptome during somatic embryogenesis in Arabidopsis thaliana. PLoS ONE 8:e69261
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652
Hakman I, Hallberg H, Palovaara J (2009) The effect of the polar auxin transport inhibitor NPA on embryo morphology and expression of an auxin efflux facilitator protein PIN during Picea abies somatic embryo development. Tree Physiol 29:483–496
Hedman H, Zhu T, von Arnold S, Sohlberg JJ (2013) Analysis of the WUSCHEL-RELATED HOMEOBOX gene family in the conifer Picea abies reveals extensive conservation as well as dynamic patterns. BMC Plant Biol 13:89
International Union of Conservation of Nature Red List of Threatened Species (2013) http://www.iucnredlist.org/details/32975/0. Accessed 03 March 2013
Jin F, Hu L, Yuan D, Xu J, Gao W, He L, Yang X, Zhang X (2013) Comparative transcriptome analysis between somatic embryos (SEs) and zygotic embryos in cotton: evidence for stress response functions in SE development. Plant Biotechnol J 2013:1–13
Jin JP, Zhang H, Kong L, Gao G, Luo JC (2014) PlantTFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors. Nucleic Acids Res 42:D1182–D1187
Jo L, dos Santos ALW, Bueno CA, Barbosa HR, Floh EIS (2014) Proteomic analysis and polyamines, ethylene and reactive oxygen species levels of Araucaria angustifolia (Brazilian pine) embryogenic cultures with different embryogenic potential. Tree Physiol 34:94–104
Lai Z, Lin Y (2013) Analysis of the global transcriptome of longan (Dimocarpus longan Lour.) embryogenic callus using illumina paired-end sequencing. BMC Genom 14:561
Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359
Larsson E, Sitbon F, Ljung K, von Arnold S (2008) Inhibited polar auxin transport results in aberrant embryo development in Norway spruce. New Phytol 177:356–366
Larsson E, Sundström JF, Sitbon F, von Arnold S (2012) Expression of PaNACO1, a Picea abies CUP-SHAPED COTYLEDON orthologue, is regulated by polar auxin transport and associated with differentiation of the shoot apical meristem and formation of separated cotyledons. Ann Bot Lond 110:923–934
Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658–1659
Mackay J, Dean JFD, Plomion C, Peterson DG, Cánovas FM, Pavy N, Ingvarsson PK, Savolainen O, Guevara MA, Fluch S (2012) Towards decoding the conifer giga-genome. Plant Mol Biol 80:555–569
Merkle SA, Dean JFD (2000) Forest biotechnology. Curr Opin Biotech 11:298–302
Nystedt B, Street NR, Wetterbom A, Zuccolo A, Lin YC, Scofield DG, Vezzi F, Delhomme N, Giacomello S, Alexeyenko A (2013) The Norway spruce genome sequence and conifer genome evolution. Nature 497:480–484
Ralph SG, Chun HJ, Kolosova N, Cooper D, Oddy C, Ritland CE, Kirkpatrick R, Moore R, Barber S, Holt RA (2008) A conifer genomics resource of 200,000 spruce (Picea spp.) ESTs and 6,464 high-quality, sequence-finished full-length cDNAs for Sitka spruce (Picea sitchensis). BMC Genom 14:484
Rigault P, Boyle B, Lepage P, Cooke JEK, Bousquet J, MacKay JJ (2011) A white spruce gene catalog for conifer genome analyses. Plant Physiol 157:14–28
Robertson G, Schein J, Chiu R, Corbett R, Field M, Jackman SD, Mungall K, Lee S, Okada HM, Qian JQ (2011) De novo assembly and analysis of RNA-seq data. Nat Methods 7:909–912
Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11:R25
Robinson MD, Smyth GK (2008) Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics 9:321–332
Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140
Rutledge RG, Stewart D, Caron S, Overton C, Boyle B, Mackay J, Klimaszewska K (2013) Potential link between biotic defense activation and recalcitrance to induction of somatic embryogenesis in shoot primordial from adult trees of with spruce (Picea glauca). BMC Plant Biol 13:116
Sangwan RS, Tripathi S, Singh J, Narnoliya LK, Sangwan NS (2013) De novo sequencing and assembly of Centella asiatica leaf transcriptome for mapping of structural, functional and regulatory genes with special reference to secondary metabolism. Gene 525:58–76
Schlögl PS, dos Santos ALW, do Nascimento VL, Floh EIS, Guerra MP (2012) Gene expression during early somatic embryogenesis in Brazilian pine (Araucaria angustifolia (Bert) O. Ktze). Plant Cell, Tissue Organ Cult 108:173–180
Shi CY, Yang H, Wei CL, Yu O, Zhang ZZ, Jiang CJ, Sun J, Li YY, Chen Q, Xia T (2011) Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds. BMC Genom 12:131
Stasolla C, Bozhkov PV, Chu TM, Zyl LV, Egertsdotter U, Suarez MF, Craig DC, Wolfinger RD, von Arnold S, Sederoff RR (2004) Variation in transcripts abundance during somatic embryogenesis in Gymnosperms. Tree Physiol 24:1073–1085
Steiner N, Santa-Catarina C, Andrade JBR, Balbuena TS, Guerra MP, Handro W, Floh EIS, Silveira V (2008) Araucaria angustifolia biotechnology. Funct Plant Sci Biotech 2:20–28
Steiner N, Santa-Catarina C, Guerra MP, Cutri L, Dornelas MC, Floh EIS (2012) A gymnosperm homolog of somatic embryogenesis receptor-like kinase-1 (SERK1) is expressed during somatic embryogenesis. Plant Cell, Tissue Organ Cult 109:41–50
Ueche OG (2012) Pine transcriptomics—RNA-Seq data analysis of Scots pine (Pinus sylvestris) seedlings subjected to a wounding experiment. Dissertation, University of Helsinki
Van Verk MC, Hickman R, Pieterse CMJ, Van Wees S (2013) RNA-Seq: revelation of the messengers. Trends Plant Sci 18:175–179
Vega-Bartol JJ, Simões M, Lorenz WW, Rodrigues AS, Alba R, Dean JFD, Miguel CLM (2013) Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster. BMC Plant Biol 13:123
Vestman D, Larsson E, Uddenberg D, Cairney J, Claphan D, Sundberg E, von Arnold S (2011) Important process during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression. Tree Genet Genomes 7:347–362
Wang Y, Hua W, Wang J, Hannoufa A, Xu Z, Wang Z (2013) Deep sequencing of Lotus corniculatus L. reveals key enzymes and potential transcription factors related to the flavonoid biosynthesis pathway. Mol Genet Genomics 288:131–139
Xie C, Li B, Xu Y, Ji D, Chen C (2013) Characterization of the global transcriptome for Pyropia haitanensis (Bangiales, Rhodophyta) and development of cSSR markers. BMC Genom 14:107
Xu H, Zhang W, Gao Y, Zhao Y, Guo L, Wang J (2012) Proteomic analysis of embryo development in rice (Oryza sativa). Planta 235:687–701
Zhang Y, Zhang S, Han S, Li X, Qi L (2012) Transcriptome profiling and in silico analysis of somatic embryos in Japanese larch (Larix leptolepis). Plant Cell Rep 31:1637–1657
Zhao QY, Wang Y, Kong YM, Luo D, Li X, Hao P (2011) Optimizing de novo transcriptome assembly from short-read RNA-Seq data: a comparative study. BMC Bioinformatics 12:2–12
Zheng W, Zhang X, Yang Z, Wu J, Li F, Duan L, Li F (2014) AtWuschel promotes formation of the embryogenic callus in Gossypium hirsutum. PLoS ONE 9:e87502
Acknowledgments
P.E. was a recipient of a CAPES fellowship. B.L., S.C.S.A. and A.L.W.S. were recipients of FAPESP fellowships. M.R. and E.F. hold fellowships from CNPq. This work was partially supported by grants from FAPESP (Brazil), CNPq (Brazil), USP (Brazil) and Petrobras. The authors thank Roberta Alvares Campos for technical assistance during sample preparation, Horácio Montenegro and Marcelo Brandão for help and access to the Computer Cluster Thunder (Bioinformatics Group from the Laboratório de Biologia Molecular de Plantas, ESALQ, USP) and PlantScribe (www.plantscribe.com) for carefully editing this paper.
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Eny Iochevet Segal Floh and Magdalena Rossi have contributed equally to this work.
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Elbl, P., Lira, B.S., Andrade, S.C.S. et al. Comparative transcriptome analysis of early somatic embryo formation and seed development in Brazilian pine, Araucaria angustifolia (Bertol.) Kuntze. Plant Cell Tiss Organ Cult 120, 903–915 (2015). https://doi.org/10.1007/s11240-014-0523-3
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DOI: https://doi.org/10.1007/s11240-014-0523-3