Transcript profiling for early stages during embryo development in Scots pine

Background: Characterization 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. Results: RNA 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. Conclusion: Based 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

Transcriptional regulation os phenylalaline biosynthesis and utilization

Conifer trees divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin, an important constituent of wood. Since phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and utilization should occur simultaneously. This crucial pathway is finely regulated primarily at the transcriptional level. Transcriptome analyses indicate that the transcription factors (TFs) preferentially expressed during wood formation in plants belong to the MYB and NAC families. Craven-Bartle et al. (2013) have shown in conifers that Myb8 is a candidate regulator of key genes in phenylalanine biosynthesis involved in the supply of the phenylpropane carbon skeleton necessary for lignin biosynthesis. This TF is able to bind AC elements present in the promoter regions of these genes to activate transcription. Constitutive overexpression of Myb8 in white spruce increased secondary-wall thickening and led to ectopic lignin deposition (Bomal et al. 2008). In Arabidopsis, the transcriptional network controlling secondary cell wall involves NAC-domain regulators operating upstream Myb transcription factors. Functional orthologues of members of this network described have been identified in poplar and eucalyptus, but in conifers functional evidence had only been obtained for MYBs. We have identified in the P. pinaster genome 37 genes encoding NAC proteins, which 3 NAC proteins could be potential candidates to be involved in vascular development (Pascual et al. 2015). The understanding of the transcriptional regulatory network associated to phenylpropanoids and lignin biosynthesis in conifers is crucial for future applications in tree improvement and sustainable forest management. This work is supported by the projects BIO2012-33797, BIO2015-69285-R and BIO-474 References: Bomal C, et al. (2008) Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis. J Exp Bot. 59: 3925-3939. Craven-Bartle B, et al. (2013) A Myb transcription factor regulates genes of the phenylalanine pathway in maritime pine. Plant J, 74: 755-766. Pascual MB, et al. (2015) The NAC transcription factor family in maritime pine (Pinus pinaster): molecular regulation of two genes involved in stress responses. BMC Plant Biol, 15: 254.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Regulación coordinada de los genes implicados en la síntesis de fenilalanina en pino

Regulación coordinada de los genes implicados en la síntesis de Fenilalanina en pino Craven-Bartle B, Pascual MB, Cánovas FM, Avila C Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Campus Universitario de Teatinos, Universidad de Málaga, 29071-Málaga, España ([email protected]) Durante el ciclo vital de coníferas como el pino marítimo (Pinus pinaster Ait.) una gran cantidad de esqueletos carbonados se ven inmovilizados de forma irreversible en la madera. Este es un proceso muy costoso en términos energéticos en el que el carbono de la fotosíntesis se canaliza a través de la vía del Siquimato para la biosíntesis de los fenilpropanoides. Esta ruta metabólica fundamental está finamente regulada principalmente a través de control de la transcripción, y puesto que la fenilalanina es el precursor para la biosíntesis de los fenilpropanoides, la regulación precisa de la síntesis de fenilalanina y su utilización debe ocurrir simultáneamente. Los tres promotores de los genes que codifican las enzimas, Prefenato Aminotransferasa (PAT), Fenilalanina Amonio Liasa (PAL), y la Glutamina Sintetasa (GS1b), contienen elementos AC que participan en la activación transcripcional mediada por factores de R2R3-Myb. En este trabajo hemos examinado la capacidad de los factores de transcripción R2R3-Myb: Myb1, Myb4 y Myb8 para co-regular la expresión de PAT, PAL y GS1b. Sólo Myb8 es capaz de activar la transcripción de los tres genes. Por otra parte, la expresión de este factor de transcripción es mayor en tejidos lignificados, donde hay una gran demanda de fenilpropanoides. En un experimento de ganancia de función, hemos demostrado que Myb8 puede unirse específicamente un elemento bien conservado tipo AC-II, de ocho nucleótidos de longitud en las regiones promotoras de PAT, PAL y GS1b, activando de ese modo su expresión. Nuestros resultados muestran que Myb8 regula la expresión de estos genes implicados en el metabolismo de la fenilalanina, que se requiere para la canalización de carbono fotosintético para promover la formación de la madera. La co-localización de los tránscritos de PAT, PAL, GS1b y MYB8 en células vasculares también apoya esta conclusión. Financiado por: Proyecto de excelencia de la Junta de Andalucía (CVI-3739), Proyecto del Ministerio de Ciencia e Innovación (BIO2009-07490) y por el programa KBBE Plant (proyecto SUSTAINPINE)Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Novel Insights into Regulation of Asparagine Synthetase in Conifers

Asparagine, a key amino acid for nitrogen storage and transport in plants, is synthesized via the ATP-dependent reaction catalyzed by the enzyme asparagine synthetase (AS; EC 6.3.5.4). In this work, we present the molecular analysis of two full-length cDNAs that encode asparagine synthetase in maritime pine (Pinus pinaster Ait.), PpAS1, and PpAS2. Phylogenetic analyses of the deduced amino acid sequences revealed that both genes are class II AS, suggesting an ancient origin of these genes in plants. A comparative study of PpAS1 and PpAS2 gene expression profiles showed that PpAS1 gene is highly regulated by developmental and environmental factors, while PpAS2 is expressed constitutively. To determine the molecular mechanisms underpinning the differential expression of PpAS1, the promoter region of the gene was isolated and putative binding sites for MYB transcription factors were identified. Gel mobility shift assays showed that a MYB protein from Pinus taeda (PtMYB1) was able to interact with the promoter region of PpAS1. Furthermore, transient expression analyses in pine cells revealed a negative effect of PtMYB1 on PpAS1 expression. The potential role of MYB factors in the transcriptional regulation of PpAS1 in vascular cells is discussed

PpNAC1, un regulador principal de la biosíntesis y utilización de fenilalanina en pino

La regulación transcripcional del metabolismo de la fenilalanina es particularmente importante en las coníferas, especies de vida larga que usan grandes cantidades de carbono en la formación de madera. El factor de transcripción PpNAC1 es un regulador principal de la biosíntesis de fenilalanina y su utilización en Pinus pinaster. El análisis filogenético lo clasifica dentro del grupo de proteínas NST y se expresa predominantemente en el xilema secundario y madera de compresión de árboles adultos. El silenciamiento de PpNAC1 en P. pinaster da como resultado la alteración del patrón vascular radial del tallo y la represión de la expresión de genes asociados con la biogénesis de pared celular y metabolismo secundario. Además, ensayos de transactivación y EMSA han mostrado que PpNAC1 puede activar su propia expresión y al promotor PpMyb4. A su vez PpMyb4 es capaz de activar a PpMyb8, un regulador transcripcional de la biosíntesis de fenilalanina y lignina en pino marítimo. En conjunto, estos resultados sugieren que PpNAC1 es un ortólogo funcional de los genes de Arabidopsis SND1 y NST1 y respalda la idea de que los reguladores clave que gobiernan la formación de la pared celular secundaria podrían estar conservados entre gimnospermas y angiospermas. Identificarlos interruptores moleculares que controlan la formación de la madera es de suma importancia importancia para la biología fundamental de los árboles y allana el camino para las aplicaciones biotecnológicas en coníferas.Universidad de Málaga. Campus de excelencia Internacional Andalucía Tec

The year out

Figure S5. Distribution of up-regulated TF family members in embryos and megagametophytes. Presented data are based on TF family members differentially accumulated (FC > 2) during seed development in any of the pairwise comparisons between embryos and megagametophytes. Orange bars show the number of TFs belonging to each family in embryos and green bars in megagametophytes. TFs were classified into TF families by using the publicly available PlantTFDB v 3.0 database. Figure S6. Abundance of the ten largest TF families differentially expressed between embryos and megagametophytes during seed development shown in Fig. 5. Number of members in each TF family detected at different developmental stages in (A) embryos and (B) megagametophytes. Subordinate embryos were excluded from this analysis. (PDF 312 kb

Involvement of a Myb transcription factor in the regulation of phenylalanine pathway in maritime pine

Wood is traditionally among the most important commercial products because of the high demand that exits for its derivatives. Trees, including conifers, divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin. Although lignin and other phenolic compounds do not contain nitrogen, phenylalanine metabolism is required to channel photosynthesis-derived carbon to phenylpropanoid biosynthesis. The phenylpropane skeleton required for lignin biosynthesis is provided by the deamination of phenylalanine in the reaction catalysed by the enzyme phenylalanine ammonia-lyase (PAL). This reaction is quantitatively important in trees because lignin biosynthesis is required for wood formation, and it releases large quantities of ammonium. An efficient and coordinated pathway for the amination of prephenate and the deamination of phenylalanine should be operative in lignifying cells to provide phenylalanine for lignin biosynthesis, and to re-assimilate ammonium. We hypothesized that one way to ensure efficient photosynthetic carbon channeling for lignin and other phenylpropanoid biosynthesis, together with nitrogen recycling, would be to couple both processes in time and in space by transcriptionally regulating the genes involved in phenylalanine biosynthesis and use. The experiments described in this communication attempt to test this hypothesis. To this end, we have isolated the promoter region of the three genes involved in the phenylalanine pathway in Pinus pinaster: PAL, GS1b and PAT. We have conducted both in vitro and in vivo studies using three different Myb transcription factors: PtMyb1, PtMyb4 from P. taeda and PpMyb8 from P. pinaster. We have studied the possible coupling in space and time of gene products for the operative co-regulation of both processes in pine trees, and have proven that Myb8 is a potential candidate to be the transcriptional regulator of phenylalanine metabolism in P. pinaster vascular cells.UNIVERSIDAD DE MÁLAGA. CAMPUS DE EXCELENCIA INTERNACIONAL ANDALUCÍA TEC

NAC-MYB basedtranscriptional networkinvolved in the regulation of phenylalanine biosynthesis in P. pinaster

P2-2 NAC-MYB-BASED TRANSCRIPCIONAL NETWORK INVOLVED IN THE REGULATION OF PHENYLALANINE BIOSYNTHESIS IN P. PINASTER Mª Belén Pascual, Rafael A. Cañas, Blanca Craven-Bartle, Francisco M. Cánovas and Concepción Ávila Departamento de Biología Molecular y Bioquímica. Facultad de Ciencias. Universidad de Málaga. Campus de teatinos s/n, Málaga, Spain Email: [email protected] Conifer trees divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin, an important constituent of wood. Since phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and use should occur simultaneously. This crucial pathway is finely regulated primarily at the transcriptional level. Transcriptome analyses indicate that the transcription factors (TFs) preferentially expressed during wood formation in plants belong to the MYB and NAC families. Craven-Bartle et al. (2013) have shown that Myb8 is a candidate regulator of key genes in phenylalanine biosynthesis involved in the supply of the phenylpropane carbon skeleton necessary for lignin biosynthesis. This TF is able to bind AC elements present in the promoter regions of these genes to activate transcription. In Arabidopsis, the transcriptional network controlling secondary cell wall involves NAC-domain regulators operating upstream Myb transcription factors. We have identified in the P. pinaster genome three NAC proteins as potential candidates to be involved in vascular development. One of them, PpNAC1 is expressed both in xylem and compression wood from adult trees and has been thoroughly characterized. Its role upstream the transcriptional network involving Myb8 will be discussed. The understanding of the transcriptional regulatory network associated to phenylpropanoids and lignin biosynthesis in conifers is crucial for future applications in tree improvement and sustainable forest management.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Additional file 3: of Transcript profiling for early stages during embryo development in Scots pine

Figure S2. Number of differentially expressed transcripts (DETs) with a fold-change greater than 2 (FC > 2) identified in each pairwise comparison between embryos and megagametophytes. (A) Total number of up-regulated transcripts in embryos and megagametophytes (B) Number of up-regulated transcripts in embryos (orange) and megagametophytes (green) at different developmental stages during early seed development. Figure S3. GO enrichment analysis of up-regulated transcripts (FC > 2) in embryos, identified in any of the pairwise comparisons between embryos and megagametophytes at the four developmental stages shown in Fig. 1. Enrichment in Biological Processes was obtained using AgriGO Toolkit database (FDR < 0.05). High significant levels are represented by red squares. Figure S4. GO enrichment analysis of up-regulated transcripts (FC > 2) in megagametophytes, identified in any of the pairwise comparisons between embryos and megagametophytes at the four developmental stages shown in Fig. 1. Biological Process enrichment was obtained using AgriGO Toolkit database (FDR < 0.05). High significant levels are represented by red squares. (PDF 672 kb

Additional file 2: of Transcript profiling for early stages during embryo development in Scots pine

Table S5. List of transcripts selected for qRT-PCR analyses. Table S6. Validation of the RNA-seq expression data. (PDF 88.2 kb