SummaryMaritime pine (Pinus pinaster Ait.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species.

ABSTRACT:The metabolism of apigenin, a weak estrogenic flavonoid phytochemical, was investigated in the rat. After a single oral administration of radiolabeled apigenin, 51.0% of radioactivity was recovered in urine, 12.0% in feces, 1.2% in the blood, 0.4% in the kidneys, 9.4% in the intestine, 1.2% in the liver, and 24.8% in the rest of the body within 10 days. Sex differences appear with regard to the nature of compounds eliminated via the urinary route: immature male and female rats, like mature female rats, excreted a higher percentage of the mono-glucuronoconjugate of apigenin than the mono-sulfoconjugate of apigenin (10.0-31.6% versus 2.0-3.6%, respectively). Mature male rats excreted the same compounds in an inverse ratio (4.9% and 13.9%, respectively). Radioactivity appeared in the blood only 24 h after oral administration. Blood kinetics showed a high elimination half-time (91.8 h), a distribution volume of 259 ml, and a plasmatic clearance of 1.95 ml/h. All of the parameters calculated from these experiments suggested a slow metabolism of apigenin, with a slow absorption and a slow elimination phase. Thus, a possible accumulation of this flavonoid in the body can be hypothesized.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:The metabolism of apigenin, a low estrogenic flavonoid phytochemical, was investigated in rat using liver models both in vitro (subcellular fractions) and ex vivo (isolated perfused liver). In vitro, phase I metabolism led to the formation of three monohydroxylated derivatives: luteolin which was the major metabolite (K m ‫؍‬ 22.5 ؎ 1.5 M; V max ‫؍‬ 5.605 ؎ 0.090 nmol/min/mg protein, means ؎ S.E.M.), scutellarein, and iso-scutellarein. These oxidative pathways were mediated by cytochrome P450 monooxygenases (P450s). The use of P450 inhibitors and inducers showed that CYP1A1, CYP2B, and CYP2E1 are involved. In vitro studies of phase II metabolism indicated that apigenin underwent conjugation giving three monoglucuronoconjugates and one monosulfoconjugate. Luteolin led to the formation of four monoglucuronoconjugates, two sulfoconjugates, and one methylconjugate identified as diosmetin. Ex vivo during the apigenin perfusion of an isolated rat liver, none of the phase I metabolites could be recovered. In contrast, two monoglucuronoconjugates and one of the sulfoconjugates of apigenin already identified in vitro were recovered. Moreover, two new derivatives were isolated and identified as a diglucuronoconjugate and a glucuronosulfoconjugate. This work provides new data about the metabolism of apigenin and shows the interest value of using various experimental models in metabolic studies.

A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.

Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.