RESUMEN
Chinese tallow (Triadica sebifera) is a valuable oilseed-producing tree that can grow in a variety of conditions without competing for food production, and is a promising biofuel feedstock candidate. The fruits are unique in that they contain both saturated and unsaturated fat present in the tallow and seed layer, respectively. The tallow layer is poorly studied and is considered only as an external fatty deposition secreted from the seed. In this study we show that tallow is in fact a non-seed cellular tissue capable of triglyceride synthesis. Knowledge of lipid synthesis and storage mechanisms in tissues other than seed is limited but essential to generate oil-rich biomass crops. Here, we describe the annotated transcriptome assembly generated from the fruit coat, tallow and seed tissues of Chinese tallow. The final assembly was functionally annotated, allowing for the identification of candidate genes and reconstruction of lipid pathways. A tallow tissue-specific paralog for the transcription factor gene WRINKLED1 (WRI1) and lipid droplet-associated protein genes, distinct from those expressed in seed tissue, were found to be active in tallow, underpinning the mode of oil synthesis and packaging in this tissue. Our data have established an excellent knowledge base that can provide genetic and biochemical insights for engineering non-seed tissues to accumulate large amounts of oil. In addition to the large data set of annotated transcripts, the study also provides gene-based simple sequence repeat and single nucleotide polymorphism markers.
Asunto(s)
Euphorbiaceae/genética , Ácidos Grasos/metabolismo , Aceites de Plantas/metabolismo , Transcriptoma , Biocombustibles , Euphorbiaceae/metabolismo , Euphorbiaceae/ultraestructura , Ácidos Grasos/análisis , Frutas/genética , Frutas/metabolismo , Frutas/ultraestructura , Regulación de la Expresión Génica de las Plantas , Secuenciación de Nucleótidos de Alto Rendimiento , Metabolismo de los Lípidos , Lípidos/análisis , Anotación de Secuencia Molecular , Especificidad de Órganos , Aceites de Plantas/análisis , Proteínas de Plantas/genética , Semillas/genética , Semillas/metabolismo , Semillas/ultraestructura , Análisis de Secuencia de ADNRESUMEN
The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18â¶1 to elongation reactions beginning with 18â¶1 as substrate. These V2-based leaf assays produced â¼50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays.
Asunto(s)
Genoma de Planta , Metabolismo de los Lípidos/genética , Nicotiana/genética , Hojas de la Planta/genética , Begomovirus/genética , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Genes Virales , Ingeniería Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Secuencias Invertidas Repetidas , Hojas de la Planta/enzimología , Aceites de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , ARN Interferente Pequeño/genética , Análisis de Secuencia de ADN , Nicotiana/enzimologíaRESUMEN
Copper is required for the proliferation of endothelial cells and copper-lowering therapy reduces tumour growth in animal models. It has been reported that ATN-224, a novel copper chelator, potently inhibits the activity of the copper-dependent enzyme superoxide dismutase 1 (SOD1) in endothelial cells. We performed microarray analysis of gene expression in endothelial cells exposed to ATN-224 which revealed upregulation of stress response genes including heme-oxygenase 1 (HO-1) and differential regulation of several genes previously implicated in angiogenesis including CXCR4, ANGP2, PGES2, RHAMM, ITB4 and AQP1 (p<0.01). These changes were confirmed on qPCR. Treatment of HUVEC with ATN-224 caused increased superoxide levels, phospho-ERK signalling, nuclear NRF1 expression, HO-1 expression and induction of the anti-apoptotic proteins P21, BCL2 and BCLXL. There was also nuclear translocation of SOD1. SOD1 RNA interference replicated the effects of ATN-224 on endothelial cell function but did not cause upregulation of HO-1 or PGES2, suggesting additional mechanisms of action of ATN-224. Downregulation of AQP1, which has been shown to have a role in angiogenesis, was seen with both ATN-224 and SOD1 siRNA. AQP1 expression could be rescued after ATN-224 by added copper. RNA interference to AQP1 inhibited endothelial proliferation and migration, confirming the role of AQP1 in endothelial cell function. Therefore regulation of AQP1 may represent an important action of copper chelation therapy.