Plants synthesize and store oil mostly as triacylglycerols (TAG) in seeds that is transcriptionally controlled by WRINKLED 1 (WRI1), an APETALA2 (AP2) transcription factor. In contrast, avocado (Persea americana) mesocarp, which accumulates ~70% lipids by dry weight showed high expression of WRI2, along with WRI1 and WRI3. Among the four Arabidopsis WRI paralogs, WRI2 is nonfunctional, while the others are expressed in a tissue-specific manner. Through in silico analysis, we identified that PaWRI2 has a single intact AP2 DNA-binding domain and lacks a C-terminal intrinsically disordered region (IDR3) and PEST motif, which likely results in a relatively stable protein, compared to its Arabidopsis ortholog. We further demonstrated that avocado WRI2 is functional, unlike Arabidopsis WRI2, and accumulates TAG when transiently expressed in Nicotiana benthamiana leaves. Additionally, co-infiltration of PaWRI2 with PaWRI1 and genes encoding for terminal step in TAG assembly, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and/or phospholipid:diacylglycerol acyltransferase 1 (PDAT1) also led to further increase in the lipid content and oleic acid levels in ‘benth’ leaves. Quantitative real-time PCR (qPCR) analyses of > 40 fatty acid biosynthetic pathway genes revealed that several were distinctly up or down regulated by the expression of PaWRI2 and PaWRI1. In conclusion, avocado WRI2 is capable of transactivation of fatty acid biosynthesis genes and TAG accumulation, synergistically with DGAT1 and PDAT1, in nonseed tissues. This study provides a functional role for WRI2 in a basal angiosperm species that is likely lost in modern angiosperms and thus provides basis for mechanistic differences in the transcriptional regulation of lipid biosynthesis among different plant species and between seed and nonseed tissues.

Seeds from the desert shrub Simmondsia chinensis (jojoba) are one of the only known natural plant sources to store a majority of its oil in the form of liquid wax esters (WE) instead of triacylglycerols (TAGs) and these oils account for ~55% of the seed weight. Jojoba oil is highly valued as cosmetic additives and mechanical lubricants, yet despite its value much is still unknown about its neutral lipid biosynthetic pathways and lipid droplet packaging machinery. Here, we have used a multi-"omics" approach to study how spatial differences in lipid metabolites, gene expression, and lipid droplet proteins influence the synthesis and storage of jojoba lipids. Through these studies mass spectrometry analyses revealed that WEs are compartmentalized primarily in the cotyledonary tissues, whereas TAGs are, surprisingly, localized to the embryonic axis tissues. To study the differences in gene expression between these two tissues, a de novo transcriptome was assembled from high throughput RNAseq data. Differential gene expression analysis revealed that the Jojoba Wax Synthase, which catalyzes the formation of wax esters, and the Diacylglycerol O-Acyltransferase1, which catalyzes the final acylation of triacylglycerol synthesis, were differentially expressed in the cotyledons and embryonic axis tissues, respectively. Furthermore, through proteomic analysis of lipid droplet proteins from lipid droplets of the cotyledons and embryonic axis, it was estimated that each of these tissues contains a different proportion of the major lipid droplet proteins, oleosins, steroleosins, caleosins, and lipid droplet associated proteins. The Jojoba Olesosin1, Lipid Droplet Associated Protein 1, and Lipid Droplet Associated Protein 3, were identified as potential lipid droplet proteins that could be important for storage of wax esters. The coding sequences of these genes were transiently expressed in N. benthamiana leaves individually, and with co-expression of Mus musculus diacylglycerol acyltransferase 2, and in all cases were able to induce neutral lipid accumulation. These data also suggest a Lipid Droplet Associated Protein 1 has a specialized role for wax ester accumulation in the cotyledons, whereas Lipid Droplet Associated Protein 3 may have a more generalized role for the storage of triacylglycerols. These differences in compartmentation suggests that the cotyledons and embryonic axis of jojoba have evolved tissue-specific sets of genes for neutral lipid assembly and lipid droplet accumulation. It may be important to consider this tissue context for genetic engineering strategies designed to introduce genes from jojoba into other oilseed crops.

No description available.

No description available.

BACKGROUND:Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.RESULTS:A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.CONCLUSIONS:The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.

Elevated postprandial triacylglycerol concentrations ([TAG]) are associated with the development and progression of atherosclerosis, and are established as an independent risk factor for future cardiovascular disease. Considering the majority of the daytime is spent in a postprandial state typically, and the paediatric origins of atherosclerosis are well established, lifestyle interventions including manipulations of exercise energy expenditure and dietary energy intake should be initiated early in life. Therefore, this thesis aimed to investigate the postprandial metabolic responses to different exercise and energy intake manipulations in boys and girls, with concentrations of circulating TAG representing the primary outcome of interest. To achieve this, a total of 60 healthy 11 to 13 year old boys and girls were recruited into five experimental studies. The first experimental study (Chapter 4) demonstrated that a single session of high-intensity interval running (HIIR) involving 10 x 1 min intervals at 100% maximal aerobic speed (MAS) resulted in a moderate reduction in postprandial plasma [TAG] in 11 to 12 year old boys. In the second experimental study (Chapter 5), immediate replacement of the moderate-intensity exercise-induced energy deficit negated the reduction in postprandial plasma [TAG] in 11 to 13 year old boys. Furthermore, an exercise-induced energy deficit was required to promote an increase in whole-body fat oxidation. The importance of the associated energy deficit was explored further in Chapter 6, which demonstrated that a moderate-intensity exercise-induced energy deficit elicited a greater reduction in postprandial plasma [TAG] than an isoenergetic diet-induced energy deficit in 11 to 13 year old girls (21% vs. 10% respectively). Chapter 7 compared the effect of 10 x 1 min interval runs at 100% MAS (HIIR) and 5 x 1 min interval runs at 100% MAS combined with a mild reduction in habitual energy intake by 0.82 MJ (195 kcal; HIIR-ER) on postprandial metabolism in 11 to 13 year old girls. Acute manipulations of low volume HIIR and ER reduced postprandial plasma [TAG] and increased resting whole-body fat oxidation, with the magnitude of effect marginally, although not meaningfully, greater following HIIR than HIIR-ER. The final experimental chapter (Chapter 8) compared directly healthy 11 to 13 year old boys and girls postprandial TAG responses to acute HIIR. Although postprandial plasma [TAG] was substantially lower in boys compared with girls, the magnitude of reduction following HIIR was similar between the sexes (11% vs. 10% respectively). Collectively, these studies demonstrate the efficacy of acute moderate- and high-intensity exercise, and to a lesser extent energy-intake restriction, to reduce postprandial plasma [TAG] and increase resting whole-body fat oxidation in boys and girls. Furthermore, the beneficial effect of exercise on postprandial metabolism appears dependent on the maintenance of the associated energy deficit. These lifestyle interventions have the potential to provide a practical, effective and engaging stimulus to promote a healthier cardiovascular risk profile in early adolescence.

Background: Vitamin D is crucial for skeletal muscle function. 25-hidroxyvitamin D (25(OH)D) has been correlated with skeletal muscle mass and intramyocellular lipid (IMCL) content. The purpose of this study was to understand how calcitriol, the active vitamin D metabolite, directly affects myocellular size and lipid partitioning. Methods: C2C12 myotubes were treated with calcitriol (100nM) or vehicle control for 24 or 96 h. Myotube diameter and protein synthesis rate were measured to determine effects of calcitriol on myocellular size. Intramyocellular triacylglycerol (IMTG), diacylglycerol (DAG), and ceramide content were measured by LC/MS. Expression of genes involved in lipid packaging and lipolysis were measured by RT-PCR. Insulin-stimulated phosphorylated Akt (Thr 308) was determined by western blot. Results: Calcitriol did not affect myocellular size or protein synthesis rate. Calcitriol increased total DAG and ceramides in a sub-species specific manner. Calcitriol increased IMTG area, but did not affect total IMTG content. Calcitriol reduced mRNA content of diglyceride acyltransferase and increased mRNA content of lipid packaging genes. Calcitriol did not negatively affect insulin-stimulated pAkt. Conclusions: These results suggest calcitriol directly alters lipid content and packaging in skeletal muscle cells. Altering the expression of lipid packaging genes and increasing IMCL subspecies content may be mechanisms by which vitamin D improves skeletal muscle function in vivo.

Chez la plante modèle Arabidopsis, le processus de maturation de la graine et, en particulier, l’accumulation de composés de réserves (huile et protéines de réserve) sont étudiés depuis de nombreuses années. Si les voies de biosynthèse conduisant à l’accumulation de tels composés sont bien décrites, leur régulation est encore largement méconnue. Mon travail de thèse s’inscrit dans un projet de recherche dont le but est d’identifier de nouveaux régulateurs transcriptionnels de la maturation de la graine d’Arabidopsis. Après avoir réalisé une étude comparative du processus de maturation chez les deux zygotes de la graine, embryon et albumen, nous avons caractérisé un facteur de transcription appelé MYB118, exprimé spécifiquement dans l’albumen et potentiellement impliqué dans la régulation du processus de maturation. Son patron d’expression, finement caractérisé, montre un pic d’accumulation d’ARNm en début de maturation de la graine, plus spécifiquement dans l’albumen. Des études menées sur des lignées mutantes ou surexprimant LEAFY COTYELDON2 (LEC2) révèlent que l’expression de MYB118 est activée par ce régulateur maître de la maturation de la graine. Une analyse biochimique de graines myb118 montre que le contenu en huile et en protéines de réserve est doublé dans l’albumen et réduit dans l’embryon de ce mutant par comparaison aux graines sauvages. Finalement, une analyse transcriptomique effectuée sur des graines myb118 a permis d’identifier des cibles putatives dont la dérégulation pourrait expliquer le phénotype : MYB118 semble être un répresseur de l’accumulation de composés de réserve dans l’albumen. Comme la famille de facteurs de transcription à laquelle appartient MYB118 comprend de nombreux membres, nous nous sommes intéressés au patron d’expression et au rôle de ses paralogues les plus proches. L’un d’entre eux, appelé MYB115, est exprimé spécifiquement dans l’albumen chalazal et semble avoir une fonction partiellement redondante à celle de MYB118. / In the model plant Arabidopsis thaliana, seed maturation and more especially the accumulation of storage compounds such as oil and seed storage proteins (SSP) have been widely studied. Although the biosynthetic networks underlying the accumulation of such compounds are now well described, regulation of these pathways remains poorly understood. My Ph.D. project is a part of a research program aimed at identifying new transcriptional regulators of seed maturation in Arabidopsis. After a comparative analysis of maturation processes in the two zygotic tissues of the seed, namely the embryo and the endosperm, we have characterized MYB118, an endosperm-specific transcription factor, putatively involved in the regulation of this maturation process. A fine and comprehensive characterization of its expression pattern showed a peak of expression at the onset of the maturation phase in the endosperm. Expression studies carried out in LEAFY COTYLEDON2 (LEC2) mutant and over-expressing lines demonstrated that MYB118 expression is positively regulated by this master regulator. Biochemical analysis of myb118 seeds showed that oil and SSP contents were doubled in the endosperm fraction and decreased in the embryo of this mutant compared to the wild type. A transcriptomic analysis of myb118 mutant seeds point out some putative targets, the misregulation of which could explain this phenotype: MYB118 seems to be a repressor of storage compounds accumulation in the endosperm.Since MYB118 belongs to the broad family of MYB transcription factors, we investigated the expression pattern and the role of its closest paralogs. One of them, called MYB115 is expressed specifically in the chalazal endosperm and seems to have partially redundant functions with MYB118.

No description available.

Transcription factor Wrinkled (WRI) 1 is associated with triacylglycerol (TAG) biosynthesis and accumulation in plant tissues. In avocado (Persea americana), a basal angiosperm, four WRI orthologs (1-4) were identified by transcriptome studies and the gene expression of WRI1, 2 and 3 was associated with TAG accumulation in mesocarp tissue. Therefore, it is hypothesized that putative PaWRI1, 2 and 3 but not PaWRI4 are responsible for TAG synthesis in non-seed tissues. To this extent, various in silico analyses were performed to identify similarities and distinct features of putative WRI genes in basal angiosperm relative to maize and Arabidopsis, a monocot and dicot respectively. Predicted structural comparison of these orthologs is expected to reveal the distinct features of avocado WRI paralogs that are associated with the regulation of oil biosynthesis in non-seed tissues.