Modern society’s demand for oil has resulted in depletion of resources and caused higher oil prices. Therefore, natural oil resources of plants are gaining the spotlight and are expected to increase twice that of current use by 2030. Plants are able to accumulate up to 90% oil by dry weight in the form of triacylglycerol (TAG) and it is derived from fleshy part of the fruits, such as mesocarp of oil palm, avocado, and olive. In seed tissues, an acyl CoA-dependent enzyme, diacylglycerol acyltransferase (DGAT) participates in conversion of diacylglycerol (DAG) to TAG. However, TAG can also be formed by an acyl CoA- independent enzyme, phospholipid:diacylglycerol acyltransferase (PDAT). Avocados (Persea americana) store up to ~70% oil in mesocarp and I am interested in identifying the acyltransferase involved in oil biosynthesis in mesocarp tissues. Based on the transcriptome data available, I hypothesize that unlike in seeds, both DGAT and PDAT are associated with TAG biosynthesis in developing mesocarp of avocado. To test this hypothesis, I will determine 1) TAG content and composition and 2) expression levels for DGAT and PDAT genes in mesocarp and seed tissues of five stages of developing fruits of avocado (n=5). Total lipids will be extracted by isopropanol-chloroform method and analyzed for composition as methyl esters on GC-FID. Total RNA, for expression analysis, will be extracted by Trizol method and analyzed with gene-specific primers by real-time PCR. Statistical significance in change in oil content in association with gene expression during fruit development between mesocarp and seed tissues will be analyzed by ANOVA repeated measures. Comparison of temporal gene expression pattern of oil accumulation mesocarp, to that of seed, will allow us to differentiate the acyltransferase(s) specifically associated with TAG biosynthesis. The proposed research work will take the field of plant lipid biochemistry a step forward in understanding TAG synthesis in fruit tissue. Specifically, I will be able to clearly demonstrate the association of a particular acyltransferase to increasing lipid content in a non-seed (mesocarp) tissue. Understanding differences in oil regulation of a basal angiosperm (avocado) in relation to a monocot (oil palm) and a dicot (olive) also will provide additional insights into fundamental changes in TAG biosynthesis during the evolution of flowering plants. My research is part of a global project that includes a group in California, Florida and Israel. Data generated from my proposed work will be used to develop a joint Binational Agricultural and Research proposal. Therefore, my research work will subsequently contribute to development of ways to manipulation of extent and timing of oil accumulationa direct benefit to avocado growers. Furthermore, this study will be a pivotal step to understanding TAG synthesis that will lead to bioenergy crop.

Lipids are stored in a cell for providing energy. The main advantages of storing lipids over carbohydrates like glycogen is that, lipids yield more energy after oxidation because they represent the highly reduced form of carbon, needs less space and water for storage. Conservation of chemical energy in the form of biologically inert form is by storing molecules like triacylglycerol (TAG) and Steryl esters (SE). Triacylglycerol is the major storage form of energy in all eukaryotic cells. During the periods of nutritional excess and nutritional stress, all organisms like bacteria, yeast, animals, and plants can able to do the critical function of synthesizing the triacylglycerol. TAG is an energy store and a repository of essential and non-essential fatty acids and precursors for phospholipid biosynthesis. TAG synthesis mainly takes place in endoplasmic reticulum in mammals and in plants, it takes place in plastid and mitochondria. Triacylglycerol synthesis discovered by Kennedy starts with glycerol 3- phosphate. Glycerol 3-phosphate gets acylate to form lysophosphatic acid (LPA), which in turn acylate to form phosphatic acid (PA) and the reactions are catalyzed by glycerol 3-phosphate acyltransferase (GPAT) and LPA acyltransferase (LPAT) respectively. PA undergoes phosphorylation by PA phosphatase enzyme to give diacylglycerol (DAG). Further acylation of DAG gives rise to TAG and the reaction is catalyzed by diacylglycerol acyltransferase (DGAT). There are several DGAT classes were identified they are DGAT1, DGAT2, PDAT and bifunctional TAG/wax ester synthase. However all the enzymes involved in Kennedy TAG biosynthetic pathway as well as the enzymes of all different DGAT classes are membrane bound enzymes. Through our studies an another DGAT class that is soluble and cytosolic DGAT was first identified in peanut and also in yeast, Rhodotorula glutinis in which a soluble cytosolic complex itself has been identified. The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in R. glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of alternate TAG biosynthetic pathway in oleaginous yeasts. A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX4D acyltransferase motif at the N-terminal end and a lipid binding motif VX2GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

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A variety of plants are natural resources for oil and are capable of synthesizing and storing up to 90% oil (dry weight) in the form of triacylglycerols (TAGs). TAGs are commonly used as vegetable oils of which, >35% is derived from fleshy part of the fruits, such as mesocarp of oil palm, avocado, and olive. Studies on TAG synthesis in seed tissues mostly implicated an acyl CoA-dependent enzyme, diacylglycerol (DAG) acyltransferase (DGAT) to catalyze the conversion of DAG to TAG. However, recent studies on Arabidopsis and oil palm suggested participation of a phospholipid:diacylglycerol acyltransferase (PDAT), which is an acyl-CoA-independent enzyme. In avocados, which store up to 70% oil in mesocarp, I hypothesize that both DGAT and PDAT are likely involved in TAG synthesis. To test the hypothesis, I determined TAG content and composition by gas chromatography (GC) and expression levels of DGAT and PDAT genes by real-time PCR, in developing mesocarp. These data will be compared to that of seed tissues of avocado to associate gene expression levels with changes in oil accumulation. Future studies on cloning and characterization of these potential acyltransferase genes involved in TAG synthesis will allow us to develop genetic tools that may increase oil yield; a step towards meeting the consumption demand for oil that is expected to almost double by 2030.

Oil palm can accumulate up to 90% oil in its mesocarp, the highest level observed in the plant kingdom. In contrast, the closely related date palm accumulates almost exclusively sugars. To gain insight into the mechanisms that lead to such an extreme difference in carbon partitioning, the transcriptome and metabolite content of oil palm and date palm were compared during mesocarp development. Compared with date palm, the high oil content in oil palm was associated with much higher transcript levels for all fatty acid synthesis enzymes, specific plastid transporters, and key enzymes of plastidial carbon metabolism, including phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase. Transcripts representing an ortholog of the WRI1 transcription factor were 57-fold higher in oil palm relative to date palm and displayed a temporal pattern similar to its target genes. Unexpectedly, despite more than a 100-fold difference in flux to lipids, most enzymes of triacylglycerol assembly were expressed at similar levels in oil palm and date palm. Similarly, transcript levels for all but one cytosolic enzyme of glycolysis were comparable in both species. Together, these data point to synthesis of fatty acids and supply of pyruvate in the plastid, rather than acyl assembly into triacylglycerol, as a major control over the storage of oil in the mesocarp of oil palm. In addition to greatly increasing molecular resources devoted to oil palm and date palm, the combination of temporal and comparative studies illustrates how deep sequencing can provide insights into gene expression patterns of two species that lack genome sequence information.

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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.

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.