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Abstract Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1) share responsibility for triacylglycerol (TAG) biosynthesis, and their selectivities control TAG fatty acid (FA) compositions. For rational metabolic engineering of seed oils, replacing endogenous TAG biosynthesis with exogenous enzymes containing different substrate FA selectivities is desirable; however, the dgat1-1/pdat1-2 double mutant is pollen lethal. Here, we evaluated the ability of 3 DGAT1s, from phylogenetically diverse plants with distinct TAG assembly processes, to completely replace endogenous TAG biosynthesis in Arabidopsis (Arabidopsis thaliana). We transformed dgat1-1 mutant plants with expression constructs for DGAT1s from Camelina sativa, Physaria fendleri, and castor (Ricinus communis). Transgene expression was properly “contextualized” by using a previously determined minimum necessary expression unit containing the promoter/5′ UTR and first intron of native AtDGAT1; both of these DNA elements are essential for pollen expression. Next, we crossed homozygous lines with a DGAT1/DGAT1/PDAT1/pdat1-2 parent. C. sativa and P. fendleri DGAT1s restored the FA compositions and transcriptional differences of dgat1-1 to near wild-type and rescued the dgat1-1/pdat1-2 pollen lethality. R. communis DGAT1 was active in dgat1-1 seeds but produced unique oil profiles and alterations in the expression of lipid metabolic genes; it also failed to rescue dgat1-1/pdat1-2 lethality. This study confirms that the promoter and first intron of AtDGAT1 can modulate the expression of foreign DGAT1 genes to fit the correct spatiotemporal profile necessary for completely replacing endogenous TAG biosynthesis. Furthermore, it demonstrates an additional layer of unexpected enzyme incompatibility between oilseed lineages, which may complicate bioengineering approaches that seek to replace essential genes with orthologs.
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Transcriptome Analysis of ‘Philippine Lono Tall’ Coconut (Cocos nucifera L.) Endosperm Reveals Differential Expression of Genes Involved in Oil Biosynthesis
The ‘Philippine Lono Tall’ (PLNT) is a variant of the more common ‘Philippine Laguna Tall’ (LAGT), which produces fruits with soft endosperm and reported higher fat content. To understand patterns of fatty acid (FA) and oil accumulation in LAGT and PLNT fruits, transcriptomes of 6–7 month-old endosperm samples were analyzed by RNA-Seq. Quantitative PCR was performed to analyze the differential expression of selected genes related to oil biosynthesis. Further, oil samples from the PLNT endosperm were analyzed to determine their FA composition across developmental stages. A total of 416,488 contigs were de novo assembled, including 15,497 (14,356 upregulated and 1,141 downregulated) differentially expressed contigs. Several putative unigenes related to cell membrane and wall biogenesis, endosperm development, and oil biosynthesis and accumulation were identified among the assembled contigs. This first report of the complete ontogenetic FA profile revealed that medium chain fatty acids are the main components of oil from the PLNT endosperm. This pilot study is the first to suggest a molecular basis for the unique ‘Lono’ phenotype.
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- Award ID(s):
- 1759906
- PAR ID:
- 10217757
- Date Published:
- Journal Name:
- Philippine agricultural scientist
- Volume:
- 102
- ISSN:
- 1655-9711
- Page Range / eLocation ID:
- 101-118
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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