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Summary Accumulation of triacylglycerols (TAGs) is crucial during various stages of plant development. InArabidopsis, two enzymes share overlapping functions to produce TAGs, namely acyl‐CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1). Loss of function of both genes in adgat1‐1/pdat1‐2double mutant is gametophyte lethal. However, the key regulatory elements controlling tissue‐specific expression of either gene has not yet been identified.We transformed adgat1‐1/dgat1‐1//PDAT1/pdat1‐2parent with transgenic constructs containing theArabidopsis DGAT1promoter fused to theAtDGAT1open reading frame either with or without the first intron.Triple homozygous plants were obtained, however, in the absence of theDGAT1first intron anthers fail to fill with pollen, seed yield isc. 10% of wild‐type, seed oil content remains reduced (similar todgat1‐1/dgat1‐1), and non‐Mendelian segregation of thePDAT1/pdat1‐2locus occurs. Whereas plants expressing theAtDGAT1pro:AtDGAT1transgene containing the first intron mostly recover phenotypes to wild‐type.This study establishes that a combination of the promoter and first intron ofAtDGAT1provides the proper context for temporal and tissue‐specific expression ofAtDGAT1in pollen. Furthermore, we discuss possible mechanisms of intron mediated regulation and how regulatory elements can be used as genetic tools to functionally replace TAG biosynthetic enzymes inArabidopsis.
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Complete replacement of Arabidopsis oil-producing enzymes with heterologous diacylglycerol acyltransferases
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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- Award ID(s):
- 2242822
- PAR ID:
- 10647285
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Plant Physiology
- Volume:
- 199
- Issue:
- 3
- ISSN:
- 0032-0889
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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