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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.