Abstract
Producing unusual fatty acids (FAs) in crop plants has been a long-standing goal of green chemistry. However, expression of the enzymes that catalyze the primary synthesis of these unusual FAs in transgenic plants typically results in low levels of the desired FA. For example, seed-specific expression of castor (Ricinus communis) fatty acid hydroxylase (RcFAH) in Arabidopsis (Arabidopsis thaliana) resulted in only 17% hydroxy fatty acids (HFAs) in the seed oil. In order to increase HFA levels, we investigated castor phospholipid:diacylglycerol acyltransferase (PDAT). We cloned cDNAs encoding three putative PDAT enzymes from a castor seed cDNA library and coexpressed them with RcFAH12. One isoform, RcPDAT1A, increased HFA levels to 27%. Analysis of HFA-triacylglycerol molecular species and regiochemistry, along with analysis of the HFA content of phosphatidylcholine, indicates that RcPDAT1A functions as a PDAT in vivo. Expression of RcFAH12 alone leads to a significant decrease in FA content of seeds. Coexpression of RcPDAT1A and RcDGAT2 (for diacylglycerol acyltransferase 2) with RcFAH12 restored FA levels to nearly wild-type levels, and this was accompanied by a major increase in the mass of HFAs accumulating in the seeds. We show the usefulness of RcPDAT1A for engineering plants with high levels of HFAs and alleviating bottlenecks due to the production of unusual FAs in transgenic oilseeds.
Highlights
Producing unusual fatty acids (FAs) in crop plants has been a long-standing goal of green chemistry
RcFAH12 was expressed under the control of seed-specific promoters, but this resulted in maximum stable hydroxy fatty acids (HFAs) levels of only 17% 6 1% in the seed oil of Arabidopsis (Broun and Somerville, 1997; Smith et al, 2000, 2003; Lu et al, 2006) and 15% in camelina (Lu and Kang, 2008)
In order to determine which of the three putative phospholipid:diacylglycerol acyltransferase (PDAT) is the most likely candidate for being involved in HFA-TAG synthesis, we analyzed the expression of RcPDAT1A, RcPDAT1B, and RcPDAT2 in castor endosperm based on transcriptome analysis using the Roche FLX Genome Sequencer technology
Summary
Producing unusual fatty acids (FAs) in crop plants has been a long-standing goal of green chemistry. Nature produces a wide variety of unusual fatty acids (FAs), some of which are important for industry and human health Producing these unusual FAs in agronomically suitable plants has been a long-standing goal for companies and researchers involved in the field of oilseed engineering (Damude and Kinney, 2008; Dyer et al, 2008; Napier and Graham, 2010). One important class of unusual FAs are hydroxylated fatty acids (HFAs), which accumulate up to 90% of total FAs in the seeds of castor (Ricinus communis). Despite the increase in 18:1, the fae mutation did not increase HFA levels above 17%, suggesting that availability of oleic acid is not a limiting factor for HFAtriacylglycerol (TAG) synthesis in Arabidopsis (Broun and Somerville, 1997; Lu et al, 2006). Previous research indicates that investigation of the factors limiting the accumulation of HFAs in transgenic plants provides a valuable model system to investigate the pathways and regulation of FA metabolism and TAG accumulation in oilseeds (Lu et al, 2006; Burgal et al, 2008; Dyer et al, 2008)
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