Expression of castor LPAT2 enhances ricinoleic acid content at the sn-2 position of triacylglycerols in lesquerella seed

Authors: Chen, Grace; VAN ERP, HARRIE - Rothamsted Research; MARTIN-MORENO, JOSE - Rothamsted Research; Johnson, Kumiko; Boettcher, Eva; BROWSE, JOHN - Washington State University; EASTMOND, PETER - Rothamsted Research; Lin, Jiann

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2016
Publication Date: 4/6/2016
Citation: Chen, G.Q., Van Erp, H., Martin-Moreno, J., Johnson, K., Morales, J.S., Browse, J., Eastmond, P.J., Lin, J.T. 2016. Expression of castor LPAT2 enhances ricinoleic acid content at the sn-2 position of triacylglycerols in lesquerella seed. International Journal of Molecular Sciences. 17(4):507. doi: 10.3390/ijms17040507.

Interpretive Summary: The study aims to explore the in vivo function of a castor lysophosphatidic acid acyltransferase 2 (RcLPAT2) in lesquerella, an important industrial crop for hydroxy fatty acid (HFA) production. The conventional source of HFAs is from castor oil which contains ~90% ricinoleic acid (18:1OH). However, the production of HFAs from castor is hampered by the presence of the toxin ricin in its seed. Lesquerella seed is a safe source of HFAs because it seeds do not contain ricin. Unlike castor seed triacylglycerols (TAG) where most HFAs are located at all its three sn positions (sn-1, sn-2 and sn-3), lesquerella seed TAGs have most HFAs located only at the sn-1 and sn-3 positions. The exclusion of HFAs from the sn-2 position in lesquerella TAGs limits the HFA content to no more than 66%. To increase the HFA content of lesquerella seeds, we introduced a castor lysophosphatidic acid acyltransferase 2 gene (RcLPAT2) into lesquerella. Analysis of transgenic lesquerella seed TAGs revealed that RcLPAT2 was able to acylate 18:1OH to the sn-2 position of lysophosphatidic acid (LPA), and as a result the amount of trihydroxy-TAGs increased. This study demonstrates that RcLPAT2 is a target for the genetic engineering of a castor oil-producing lesquerella crop. Our findings advance the knowledge of plant lipid metabolism and open up new directions for future research not only for enhancing HFA content in lesquerella, but also for HFA production in other oilseed crops.

Technical Abstract: Lesquerella (Physaria fendelri) is a potential crop for hydroxy fatty acid (HFA) production. Its seed triacylglcerols (TAGs) contain 55–60% lesquerolic acid (20:1OH), mostly at the sn-1 and the sn-3 positions of TAG. Castor (Ricinus communis) TAGs contain 90% of ricinoleic acid (18:1OH) which is esterified to all three sn positions. In order to increase HFA levels in lesquerella seeds, a castor lysophosphatidic acid acyltransferase 2 gene (RcLPAT2) capable of acylating ricinoleic acid (18:1OH) to the sn-2 position of lysophosphatidic acid (LPA) was introduced into lesquerella under the control of the seed specific napin promoter from Brassica napus. The resulting fifteen independent T1 transgenic lines all showed a 12% to 238% increase in 18:1OH content in seed TAGs. Further analysis of a T2 line revealed a 35-fold increase of 18:1-OH esterified at the sn-2 position. Alteration of the sn-2 HFA composition increased trihydroxy-TAGs but did not affect the total HFA content. These results conclusively demonstrate that RcLPAT2 can function in lesquerella and incorporate 18:1OH at the sn-2 position of LPAs. To our knowledge this work is the first example of the genetic engineering of lipid metabolic pathways in lesquerella.