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Abstract
Ricinoleic acid, an industrially useful hydroxy fatty acid (HFA), only accumulates to high levels in the triacylglycerol fraction of castor (Ricinus communis) endosperm, even though it is synthesized on the membrane lipid phosphatidylcholine (PC) from an oleoyl ester. The acyl chains of PC undergo intense remodeling through the process of acyl editing. The identities of the proteins involved in this process, however, are unknown. A phospholipase A2 (PLA2) is thought to be involved in the acyl-editing process. We show here a role for RcsPLA2α in the acyl editing of HFA esterified to PC. RcsPLA2α was identified by its high relative expression in the castor endosperm transcriptome. Coexpression in Arabidopsis (Arabidopsis thaliana) seeds of RcsPLA2α with the castor fatty acid hydroxylase RcFAH12 led to a dramatic decrease in seed HFA content when compared with RcFAH12 expression alone in both PC and the neutral lipid fraction. The low-HFA trait was heritable and gene dosage dependent, with hemizygous lines showing intermediate HFA levels. The low seed HFA levels suggested that RcsPLA2α functions in vivo as a PLA2 with HFA specificity. Activity assays with yeast (Saccharomyces cerevisiae) microsomes showed a high specificity of RcsPLA2α for ricinoleic acid, superior to that of the endogenous Arabidopsis PLA2α. These results point to RcsPLA2α as a phospholipase involved in acyl editing, adapted to specifically removing HFA from membrane lipids in seeds.
Highlights
Ricinoleic acid, an industrially useful hydroxy fatty acid (HFA), only accumulates to high levels in the triacylglycerol fraction of castor (Ricinus communis) endosperm, even though it is synthesized on the membrane lipid phosphatidylcholine (PC) from an oleoyl ester
To identify which of the Phospholipase A enzymes (PLAs) genes are potentially involved in castor lipid metabolism, protein sequences of Arabidopsis PLAs were used in a BLASTP search against the 43 draft castor genome accessible from the J
Twelve PC-PLA1, one phosphatidic acid (PA)-PLA1, one Lecithin Cholesterol Acyltransferase (LCAT)-like3, 12 pPLA, two sPLA2, one SOBER1, and one LCAT-like4 were identified in the castor genome
Summary
Phospholipase activity has been shown to be involved in the metabolism of the uncommon FAs ricinoleate (12-OH 18:1Δ9cis), vernoleate, and the short-chain FAs caprylate and laurate in plant seeds (Bafor et al, 1991; Ståhl et al, 1995). Evidence for the involvement of sPLA2 in signaling and pathogen defense is more limited (Munnik and Testerink, 2009; Canonne et al, 2011) Their involvement in membrane trafficking, is emerging: reductions in sPLA2 expression in Arabidopsis (Arabidopsis thaliana) plants led to Golgi disruption and the inhibition of PIN auxin efflux transporter traffic to the plasma membrane in roots (Lee et al, 2010) and to membrane disruption in pollen (Kim et al, 2011). Our results suggest roles for RcsPLA2a in (1) acyl editing to generate LPC for reacylation through LPCAT activity, (2) freeing HFAs for activation to acylCoA and incorporation into TAG, and (3) contributing to membrane integrity through the removal of potentially deleterious ricinoleate from membrane lipids
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