Arabidopsis thaliana seed maturation is accompanied by the deposition of storage oil, rich in the essential v-3 polyunsaturated fatty acid a-linolenic acid (ALA). The synthesis of ALA is highly responsive to the level of FATTY ACID DESATURASE3 (FAD3) expression, which is strongly upregulated during embryogenesis. By screening mutants in LEAFY COTYLEDON1 (LEC1)-inducible transcription factors using fatty acid profiling, we identified two mutants (lec1-like and bzip67) with a seed lipid phenotype. Both mutants share a substantial reduction in seed ALA content. Using a combination of in vivo and in vitro assays, we show that bZIP67 binds G-boxes in the FAD3 promoter and enhances FAD3 expression but that activation is conditional on bZIP67 association with LEC1-LIKE (L1L) and NUCLEAR FACTOR-YC2 (NF-YC2). Although FUSCA3 and ABSCISIC ACID INSENSITIVE3 are required for L1L and bZIP67 expression, neither protein is necessary for [bZIP67:L1L:NF-YC2] to activate FAD3. We conclude that a transcriptional complex containing L1L, NF-YC2, and bZIP67 is induced by LEC1 during embryogenesis and specifies high levels of ALA production for storage oil by activating FAD3 expression.

Triacylglycerol (TAG) is a major storage reserve in many plant seeds. We previously identified a TAG lipase mutant called sugar-dependent1 (sdp1) that is impaired in TAG hydrolysis following Arabidopsis (Arabidopsis thaliana) seed germination (Eastmond, 2006). The aim of this study was to identify additional lipases that account for the residual TAG hydrolysis observed in sdp1. Mutants were isolated in three candidate genes (SDP1-LIKE [SDP1L], ADIPOSE TRIGLYCERIDE LIPASE-LIKE, and COMPARATIVE GENE IDENTIFIER-58-LIKE). Analysis of double, triple, and quadruple mutants showed that SDP1L is responsible for virtually all of the residual TAG hydrolysis present in sdp1 seedlings. Oil body membranes purified from sdp1 sdp1L seedlings were deficient in TAG lipase activity but could still hydrolyze di-and monoacylglycerol. SDP1L is expressed less strongly than SDP1 in seedlings. However, SDP1L could partially rescue TAG breakdown in sdp1 seedlings when expressed under the control of the SDP1 or 35S promoters and in vitro assays showed that both SDP1 and SDP1L can hydrolyze TAG, in preference to diacylglycerol or monoacylglycerol. Seed germination was slowed in sdp1 sdp1L and postgerminative seedling growth was severely retarded. The frequency of seedling establishment was also reduced, but sdp1 sdp1L was not seedling lethal under normal laboratory growth conditions. Our data show that together SDP1 and SDP1L account for at least 95% of the rate of TAG hydrolysis in Arabidopsis seeds, and that this hydrolysis is important but not essential for seed germination or seedling establishment.

There has been considerable interest recently in the prospect of engineering crops to produce triacylglycerol (TAG) in their vegetative tissues as a means to achieve a step change in oil yield. Here, we show that disruption of TAG hydrolysis in the Arabidopsis (Arabidopsis thaliana) lipase mutant sugar-dependent1 (sdp1) leads to a substantial accumulation of TAG in roots and stems but comparatively much lower TAG accumulation in leaves. TAG content in sdp1 roots increases with the age of the plant and can reach more than 1% of dry weight at maturity, a 50-fold increase over the wild type. TAG accumulation in sdp1 roots requires both ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and PHOSPHATIDYLCHOLINE: DIACYLGLYCEROL ACYLTRANSFERASE1 and can also be strongly stimulated by the provision of exogenous sugar. In transgenic plants constitutively coexpressing WRINKLED1 and DGAT1, sdp1 also doubles the accumulation of TAG in roots, stems, and leaves, with levels ranging from 5% to 8% of dry weight. Finally, provision of 3% (w/v) exogenous Suc can further boost root TAG content in these transgenic plants to 17% of dry weight. This level of TAG is similar to seed tissues in many plant species and establishes the efficacy of an engineering strategy to produce oil in vegetative tissues that involves simultaneous manipulation of carbohydrate supply, fatty acid synthesis, TAG synthesis, and also TAG breakdown.

SummaryIncreasing the productivity of oilseed crops is an important challenge for plant breeders and biotechnologists. To date, attempts to increase oil production in seeds via metabolic pathway engineering have focused on boosting synthetic capacity. However, in the tissues of many organisms, it is well established that oil levels are determined by both anabolism and catabolism. Indeed, the oil content of rapeseed (Brassica napus L.) has been reported to decline by approximately 10% in the final stage of development, as the seeds desiccate. Here, we show that RNAi suppression of the SUGAR-DEPENDENT1 triacylglycerol lipase gene family during seed development results in up to an 8% gain in oil yield on either a seed, plant or unit area basis in the greenhouse, with very little adverse impact on seed vigour. Suppression of lipolysis could therefore constitute a new method for enhancing oil yield in oilseed crops.