Maurice M. Moloney

Structural requirements of oleosin domains for subcellular targeting to the oil body.

We have investigated the protein domains responsible for the correct subcellular targeting of plant seed oleosins. We have attempted to study this targeting in vivo using “tagged” oleosins in transgenic plants. Different constructs were prepared lacking gene sequences encoding one of three structural domains of natural oleosins. Each was fused in frame to the Escherichia coli uid A gene encoding [beta]-glucuronidase (GUS). These constructs were introduced into Brassica napus using Agrobacterium-mediated transformation. GUS activity was measured in washed oil bodies and in the soluble protein fraction of the transgenic seeds. It was found that complete Arabidopsis oleosin-GUS fusions undergo correct subcellular targeting in transgenic Brassica seeds. Removal of the C-terminal domain of the Arabidopsis oleosin comprising the last 48 amino acids had no effect on overall subcellular targeting. In contrast, loss of the first 47 amino acids (N terminus) or amino acids 48 to 113 (which make up a lipophilic core) resulted in impaired targeting of the fusion protein to the oil bodies and greatly reduced accumulation of the fusion protein. Northern blotting revealed that this reduction is not due to differences in mRNA accumulation. Results from these measurements indicated that both the N-terminal and central oleosin domain are important for targeting to the oil body and show that there is a direct correlation between the inability to target to the oil body and protein stability.

A 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl-CoA and phosphatidylcholine

The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana. Biochemical analysis of T(2) and T(3) A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2(cisDelta9,12) and 18:3(cisDelta9,12,15)) at the expense of very long monounsaturated FA (20:1(cisDelta11)) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from DeltaYOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase (AthLPCAT) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro. These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.

Expression and recovery of biologically active recombinant Apolipoprotein AIMilano from transgenic safflower (Carthamus tinctorius) seeds

Apolipoprotein AI Milano (ApoAI(Milano) ) was expressed as a fusion protein in transgenic safflower seeds. High levels of expression corresponding to 7 g of ApoAI(Milano) per kilogram of seed have been identified in a line selected for commercialization. The ApoAI(Milano) fusion protein was extracted from seed using an oilbody-based process and matured in vitro prior to final purification. This yielded a Des-1,2-ApoAI(Milano) product which was confirmed by biochemical characterization including immunoreactivity against ApoAI antibodies, isoelectric point, N-terminal sequencing and electrospray mass spectrometry. Purified Des-1,2-ApoAI(Milano) readily associated with dimyristoylphosphatidylcholine in clearance assays comparable to Human ApoAI. Its biological activity was assessed by cholesterol efflux assays using Des-1,2-ApoAI(Milano) :1-palmitoyl-2-oleoyl phosphatidylcholine complexes in vitro and in vivo. This study has established that high levels of biologically functional ApoAI(Milano) can be produced using a plant-based expression system.

High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds

Gamma linolenic acid (GLA; C18:3Δ6,9,12 cis), also known as γ-Linolenic acid, is an important essential fatty acid precursor for the synthesis of very long chain polyunsaturated fatty acids and important pathways involved in human health. GLA is synthesized from linoleic acid (LA; C18:2Δ9,12 cis) by endoplasmic reticulum associated Δ6-desaturase activity. Currently sources of GLA are limited to a small number of plant species with poor agronomic properties, and therefore an economical and abundant commercial source of GLA in an existing crop is highly desirable. To this end, the seed oil of a high LA cultivated species of safflower (Carthamus tinctorius) was modified by transformation with Δ6-desaturase from Saprolegnia diclina resulting in levels exceeding 70% (v/v) of GLA. Levels around 50% (v/v) of GLA in seed oil was achieved when Δ12-/Δ6-desaturases from Mortierella alpina was over-expressed in safflower cultivars with either a high LA or high oleic (OA; C18:1Δ9 cis) background. The differences in the overall levels of GLA suggest the accumulation of the novel fatty acid was not limited by a lack of incorporation into the triacylgylcerol backbone (>66% GLA achieved), or correlated with gene dosage (GLA levels independent of gene copy number), but rather reflected the differences in Δ6-desaturase activity from the two sources. To date, these represent the highest accumulation levels of a newly introduced fatty acid in a transgenic crop. Events from these studies have been propagated and recently received FDA approval for commercialization as Sonova™400.

Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds

An acyl-CoA-binding protein changes the acyl-CoA and oil composition in seeds of transgenic Arabidopsis. Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1cisΔ11) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2cisΔ9,12; 17.9%–44.4% and 7%–13.2%, respectively) and decreases in 20:1cisΔ11 (38.7%–60.7% and 13.8%–16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3cisΔ9,12,15) in both the acyl-CoA pool and seed oil of the former (48.4%–48.9% and 5.3%–10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil.

Phytosynthetics: Transgenic Plants as the Primary Source of Industrial and Medical Feedstocks in the 21st Century

With the completion of the sequencing of the Arabidopsis genome in 2000, and the success of numerous sequencing projects, including the human genome, we have entered a new era of plant biotechnology. First, we are beginning to understand at a functional level the roles of whole classes of genes and their protein products. Second, we have at our disposal an enormous resource of genes, including those specifying developmental patterns and those encoding entire metabolic pathways, which can be mobilized into transgenic plants. As a result, we have moved out of the “observational” phase of plant biology into a “synthetic” phase in which we can design plants to perform valuable functions for industry, medicine and the environment. Over the next 25 years, driven by the depletion of fossil fuel supplies and attempts to limit greenhouse gas emission, we will be obliged to develop plants capable of supplying oleochemical feedstocks, biodegradeable structural materials and a wide variety of pharmaceuticals and nutraceuticals. This will require sophisticated approaches to the genetic manipulation of plants and the processing of plant-derived products. In this presentation, the achievements of plant biotechnology and its potential for the sustainable production of oleochemicals, biodegradeable plastics, high-value proteins and complex organic compounds will be evaluated. The necessity to meet the needs for these products using plant resources will be supported using technological, economic and environmental criteria.