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.

Production of biologically active hirudin in plant seeds using oleosin partitioning.

A plant oleosin was used as a 'carrier' for the production of the leech anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusion protein was expressed and accumulated in seeds. Seed-specific expression of the oleosin-hirudin fusion mRNA was directed via an Arabidopsis oleosin promoter. The fusion protein was correctly targeted to the oil body membrane and separated from the majority of other seed proteins by flotation centrifugation. Recombinant hirudin was localized to the surface of oil bodies as determined by immunofluorescent techniques. The oleosin-hirudin fusion protein accumulated to ca. 1% of the total seed protein. Hirudin was released from the surface of the oil bodies using endoprotease treatment. Recombinant hirudin was partially purified through anion exchange chromatography and reverse-phase chromatography. Hirudin activity, measured in anti-thrombin units (ATU), was observed in seed oil body extracts, but only after the proteolytic release of hirudin from its oleosin 'carrier'. About 0.55 ATU per milligram of oil body protein was detected in cleaved oil body preparations. This activity demonstrated linear dose dependence. The oleosin fusion protein system provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for purification of the desired polypeptide.

Plant seed oil-bodies as carriers for foreign proteins.

Plant seeds frequently store oils (triglycerides) in discrete organelles called oil-bodies. These are normally surrounded by a phospholipid half-unit membrane equipped with specialized proteins called oleosins. Oleosins are highly lipophilic proteins, are expressed at high levels in many seeds and are specifically targeted to oil-bodies. We have investigated the potential of oleosins to act as carriers for recombinant proteins by the production of translational fusions between oleosins and genes encoding proteins foreign to plant cells. We have shown that a fusion comprising a complete oleosin coding domain and a beta-glucuronidase coding sequence may be expressed specifically in the seeds of the oilseed crop plant, Brassica napus, and its product is correctly targeted with approximately 80% of the activity partitioning with oil-bodies. Recombinant oil-bodies may be used to facilitate separation of a recombinant protein from other cellular proteins. Using this approach, the desired protein may be cleaved from the oil-bodies using an endoprotease and further purified. Alternatively, a fusion protein which is enzymatically active and resides on the oil-bodies may be used directly in heterogeneous catalysis. In this application, after a round of catalysis the oil-bodies may be recovered and re-used several times without loss of activity. Thus the oil-bodies act as an immobilization matrix. The fusion protein is stable in dry seeds for long periods and when extracted has a half-life of 3-4 weeks on oil-bodies. Finally, the production of these recombinant oil-bodies is extremely inexpensive, offering a novel route to the manufacture of recombinant proteins.

Regulation of an Arabidopsis oleosin gene promoter in transgenic Brassica napus.

Progressive deletions of the 5'-flanking sequences of an Arabidopsis oleosin gene were fused to beta-glucuronidase (GUS) and introduced into Brassica napus plants using Agrobacterium-mediated transformation. The effect of these deletions on the quantitative level of gene expression, organ specificity and developmental regulation was assessed. In addition, the influence of abscisic acid (ABA), jasmonic acid (JA), sorbitol and a combined ABA/sorbitol treatment on gene expression was investigated. Sequences that positively regulate quantitative levels of gene expression are present between -1100 to -600 and -400 to -200 of the promoter. In addition, sequences present between -600 and -400 down-regulate quantitative levels of expression. In transgenic B. napus plants, the oleosin promoter directs seed-specific expression of GUS which is present at early stages of seed development and increases throughout seed maturation. Sequences present between -2500 and -1100 of the promoter are involved in modulating the levels of expression at early stages of embryo development. Histochemical staining of embryos demonstrated that expression is uniform throughout the tissues of the embryo. Sequences involved in the response to ABA and sorbitol are present between -400 and -200. The induction of GUS activity by a combined ABA/sorbitol treatment is additive suggesting that ABA is not the sole mediator of osmotically induced oleosin gene expression. A response to JA was only observed when the oleosin promoter was truncated to -600 suggesting that the reported effect of JA on oleosin gene expression may be at a post-transcriptional level.

Oilbody Proteins in Microspore-Derived Embryos of Brassica napus: Hormonal, Osmotic, and Developmental Regulation of Synthesis.

A number of treatments were tested for their ability to affect the synthesis of oilbody proteins in microspore-derived embryos of rapeseed (Brassica napus). Synthesis of the oilbody proteins was determined by [(35)S]methionine incorporation in vivo and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of washed oilbody fractions. Oilbody proteins of approximately 19, 23, and 32 kilodaltons were found to be prominent. These proteins showed differential patterns of regulation. The 19 and 23 kilodalton proteins (oleosins) were greatly enhanced by treatments with abscisic acid, jasmonic acid, and osmotic stress imposed using sorbitol (12.5%). Synthesis of the 32 kilodalton protein was inhibited by abscisic acid and by sorbitol (12.5%), but unaffected by jasmonates. The strong promotion of synthesis of the 19 and 23 kilodalton oilbody proteins appeared to be specific as they are not seen with gibberellic acid treatment or with a stress such as heat shock. Time course experiments revealed that the abscisic acid stimulation of oleosin synthesis is quite rapid (less than 2 hours), reaching a maximum at 6 to 8 hours. The response of the oleosins to abscisic acid is found in all stages of embryogenesis, with a major increase in synthetic rates even in globular embryos on abscisic acid treatment. This suggests that these proteins may accumulate much earlier in embryogenesis than has previously been believed. The 32 kilodalton oilbody-associated protein appears different from the oleosins in several ways, including its distinct pattern of regulation and its unique property, among the oilbody proteins, of undergoing phosphorylation.

Effects of jasmonic Acid on embryo-specific processes in brassica and linum oilseeds.

A number of effects on embryogenesis of the putative phytohormone jasmonic acid (JA), and its methyl ester (MeJA), were investigated in two oilseed plants, repeseed (Brassica napus) and flax (Linum usitatissimum). Results from treatments with JA and MeJA were compared with those of a known effector of several aspects of embryogenesis, abscisic acid (ABA). Jasmonic acid was identified by gas chromatography-mass spectrometry as a naturally occurring substance in both plant species during embryo development. Both JA and MeJA can prevent precocious germination of B. napus microspore embryos and of cultured zygotic embryos of both species at an exogenous concentration of >1 micromolar. This dose-response was comparable with results obtained with ABA. Inhibitory effects were also observed on seed germination with all three growth regulators in rapeseed and flax. A number of molecular aspects of embryogenesis were also investigated. Expression of the B. napus storage protein genes (napin and cruciferin) was induced in both microspore embryos and zygotic embryos by the addition of 10 micromolar JA. The level of napin and cruciferin mRNA detected was similar to that observed when 10 micromolar ABA was applied to these embryos. For MeJA only slight increases in napin or cruciferin mRNA were observed at concentrations of 30 micromolar. Several oilbody-associated proteins were found to accumulate when the embryos were incubated with either JA or ABA in both species. The MeJA had little effect on oilbody protein synthesis. The implications of JA acting as a natural regulator of gene expression in zygotic embryogenesis are discussed.

Cloning and sequencing of the gene encoding cytochrome c553 from Desulfovibrio vulgaris Hildenborough.

The gene encoding cytochrome c553 from Desulfovibrio vulgaris Hildenborough was cloned by using two synthetic deoxyoligonucleotide probes. The amino acid sequence derived from the sequence of the gene differs from that reported by Bruschi and LeGall (Biochim. Biophys. Acta 271:48-60, 1972). Renewed protein sequencing confirmed the correctness of the DNA-derived sequence. The gene sequence indicates cytochrome c553 to be synthesized as a precursor protein with an NH2-terminal signal sequence of 24 residues.

Effects of atenolol and propranolol when added to long-term antihypertensive diuretic therapy.

The antihypertensive effects of atenolol and propranolol were compared in a double-blind crossover study of 19 patients with essential hypertension (World Health Organization, I and II) who were receiving long-term diuretic treatment (chlorthalidone, 50 mg daily) during the study. After a 3-wk placebo period, a beta-adrenergic antagonist was administered once daily (atenolol, 50 mg daily, or propranolol, 80 mg daily) for a week. If the MAP was more than 108 mm Hg at the end of the week, dosage of the beta-blocker was doubled the following week; when necessary, doubling was repeated to a maximum dose of 640 mg propranolol and 400 mg atenolol daily. Fifty milligrams atenolol had a greater effect than 80 mg propranolol and was as effective as 160 mg propranolol. The dose-response curve flattened off after 160 mg propranolol and 50 mg atenolol daily. The two highest doses of atenolol lowered MAP more than the highest doses of propranolo. Heart rate slowing was the same for both drugs and did not correlate with the fall in blood pressure. PRA was suppressed by all doses of propranolol, whereas atenolol suppressed PRA only at the 2 highest doses, (200 and 400 mg daily). With the lower propranolol doses, the percent MAP change correlated weakly with the percent PRA change (80 mg--r = 0.41, p less than 0.1; 160 mg--r = 0.64, p less than 0.05). Side effects were minimal, and were noted only with 640 mg propranolol; with this exception, the percentage of patients with no complaints rose when placebo was replaced by beta-blockers.