EXPRESSION AND SUBCELLULAR TARGETING OF A SOYBEAN OLEOSIN IN TRANSGENIC RAPESEED - IMPLICATIONS FOR THE MECHANISM OF OIL-BODY FORMATION IN SEEDS

Two genomic clones, encoding isoforms A and B of the 24 kDa soybean ol eosin and containing 5 kbp and 1 kbp, respectively, of promoter sequen ce, were inserted separately into rapeseed plants. T-2 seeds from five independent transgenic lines, three expressing isoform A and two expr essing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exh ibited the same patterns of mRNA and protein accumulation as the resid ent rapeseed oleosins, i.e. their expression was absolutely seed-speci fic and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bo dies, despite the absence of a soybean partner isoform. The soybean pr otein accumulated in young embryos mainly as a 23 kDa polypeptide, whe reas a 24 kDa protein predominated later in development. The ratio of rapeseed:soybean oleosin in the transgenic plants was about 5:1 to 6:1 , as determined by SDS-PAGE and densitometry. Accumulation of these re latively high levers of soybean oleosin protein did not affect the amo unt of endogenous rapeseed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogeno us 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remai nder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membra ne fraction of buoyant density 1.10-1.14 g ml(-1), which partially ove rlapped with several endoplasmic reticulum (ER) markers. Unlike oleosi ns associated with oil bodies, none of the membrane-associated oleosin s could be immunoprecipitated in the presence of protein A-Sepharose, indicating a possible conformational difference between the two pools of oleosin. Complementary electron microscopy-immunocytochemical studi es of transgenic rapeseed revealed that all oil bodies examined could be labelled with both the soybean or rapeseed anti-oleosin antibodies, indicating that each oil body contained a mixed population of soybean and rapeseed oleosins. A small but significant proportion of both soy bean and rapeseed oleosins was located on ER membranes in the vicinity of oil bodies, but none were detected on the bulk ER cisternae. This is the first report of apparent targeting of oleosins via ER to oil bo dies in vivo and of possible associated conformational/processing chan ges in the protein. Although oil-body formation per se can occur indep endently of oleosins, it is proposed that the relative net amounts of oleosin and oil accumulated during the course of seed development are a major determinant of oil-body size in desiccation-tolerant seeds.