Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds

Cristina Sarmiento, Joanne H E Ross, Eliot M Herman, Denis J. Murphy

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp end 1 kbp, respectively, of promoter sequence, were inserted separately into repassed plants. T2 seeds from five independent transgenic lines, three expressing isoform A end two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed-specific and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas 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 relatively high levels of soybean oleosin protein did not affect the amount of endogenous repassed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remainder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10-1.14 g ml-1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with oil bodies, none of the membrane-associated oleosins 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 studies of transgenic repassed revealed that all oil bodies examined could be labelled with both the soybean or repassed anti-oleosin antibodies, indicating that each oil body contained a mixed population of soybean and repassed oleosins. A small but significant proportion of both soybean 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 bodies in vivo and of possible associated conformational/processing changes in the protein. Although oil-body formation per se can occur independently 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.

Original languageEnglish (US)
Pages (from-to)783-796
Number of pages14
JournalPlant Journal
Volume11
Issue number4
DOIs
StatePublished - Apr 1997
Externally publishedYes

Fingerprint

oleosin
Brassica rapa
lipid bodies
rapeseed
Soybeans
Seeds
genetically modified organisms
soybeans
seeds
Endoplasmic Reticulum
Protein Isoforms
Soybean Proteins
Transgenes
endoplasmic reticulum
Membranes
Lipid Droplets
Desiccation
Proteins
Density Gradient Centrifugation
Cotyledon

ASJC Scopus subject areas

  • Plant Science

Cite this

Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds. / Sarmiento, Cristina; Ross, Joanne H E; Herman, Eliot M; Murphy, Denis J.

In: Plant Journal, Vol. 11, No. 4, 04.1997, p. 783-796.

Research output: Contribution to journalArticle

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title = "Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds",
abstract = "Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp end 1 kbp, respectively, of promoter sequence, were inserted separately into repassed plants. T2 seeds from five independent transgenic lines, three expressing isoform A end two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed-specific and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas 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 relatively high levels of soybean oleosin protein did not affect the amount of endogenous repassed oleosin. Immunoblotting studies showed that about 95{\%} of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remainder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10-1.14 g ml-1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with oil bodies, none of the membrane-associated oleosins 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 studies of transgenic repassed revealed that all oil bodies examined could be labelled with both the soybean or repassed anti-oleosin antibodies, indicating that each oil body contained a mixed population of soybean and repassed oleosins. A small but significant proportion of both soybean 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 bodies in vivo and of possible associated conformational/processing changes in the protein. Although oil-body formation per se can occur independently 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.",
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