Lipid and protein accumulation in developing seeds of three lupine species: Lupinus luteus L., Lupinus albus L., and Lupinus mutabilis Sweet

Abstract

A comparative study was carried out on the dynamics of lipid accumulation in developing seeds of three lupine species. Lupine seeds differ in lipid content; yellow lupine (Lupinus luteus L.) seeds contain about 6%, white lupine (Lupinus albus L.) 7-14%, and Andean lupine (Lupinus mutabilis Sweet) about 20% of lipids by dry mass. Cotyledons from developing seeds were isolated and cultured in vitro for 96 h on Heller medium with 60 mM sucrose (+S) or without sucrose (-S). Each medium was additionally enriched with 35 mM asparagine or 35 mM NaNO3. Asparagine caused an increase in protein accumulation and simultaneously decreased the lipid content, but nitrate increased accumulation of both protein and lipid. Experiments with [1-14C]acetate and [2-14C]acetate showed that the decrease in lipid accumulation in developing lupine seeds resulted from exhaustion of lipid precursors rather than from degradation or modification of the enzymatic apparatus. The carbon atom from the C-1 position of acetate was liberated mainly as CO2, whereas the carbon atom from the C-2 position was preferentially used in anabolic pathways. The dominant phospholipid in the investigated lupine seed storage organs was phosphatidylcholine. The main fatty acid in yellow lupine cotyledons was linoleic acid, in white lupine it was oleic acid, and in Andean lupine it was both linoleic and oleic acids. The relationship between stimulation of lipid and protein accumulation by nitrate in developing lupine cotyledons and enhanced carbon flux through glycolysis caused by the inorganic nitrogen form is discussed.

Fig. 1.

Total lipid level in whole seeds from five developmental stages.

Fig. 2.

Soluble protein content in whole seeds from five developmental stages.

Fig. 3.

Electron micrographs of cotyledons isolated from seeds from developmental stage III (used for preparation of in vitro culture). CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; OB, oil body; S, starch; SP, storage protein; V, vacuole.

Fig. 4.

Total lipid level in cotyledons isolated from seeds from developmental stage III grown in vitro for 96 h. Control, cotyledons isolated from seeds, used for preparation of in vitro culture; +S, medium with 60 mM sucrose; –S, medium without sucrose; +Asn, medium with 35 mM asparagine; +NO₃^–, medium with 35 mM NaNO₃. Statistical significance at *P <0.05; **P <0.01.

Fig. 5.

Electron micrographs of yellow lupine cotyledons isolated from seeds from developmental stage III, grown in vitro for 96 h. +S, medium with 60 mM sucrose; –S, medium without sucrose; +Asn, medium with 35 mM asparagine; +NO₃^–, medium with 35 mM NaNO₃. CW, cell wall; M, mitochondrion; OB, oil body; S, starch; SP, storage protein; V, vacuole.

Fig. 6.

Electron micrographs of white lupine cotyledons isolated from seeds from developmental stage III, grown in vitro for 96 h. +S, medium with 60 mM sucrose; –S, medium without sucrose; +Asn, medium with 35 mM asparagine; +NO₃^–, medium with 35 mM NaNO₃. CW, cell wall; M, mitochondrion; OB, oil body; S, starch; SP, storage protein; V, vacuole.

Fig. 7.

Electron micrographs of Andean lupine cotyledons isolated from seeds from developmental stage III, grown in vitro for 96 h. +S, medium with 60 mM sucrose; –S, medium without sucrose; +Asn, medium with 35 mM asparagine; +NO₃^–, medium with 35 mM NaNO₃. CW, cell wall; M, mitochondrion; OB, oil body; S, starch; SP, storage protein; V, vacuole.

Fig. 8.

Soluble protein content in cotyledons isolated from seeds from developmental stage III, grown in vitro for 96 h. Control, cotyledons isolated from seeds used for preparation of in vitro culture; +S, medium with 60 mM sucrose; –S, medium without sucrose; +Asn, medium with 35 mM asparagine; +NO₃^–, medium with 35 mM NaNO₃. Statistical significance at *P <0.05; **P <0.01.

Fig. 9.

Radioactivity of ¹⁴CO₂ liberated by cotyledons isolated from seeds from developmental stage III, grown in vitro for 96 h and incubated for 120 min in solutions of [1-¹⁴C]acetate (C-1) and [2-¹⁴C]acetate (C-2). Radioactivity of added acetate: 925 kBq. +S, medium with 60 mM sucrose; −S, medium without sucrose; +Asn, medium with 35 mM asparagine; +NO₃⁻, medium with 35 mM NaNO₃. Data are from one (representative) breeding season. Statistical significance at *P <0.05; **P <0.01.

Fig. 10.

Radioactivity of the lipid fraction (2 ml of chloroform:methanol) of cotyledons isolated from seeds from developmental stage III, grown in vitro for 96 h and incubated for 120 min in solutions of [1-¹⁴C]acetate (C-1) and [2-¹⁴C]acetate (C-2). Radioactivity of added acetate: 925 kBq. +S, medium with 60 mM sucrose; −S, medium without sucrose; +Asn, medium with 35 mM asparagine; +NO₃⁻, medium with 35 mM NaNO₃. Data are from one (representative) breeding season. Statistical significance at *P <0.05; **P <0.01.

Fig. 11.

Phospholipid content in cotyledons isolated from seeds from developmental stage III, grown in vitro for 96 h. Control, cotyledons isolated from seeds used for preparation of in vitro culture; +S, medium with 60 mM sucrose; −S, medium without sucrose; +Asn, medium with 35 mM asparagine; +NO₃⁻, medium with 35 mM NaNO₃. Statistical significance at *P < 0.05; **P < 0.01.

Fig. 12.

Fatty acid composition (percentage of total integrated peaks on chromatographs) in cotyledons isolated from seeds from developmental stage III, grown in vitro for 96 h. Control, cotyledons isolated from seeds used for preparation of in vitro culture; +S, medium with 60 mM sucrose; –S, medium without sucrose; +Asn, medium with 35 mM asparagine; +NO₃^–, medium with 35 mM NaNO₃. Statistical significance at *P <0.05.