The lipoxygenase-dependent oxygenation of lipid body membranes is promoted by a patatin-type phospholipase in cucumber cotyledons

Abstract

Oilseed germination is characterized by the mobilization of storage lipids as a carbon and energy source for embryonic growth. In addition to storage lipid degradation in germinating oilseeds via the direct action of a triacylglycerol lipase (TGL) on the storage lipids, a second degradation pathway that is dependent on a specific lipid body trilinoleate 13-lipoxygenase (13-LOX) has been proposed in several plant species. The activity of this specific 13-LOX leads first to the formation of ester lipid hydroperoxides. These hydroperoxy fatty acids are then preferentially cleaved off by a TGL and serve as a substrate for glyoxysomal β-oxidation. As a prerequisite for triacylglycerol (TAG) mobilization, a partial degradation of the phospholipid monolayer and/or membrane proteins of the oil body has been discussed. Evidence has now been found for both processes: partial degradation of the proteins caleosin and oleosin was observed and simultaneously a patatin-like protein together with transient phospholipase (PLase) activity could be detected at the oil body membranes during germination. Moreover, in vitro experiments with isolated oil bodies from mature seeds revealed that the formation of 13-LOX-derived lipid peroxides in lipid body membranes is increased after incubation with the purified recombinant patatin-like protein. These experiments suggest that in vivo the degradation of storage lipids in cucumber cotyledons is promoted by the activity of a specific oil body PLase, which leads to an increased decomposition of the oil body membrane by the 13-LOX and thereby TAGs may be better accessible to LOX and TGL.

Fig. 1.

Time course of lipid body-associated proteins during germination of cucumber. Proteins were prepared from isolated lipid bodies of cotyledons harvested at the time points indicated, separated by SDS–PAGE, and analysed by western blotting using anti-CsLbLOX, anti-CsPAT, anti-caleosin, and anti-oleosin antiserum.

Fig. 2.

Intracellular localization of CsLbLOX and CsPAT. Transmission electron microscope images of cotyledons from 72 h germinated etiolated seedlings. Immunogold labelling with antibodies against CsLbLOX (A, B) and CsPAT (C, D) with significant localization of the 5 nm gold particles at the membrane of the lipid bodies. Cy, cytoplasm; LB, lipid body. Scale bars represent 200 μm.

Fig. 3.

Analysis of PLase A₂ and TGL activity. (A) Analysis of the pH optimum of recombinant CsPAT acting on PC. PLase A₂ activity was measured by incubation of affinity-purified CsPAT with 2-linoleoyl-PC at various pH values, followed by determining the release of linoleic acid using reversed-phase HPLC. Relative activity was calculated based on the highest value at pH 8. (B) PLase A₂ activity at isolated lipid bodies incubated with ¹⁴C-labelled PC. (C) TGL activity at isolated lipid bodies incubated with ¹⁴C-labelled triolein. Activity was detected by TLC and quantified by 2D densitometry. Activities are given per mg lipid body fresh weight (B and C). Data represent the mean of two independent experiments.

Fig. 4.

Profile of lipid body membrane phospholipids during germination. Phospholipids were extracted from isolated lipid bodies and subjected to TLC. Individual phospholipids were quantified by 2D densitometry: (A) PC; (B) lyso-PC; (C) PE, PI, and PS. Amounts are given in μg mg⁻¹ lipid body fresh weight. Data shown are the means of three independent experiments. For PS, data from two experiments are available. The standard deviation is given. *P=0.098;**P=0.037 by Student's t-test.

Fig. 5.

Analysis of LOX activity, product accumulation, and substrate consumption during germination. (A) LOX activity at isolated lipid bodies measured polarographically with an oxygen electrode. (B) The LA-derived LOX product 13-H(P)OD determined by transmethylation of the lipid extract from isolated lipid bodies, purification via reversed-phase HPLC, and subsequent separation by straight-phase HPLC. (C) LOX substrate LA in TAGs of lipid bodies analysed by transmethylation of lipid extract from isolated lipid bodies and following GC. Amounts are given in nmol mg⁻¹ lipid body fresh weight (B and C). Data shown are the means of three (A and B) and four independent experiments (C), respectively. The standard deviation is given. *P=0.037 by Student's t-test.

Fig. 6.

TLC analysis of neutral lipids of isolated lipid bodies during germination. Lipid extracts of lipid bodies were fractionated by Strata SI-1 silica columns. Neutral lipids were separated by TLC, visualized in aqueous CuSO₄ with subsequent heating, and identified by co-migration of standards as indicated in the Materials and methods. Oxygenated TAG species were quantified by 2D densitometry. (A) TLC of neutral lipids. (B) Content of oxygenated TAG species during germination. The amount is given in μg mg⁻¹ lipid body fresh weight. Data shown are the means of three independent experiments. At the 96 h time point, data from one experiment are available. The standard deviation is given. TAG, triacylglycerol; DAG, diacylglycerol; 3oxTAG, trihydro(pero)xy derivative of TAG; 2oxTAG, dihydro(pero)xy derivative of TAG; 1oxTAG, monohydro(pero)xy derivative of TAG; 1epoxyTAG, monoepoxy derivative of TAG. *P=0.012, 0.049, and 0.001 by Student's t-test.

Fig. 7.

In vitro analysis of the interaction of recombinant CsLbLOX and recombinant CsPAT. Lipid bodies (Lb) of seeds imbibed for two hours were incubated with CsLbLOX and/or C-terminal His-tagged CsPAT (CsPATC), as indicated, for 120 min. Lipids were extracted, separated into phospho- and neutral lipids, lipid hydroperoxides were reduced, transmethylated, and the 13-hydro(pero)xylinoleic acid/linoleic acid ratio (13-H(P)OD/LA ratio) was determined by reversed-phase HPLC. Ratios of the TAG fraction are shown as grey bars and ratios of the phospholipid fraction are shown as black bars. Data shown represent the mean of three independent experiments. The standard deviation is given. *P=0.00009 by Student's t-test.