A novel role for oleosins in freezing tolerance of oilseeds in <i>Arabidopsis thaliana</i>

<jats:title>Summary</jats:title><jats:p>Oil bodies in seeds of higher plants are surrounded with oleosins. Here we demonstrate a novel role for oleosins in protecting oilseeds against freeze/thaw‐induced damage of their cells. We detected four oleosins in oil bodies isolated from seeds of <jats:italic>Arabidopsis thaliana</jats:italic>, and designated them OLE1, OLE2, OLE3 and OLE4 in decreasing order of abundance in the seeds. For reverse genetics, we isolated oleosin‐deficient mutants (<jats:italic>ole1</jats:italic>, <jats:italic>ole2</jats:italic>, <jats:italic>ole3</jats:italic> and <jats:italic>ole4</jats:italic>) and generated three double mutants (<jats:italic>ole1 ole2</jats:italic>, <jats:italic>ole1 ole3</jats:italic> and <jats:italic>ole2 ole3</jats:italic>). Electron microscopy showed an inverse relationship between oil body sizes and total oleosin levels. The double mutant <jats:italic>ole1 ole2</jats:italic>, which had the lowest levels of oleosins, had irregular enlarged oil‐containing structures throughout the seed cells. Germination rates were positively associated with oleosin levels, suggesting that defects in germination are related to the expansion of oil bodies due to oleosin deficiency. We found that freezing followed by imbibition at 4°C abolished seed germination of single mutants (<jats:italic>ole1</jats:italic>, <jats:italic>ole2</jats:italic> and <jats:italic>ole3</jats:italic>), which germinated normally without freezing treatment. The treatment accelerated the fusion of oil bodies and the abnormal‐positioning and deformation of nuclei in <jats:italic>ole1</jats:italic> seeds, which caused seed mortality. In contrast, <jats:italic>ole1</jats:italic> seeds that had undergone freezing treatment germinated normally when incubated at 22°C instead of 4°C, because degradation of oils abolished the acceleration of fusion of oil bodies during imbibition. Taken together, our findings suggest that oleosins increase the viability of over‐wintering oilseeds by preventing abnormal fusion of oil bodies during imbibition in the spring.</jats:p>