Involvement of caleosin in oil body biogenesis and degradation

Caleosins are proteins embedded in the half unit membranes of oil bodies in many plant seeds. Caleosin's function in oil bodies has yet to be determined. Early in Brassica napus seed development CLO1 is associated with the microsomal membrane fraction, whereas in later stages of development and mature seeds it is associated solely with oil bodies. These observations raised the possibility that CLO1 might participate in creating microdomains in the ER from which oil bodies bud. Immunofluorescent and immunocytochemical microscopy of seeds from two separate Atclo1 insertion mutants determined that caleosin is not involved in oil body formation.; However, previous micrographs showing caleosin-labeled oil bodies inside vacuoles led us to hypothesize that caleosin may be involved in facilitating the uptake of oil bodies across the tonoplast to the interior of vacuoles. In wild type embryo cells, two experimental approaches documented uptake of oil bodies and vacuole membrane into vacuoles during germination. Transmission electron micrographs of high pressure-rapid frozen tissue demonstrated oil bodies surrounded by tonoplast inside vacuoles, and immunofluorescence studies demonstrated both alpha- and delta-tonoplast intrinsic proteins as well as the oil body proteins caleosin and oleosin within vacuoles. To study the role of caleosin in the oil body mobilization during germination, the germination of seeds from the Atclo1 mutants were examined. Embryo cells from the caleosin insertion mutant lines contained twice as many oil bodies in the cytoplasm, and smaller, distorted vacuoles that lacked internal oil bodies. These results support the concept that oil bodies are taken up into vacuoles during germination by a process similar to autophagy, and that caleosin plays an essential role in that process. Thus, caleosin is the first plant protein to be associated functionally with autophagy into vacuoles.