Abstract
Stromules are stroma-containing tubules that have been observed to emanate from the main plastidic body in vivo. These structures have been shown to require cytoskeletal components for movement. Though numerous studies have shown a close association with the endoplasmic reticulum, nucleus, mitochondria, and other plastids, the mechanism of formation and their overall function remain unknown. A limiting factor in studying these structures has been the lack of a reconstituted system for in vitro stromule formation. In this study, stromule formation was induced in vitro by adding a plant extract fraction that is greater than 100 kDa to a population of isolated chloroplasts. Kinetic measurements show that stromule formation occurs within ~10 seconds after the addition of the plant extract fraction. Heat inactivation and apyrase treatment reveal that the stromule stimulating compound found in the extract fraction is a protein or protein complex 100 kDa or greater. The formation of the stromules in vitro with isolated chloroplasts and a concentrated fraction of cell extract opens an avenue for the biochemical dissection of this process that has heretofore been studied only in vivo.
Highlights
Plastids are double membrane enclosed organelles that carry out essential functions in plants
Chloroplasts that were treated with a concentrated plant extract fraction prepared by using a 100 kDa molecular weight cutoff filter displayed a significant change in their morphology (Fig 1A– 1C)
The obvious advantages to application of in vitro techniques to the elucidation of aspects of stromule formation prompted us to attempt to induce stromule development using isolated chloroplasts. To this end we monitored via confocal microscopy the response to various additions of isolated chloroplasts from N. benthamiana in which Cerulean is expressed in the stroma
Summary
Plastids are double membrane enclosed organelles that carry out essential functions in plants. These tasks range from manufacturing and storing starch in leucoplasts to conversion of light energy into sugars as seen in chloroplasts. The adoption of fluorescent proteins by cell biologists has allowed researchers to monitor the structural changes that organelles undergo in vivo. While tubular projections emanating from plastids had been noted as long as 107 years ago [1], they did not receive much attention until they were observed using a stromal protein fused to GFP [2]. Plastids without chlorophyll and those found in areas of low plastid density display a higher frequency of stromule formation [3, 10]. Temperature, or pathogenic stresses exhibit a higher
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