Abstract
Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with 35S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.
Highlights
Chloroplasts are highly abundant organelles that exist in the green tissues of plants
For chloroplasts isolated from plants grown under normal conditions, there was no obvious difference in PsaD import between sp[1] and WT3
While we observed the accumulation of the mature protein form in a time-dependent manner with both genotypes, the rate of import was significantly lower for WT chloroplasts than for sp[1] chloroplasts (Figure 1), which is consistent with our previous results 3, and reveals an important role for the SP1 protein in regulating the chloroplast import of PsaD under stress conditions
Summary
Chloroplasts are highly abundant organelles that exist in the green tissues of plants They are well-known for their critical role in photosynthesis, a process that uses light energy to convert carbon dioxide to sugar and support almost all life on earth 1. Recent work has indicated that chloroplast protein import is actively regulated, and so is able to exert an important level of control over the chloroplast proteome It was reported in 2015 that protein import can respond to abiotic stress through the direct regulation of the abundance of the translocon at the outer envelope membrane of chloroplasts (TOC) by the ubiquitin-proteasome system 3. To study the regulation of protein import by stress, we have modified our routine chloroplast isolation method as we will describe here. Www.jove.com addition to the chloroplast isolation protocol, we present our routine method for in vitro protein import, which has proven to be robust and is widely used 3,9-12
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