Here we describe a new method to study protein import into isolated chloroplasts under stress. The method is rapid and straightforward, and can be applied to study the consequences of different stress conditions for chloroplast protein import, and the corresponding regulatory mechanisms.
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.
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 thus support almost all life on earth 1. In addition, chloroplasts (and the broader family of related organelles called plastids) play many other vital roles in plants, including the biosynthesis of amino acids, lipids, pigments, and the sensing of environmental signals such as gravity and pathogen challenge. Photosynthesis generates reactive oxygen species (ROS) as by-products, which under certain circumstances have useful roles, but if overproduced can cause damaging or even lethal effects. The overproduction of ROS is particularly promoted by adverse environmental conditions, and thus chloroplasts are closely linked to responses to abiotic stresses, such as salinity and osmotic stresses 2.
Chloroplasts have a complex structure. Each chloroplast is surrounded by a double-membrane outer layer called the envelope, which consists of outer and inner membranes. Internally, there is another membrane system called the thylakoids, where the light reactions of photosynthesis take place. Between the two membrane systems there is an aqueous compartment call the stroma, which is involved in carbon fixation. A chloroplast may contain up to ~3,000 different proteins, and the vast majority of these proteins are synthesized in the cytosol in precursor form and need to be imported into the organelle through dedicated protein translocons in the envelope membranes 1. Interestingly, 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. For example, 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.
Using purified chloroplasts and in vitro synthesized precursor proteins, protein import can be reconstituted in vitro 4,5. Thus, in vitro methods can be used to assess the rates of import in different mutant plants 6, which has been a critical approach for the analysis of putative components of the protein import machinery and for discovering the mechanisms underlying protein import and its regulation. Moreover, chloroplasts can be processed with further fractionation or protease digestion, following in vitro import, which can facilitate studies on the sub-organellar localization and topology of chloroplast proteins 7,8.
To study the regulation of protein import by stress, we have modified our routine chloroplast isolation method as we will describe here. Importantly, chloroplasts were isolated from plants that had been grown on standard Murashige and Skoog (MS) agar medium for 8 days and then transferred into liquid MS medium supplemented with stressor, providing a relatively short, controlled stress treatment. The yield and competency of chloroplasts isolated from such stress-treated plants are compatible with the downstream in vitro protein import assay 3. In 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.
1. Growth of Arabidopsis Plants and the Stress Treatment
2. Making a Precursor Protein by In Vitro Transcription/Translation
Note: This protocol assumes the use of Arabidopsis photosystem I subunit D precursor (pPsaD) as the template/preprotein, but the method is compatible with others.
3. Chloroplast Isolation
4. Analysis of the Yield and Intactness of Chloroplasts
5. Chloroplast Protein Import
An example chloroplast protein import experiment with 3 time-points is shown in Figure 1. PsaD is an ~18 kD component of photosystem I exposed to the stroma, with a precursor form of ~23 kD16. PsaD was selected here for the in vitro protein import assay because its steady-state levels are elevated in the sp1 mutant, relative to WT, under stress conditions, suggesting a change in its import efficiency in the mutant 3. The sp1 mutant carries a defect in an important regulator of the chloroplast protein import machinery — the SP1 protein 3. For chloroplasts isolated from plants grown under normal conditions, there was no obvious difference in PsaD import between sp1 and WT (data not shown)3. However, using the methods described here to assess PsaD import into chloroplasts isolated from osmotically stressed plants, a clear difference was detected. 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 sp1 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.
Figure 1. A Protein Import Assay Conducted using Chloroplasts Isolated from Plants Grown under Osmotic Stress Conditions. Chloroplasts were isolated from a 10-day-old WT (Col-0) and a sp1 mutant Arabidopsis plants grown under stress conditions (200 mM mannitol) for 2 d. (A) Protein import was conducted using [35S]-methionine-labeled pPsaD and allowed to proceed for 4, 8 and 12 min before analysis by SDS-PAGE and phosphor imaging. In parallel, the pPsaD 10% input control comprising in vitro translated protein (IVT) was analyzed. The precursor (pre) and mature (mat) forms of pPsaD are indicated, while in between there are two bands that likely correspond to truncated or proteolyzed translation products because their intensity did not change during the time course (*). (B) To compare the rates of import into chloroplasts from WT and sp1 plants grown under stress conditions, the intensity of each band corresponding to imported mature protein in A was quantified. All data are expressed as percentages of the amount of imported protein in chloroplasts from WT plants after 12 min. Please click here to view a larger version of this figure.
We recently showed that chloroplast protein import can be actively regulated by stress, which is critical for chloroplast function and plant survival 3. In that study, to monitor such regulation, we modified our chloroplast isolation and in vitro import assay procedures in order to enable assessment of the import capacity of plants grown under stress conditions. The results indicated an important role for SP1 in chloroplast protein import regulation.
Conventional in vitro import assays use plants grown on standard MS agar medium6,17,18. In the case of the sp1 mutant described here, such conventional assays did not reveal any differences in protein import relative to the WT3. However, the role of SP1 in regulating protein import is clearly revealed when protein import is assessed under stress conditions using the methods described herein (Figure 1). While it may not be possible to directly compare import data from the stress conditions assay with those obtained from conventional assays (as the methods employ plants grown in liquid culture and on agar medium, respectively), comparisons between stress and non-stress conditions are feasible provided that the plants are all grown in the same liquid culture medium, with or without stressors.
Compared with the stress treatment on agar medium, liquid culture is more convenient for the treatment of the large numbers of plants needed for in vitro import assays. Moreover, it facilitates the uniform application of the stressor to all plants, which is particularly important for short-term stress treatments. The method presented here has been applied to study the osmotic stress using mannitol treatment, but could easily be adapted to a wide range of other stresses; for example, short-term salt stress and oxidative stress, for which the corresponding stressors could be similarly applied via liquid MS medium. For other types of stresses, we suggest the degree of stress is first optimized; overly severe treatments may have adverse effects on the yield and/or import competence of the isolated organelles.
There are several important steps within the protocol to which one should pay special attention, as detailed below.
The optimal agar concentration for the MS medium can differ (0.6 – 0.9%, w/v) depending on the manufacturer. Thus, it is recommended to empirically optimize the agar concentration before commencing experiments. The medium should not be so soft that it sticks to the tissue at the harvesting step (step 1.9), neither should it be so hard that it inhibits plant root development. The sucrose concentration can also be adjusted according to the plants used. When working with particularly sick mutants, MS medium supplemented with 2 – 3% (w/v) sucrose may help plants to grow better. When applying stress treatments, it is not good to transfer very old plants to the liquid medium (e.g., >14 days old). This is because the more developed roots of older plants are more easily damaged during transferral.
With regard to the transcription/translation system, there are 2 major systems: those based on wheat germ, and those based on rabbit reticulocyte. These kits may use different templates, such as linearized plasmids, unlinearized plasmids, or PCR products. The kits are also specific for the T3, T7, and SP6 promoters. Note that the kit we recommend here is only suitable for use with PCR products and the T7 promoter. However, for unknown reasons, some radiolabeled preproteins made with this system might not work efficiently in the import assay; in such instances, one may consider trying a wheat germ extract system or a reticulocyte lysate system meant for plasmid templates. One may also improve the result of the transcription/translation reaction by modifying the reaction conditions according to the manufacturer's handbook.
It is important to start chloroplast isolation early in the morning (or early in the light cycle of the growth chamber) in order to avoid accumulation of starch inside the chloroplasts due to photosynthesis, which can hinder the isolation of intact organelles. The chloroplast isolation procedure has to be done quickly without any unnecessary delays, and the isolated chloroplasts must always be kept cold. This is to mitigate against the observation that isolated chloroplasts will gradually lose their viability, which is not good. If using newly thawed CIB or HMS buffer, be sure to mix the buffer well before use to obtain a homogeneous solution. The optimal conditions for the homogenization of plant material have been established empirically, and can vary if a different tissue homogenizer is used.
If the different plant genotypes contain similar chlorophyll levels, chlorophyll quantification can be used as an alternative way to normalize the samples before conducting the import assays. Chlorophyll can be determined spectrophotometrically following extraction of a sample of the isolated chloroplasts in 80% (v/v) aqueous acetone 19,20. However, if the import rates of plants showing different chlorophyll contents (e.g., mutants with chlorotic phenotypes) are to be compared, use chloroplast number counting to normalize the chloroplast samples in the import assays. It is particularly important to resuspend the chloroplasts thoroughly in step 3.11. Insufficient resuspension may leave aggregates of chloroplasts, which will make it difficult to count numbers accurately and thus will hinder the correct loading in import reactions. If severe aggregation is seen under the microscope (e.g., aggregates with >10 chloroplast joined together), continue shaking the chloroplast sample on ice until the aggregates are removed. It is easier to resuspend the chloroplasts in a smaller volume of buffer.
It is important to be aware that protein import reactions (Section 5) must be conducted with appropriate precautions because of their radioactive nature. Necessary precautions include: wearing disposable gloves, laboratory clothing and safety glasses, monitoring and decontaminating the working surface and equipment, and disposing of all radioactive waste in an approved waste container. Also keep in mind that the correct osmotic pressure is critical for maintaining the intactness of chloroplasts during the import reactions, and this is chiefly maintained by the HMS buffer. Because 10x HMS is viscous, it should first be warmed up to RT, and then thoroughly mixed, and applied using a cut pipette tip to ensure measurement of accurate volumes. In the import reaction, cold methionine is added to inhibit the incorporation of free radiolabeled methionine into unrelated chloroplast proteins through organellar translation during the incubation step, while BSA is used to minimize proteolysis by acting as a substrate for proteases.
The authors have nothing to disclose.
This work was supported by a grant to PJ from the Biotechnology and Biological Sciences Research Council (BBSRC; grant ref. BB/K018442/1).
Murashige and Skoog basal salt | Melford | M0221 | |
phytoagar | Melford | P1003 | |
2-(N-morpholino) ethane sulfonic acid (MES) | Melford | B2002 | |
triton X-100 | Fisher | BPE151-500 | |
surgical tape (e.g., Micropore, 3M) | 3M | 1530-1 | |
filter paper | Fisher | FB59023 | |
Percoll | Fisher | 10607095 | |
ethylene glycol tetraacetic acid (EGTA) | Sigma | E4378 | |
ethylenediaminetetraacetic acid (EDTA) | Fisher | D/0700/53 | |
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) | Melford | B2001 | |
filtration cloth (Miracloth) | Calbiochem | 475855 | |
50 mL centrifuge tube | Fisher | CFT-595-040M | |
250 mL centrifuge bottle | Fisher | CFT-891-V | |
Polytron (e.g., Kinematica PT10-35) | Fisher | 11010070 | |
Polytron probe (e.g., Kinematica PTA20S) | Fisher | 11030083 | |
centrifuge (e.g., Beckman Coulter Avanti JXN-26, for 50 and 250 mL tubes) | Beckman Coulter | B34182 | |
fixed-angle rotor for 250 mL bottles (e.g., JLA-16.250) | Beckman Coulter | 363930 | |
fixed-angle rotor for 50 mL tubes (e.g., JA-25.50) | Beckman Coulter | 363058 | |
swinging-bucket rotor for 50 mL tubes (e.g., JS-13.1) | Beckman Coulter | 346963 | |
radiolabeled [35S] methionine | Perkin Elmer | NEG072002MC | |
rabbit reticulocyte lysate based cell-free translation system (TNT T7 Quick kit for PCR DNA) | Promega | L5540 | |
phase-contrast microscope (e.g., Nikon Eclipse 80i) | Nikon | unavailable | |
haemocytometer (Improved Neubauer BS748 chamber) | Hawksley Technology | AC1000 | 0.1 mm depth, 1/400 mm2 |
cover glasses | VWR | 16004-094 | 22 mm × 22 mm, thickness 0.13-0.17 mm |
1.5 mL microfuge tubes | Sarstedt | 72.690.001 | |
2 mL microfuge tubes | Starlab | S1620-2700 | |
microfuge (e.g., Eppendorf 5415D) | Eppendorf | unavailable | |
Nanodrop 2000 spectrophotometer or similar | Thermo Fisher | SPR-700-310L | |
gluconic acid (potassium salt) | Fisher | 22932-2500 | |
bovine serum albumin (BSA) | Sigma | A7906 | |
MgATP | Sigma | A9187 | |
methionine | Sigma | M6039 | |
bromophenol blue | Fisher | B/P620/44 | |
glycerol | Fisher | G/0650/17 | |
SDS | Fisher | S/5200/53 | |
Tris Base | Melford | B2005 | |
dithiothreitol (DTT) | Melford | MB1015 | |
image analysis software (e.g., Aida Image Analyzer) | Raytest | unavailable |