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Here we describe a protocol to express proteins into protoplasts by using PEG-mediated transformation method. The method provides easy expression of proteins of interest, and efficient investigation of protein localization and the import process for various experimental conditions in vivo.
The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many secondary metabolites and lipids. Chloroplasts require a large number of proteins for these various physiological processes. Over 95% of chloroplast proteins are nucleus-encoded and imported into chloroplasts from the cytosol after translation on cytosolic ribosomes. Thus, the proper import or targeting of these nucleus-encoded chloroplast proteins to chloroplasts is essential for the proper functioning of chloroplasts as well as the plant cell. Nucleus-encoded chloroplast proteins contain signal sequences for specific targeting to chloroplasts. Molecular machinery localized to the chloroplast or cytosol recognize these signals and carry out the import process. To investigate the mechanisms of protein import or targeting to chloroplasts in vivo, we developed a rapid, efficient protoplast-based method to analyze protein import into chloroplasts of Arabidopsis. In this method, we use protoplasts isolated from leaf tissues of Arabidopsis. Here, we provide a detailed protocol for using protoplasts to investigate the mechanism by which proteins are imported into chloroplasts.
The chloroplast is one of the most important organelles in plants. One of the main functions of chloroplasts is to carry out photosynthesis1. Chloroplasts also carry out many other biochemical reactions for the production of fatty acids, amino acids, nucleotides, and numerous secondary metabolites1,2. For all of these reactions, chloroplasts require a large number of different types of proteins. However, the chloroplast genome contains only approximately 100 genes3,4. Therefore, chloroplasts must import the majority of their pro....
1. Growth of Arabidopsis Plants
The import of proteins into chloroplasts can be examined using two approaches: fluorescence microscopy and immunoblot analysis after SDS-PAGE-mediated separation. Here, we used RbcS-nt:GFP, a fusion construct encoding the 79 N-terminal amino acid residues of RbcS containing the transit peptide fused to GFP. When proteins are imported into chloroplasts, green fluorescence signals from the target protein RbcS-nt:GFP should merge with the red fluorescent signals from chlorophyll auto-fluores.......
We provided a detailed protocol for the use of protoplasts of Arabidopsis to study protein import into chloroplasts. This method is powerful for investigating the protein import process. This simple, versatile technique is useful for examining the targeting of the intended cargo proteins to chloroplasts. Using this method, protoplasts are prepared from leaf tissues of Arabidopsis11,12 which can be obtained from plants at many different growth st.......
This work was carried out with the supports of Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ010953012018), Rural Development Administration, and the National Research Foundation (Korea) grant funded by the Ministry of Science and ICT (No. 2016R1E1A1A02922014), Republic of Korea.
....Name | Company | Catalog Number | Comments |
GAMBORG B5 MEDIUM INCLUDING VITAMINS | Duchefa Biochemie | G0210.0050 | |
SUCROSE | Duchefa Biochemie | S0809.5000 | |
MES MONOHYDRATE | Duchefa Biochemie | M1503.0250 | |
Agar, powder | JUNSEI | 24440S1201 | |
Micropore Surgical tape | 3M | 1530-0 | |
Surgical blade stainless No.10 | FEATHER | Unavailable | |
Conical Tube, 50ml | SPL LIFE SCIENCES | 50050 | |
Macerozyme R-10 | YAKULT PHARMACEUTICAL IND. | Unavailable | |
Cellulase ONOZUKA R-10 | YAKULT PHARMACEUTICAL IND. | Unavailable | |
ALBUMIN, BOVINE (BSA) | VWR | 0332-100G | |
D-Mannitol | SIGMA | M1902-1KG | |
CALCIUM CHLORIDE, DIHYDRATE | MP BIOMEDICALS | 0219463505-5KG | |
Twister | VISION SCIENTIFIC | VS-96TW | |
Screen cup for CD-1 | SIGMA | S1145 | |
Screens for CD-1 | SIGMA | S3895 | |
Petri Dish | SPL LIFE SCIENCES | 10090 | |
Pasteur pipette | HILGENBERG | 3150102 | |
LABORATORY CENTRIFUGE / BENCH-TOP | VISION SCIENTIFIC | VS-5500N | |
Sodium chloride | JUNSEI | 19015S0350 | |
Potassium chloride | SIGMA | P3911-1KG | |
D-GLUCOSE, ANHYDROUS | BIO BASIC | GB0219 | |
Potassium Hydroxide | DUKSAN | 40 | |
Calcium nitrate tetrahydrate | SIGMA | C2786-500G | |
Poly(ethylene glycol) | SIGMA | P2139-2KG | |
Magnesium chloride hexahydrate | SIGMA | M2393-500G | |
Tube 13ml, 100x16mm, PP | SARSTEDT | 55.515 | |
Microscope slides | MARIENFELD | 1000412 | |
Microscope Cover Glasses | MARIENFELD | 101030 | |
Counting Chamber | MARIENFELD | 650030 | |
Axioplan 2 Imaging Microscope | Carl Zeiss | Unavailable | |
Micro tube 1.5ml | SARSTEDT | 72.690.001 | |
2-Mercaptoethanol | SIGMA | M3148-250ML | |
Sodium Dodecyl Sulfate (SDS), Proteomics Grade | VWR | M107-500G | |
TRIS, Ultra Pure Grade | VWR | 0497-5KG | |
DTT (DL-Dithiothreitol), Biotechnology Grade | VWR | 0281-25G | |
Bromophenol blue sodium salt ACS | VWR | 0312-50G | |
Glycerol | JUNSEI | 27210S0350 | |
Living Colors A.v. Monoclonal Antibody (JL-8) | TAKARA | 632381 |
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