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Biochemistry

Studying Protein Import into Chloroplasts Using Protoplasts

Published: December 10th, 2018

DOI:

10.3791/58441

1Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, 2Department of Life Sciences, Pohang University of Science and Technology

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....

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1. Growth of Arabidopsis Plants

  1. Prepare 1 L Gamborg B5 (B5) medium by adding 3.2 g of B5 medium including vitamins, 20 g of sucrose, 0.5 g of 2-(N-morpholino) ethane sulfonic acid (MES) to approximately 800 mL of deionized water, and adjust the pH to 5.7 with potassium hydroxide (KOH). Add more deionized water bring the total volume to 1 L. Add 8 g of phytoagar and autoclave for 15 min at 121 °C.
  2. Allow the medium to cool down to 55 °C and pour 20 – 25 mL of the B5 medium into a Petri.......

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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.......

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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.......

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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.

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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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