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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
GAMBORG B5 MEDIUM INCLUDING VITAMINSDuchefa BiochemieG0210.0050
SUCROSEDuchefa BiochemieS0809.5000
MES MONOHYDRATEDuchefa BiochemieM1503.0250
Agar, powderJUNSEI24440S1201
Micropore Surgical tape3M1530-0
Surgical blade stainless No.10FEATHERUnavailable
Conical Tube, 50mlSPL LIFE SCIENCES50050
Macerozyme R-10YAKULT PHARMACEUTICAL IND.Unavailable
Cellulase ONOZUKA R-10YAKULT PHARMACEUTICAL IND.Unavailable
ALBUMIN, BOVINE (BSA)VWR0332-100G
D-MannitolSIGMAM1902-1KG
CALCIUM CHLORIDE, DIHYDRATEMP BIOMEDICALS0219463505-5KG
TwisterVISION SCIENTIFICVS-96TW
Screen cup for CD-1SIGMAS1145
Screens for CD-1SIGMAS3895
Petri DishSPL LIFE SCIENCES10090
Pasteur pipetteHILGENBERG3150102
LABORATORY CENTRIFUGE / BENCH-TOPVISION SCIENTIFICVS-5500N
Sodium chlorideJUNSEI19015S0350
Potassium chlorideSIGMAP3911-1KG
D-GLUCOSE, ANHYDROUSBIO BASICGB0219
Potassium HydroxideDUKSAN40
Calcium nitrate tetrahydrateSIGMAC2786-500G
Poly(ethylene glycol)SIGMAP2139-2KG
Magnesium chloride hexahydrateSIGMAM2393-500G
Tube 13ml, 100x16mm, PPSARSTEDT55.515
Microscope slidesMARIENFELD1000412
Microscope Cover GlassesMARIENFELD101030
Counting ChamberMARIENFELD650030
Axioplan 2 Imaging MicroscopeCarl ZeissUnavailable
Micro tube 1.5mlSARSTEDT72.690.001
2-MercaptoethanolSIGMAM3148-250ML
Sodium Dodecyl Sulfate (SDS), Proteomics GradeVWRM107-500G
TRIS, Ultra Pure GradeVWR0497-5KG
DTT (DL-Dithiothreitol), Biotechnology GradeVWR0281-25G
Bromophenol blue sodium salt ACSVWR0312-50G
GlycerolJUNSEI27210S0350
Living Colors A.v. Monoclonal Antibody (JL-8)TAKARA632381

References

  1. Jarvis, P., Lopez-Juez, E. Biogenesis and homeostasis of chloroplasts and other plastids. Nature Reviews Molecular Cell Biology. 14 (12), 787-802 (2013).
  2. Neuhaus, H. E., Emes, M. J. Nonphotosynthetic Metabolism in Plastids.

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