Studying Protein Import into Chloroplasts Using Protoplasts

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 metabolites^1,2. For all of these reactions, chloroplasts require a large number of different types of proteins. However, the chloroplast genome contains only approximately 100 genes^3,4. 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.......

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 Arabidopsis^11,12 which can be obtained from plants at many different growth st.......

Materials

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