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 proteins from the cytosol. In fact, most chloroplast proteins were shown to be imported from the cytosol after translation^4,5,6. Plant cells require specific mechanisms to import proteins from the cytosol to chloroplasts. However, although these protein import mechanisms have been investigated for the past several decades, we still do not fully understand them at the molecular level. Here, we provide a detailed method for preparing protoplasts and exogenously expressing genes in protoplasts. This method could be valuable for elucidating the molecular mechanisms underlying protein import into chloroplasts in detail.

Protein import can be studied using many different approaches. One of these methods involves the use of an in vitro protein import system^7,8. Using this approach, in vitro-translated protein precursors are incubated with purified chloroplasts in vitro, and protein import is analyzed by SDS-PAGE followed by western blot analysis. The advantage of this approach is that each step of protein import into chloroplasts can be studied in detail. Thus, this method has been widely used to define the components of the protein import molecular machinery and to dissect sequence information for transit peptides. More recently, another approach involving the use of protoplasts from leaf tissues was developed and it has become widely used to study protein import into chloroplasts^9,10. The advantage of this approach is that protoplasts provide a cellular environment that is closer to that of intact cells than the in vitro system. Thus, the protoplast system allows us to address many additional aspects of this process, such as the associated cytosolic events and how the specificity of targeting signals is determined. Here, we present a detailed protocol for the use of protoplasts to study protein import into chloroplasts.

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 dish (9 cm in diameter, 1.5 cm in height) at a clean bench. Dry plates for 2 – 3 days, pack them with clean wrap, and keep them in a refrigerator until use.
3. Sterilize Arabidopsis thaliana seeds with 1 mL of 70% (v/v) ethanol in a centrifuge tube (e-tube) by continuously shaking for 2 – 3 min. Remove the supernatant, add 1 mL of 1% (v/v) sodium hypochlorite solution, and shake continuously for 2 – 3 min. Prepare 100 – 150 seeds per Petri dish.
   1. Remove the supernatants and wash the seeds with 1 mL of distilled water. Repeat this step 4x. Place the sterilized seeds at 4 °C for 3 days to synchronize seed germination.
4. Sow 100 – 150 seeds onto B5 plates using a micropipette and seal the plate with surgical tape.
5. Grow plants in a growth room with a 16 h/8 h light/dark cycle at 100 µmol·m^-2·s^-1 light intensity, 70% relative humidity and 20 °C temperature for 2 weeks.

2. Preparation of the Plasmid

NOTE: High-purity plasmids should be used for transformation; the use of a commercial plasmid purification kit is recommended.

1. Purify the plasmid (RbcS-nt:GFP) using a DNA isolation kit. To concentrate DNA, perform ethanol precipitation in a 1.5 mL tube and dissolve the DNA pellet in 100 µL of distilled water. Determine the concentration of the plasmid using a spectrophotometer at 260 nm. Dilute the plasmid to a final concentration of ~ 2 µg/µL. Keep the plasmid at -20 °C until use.

3. Isolation of Protoplasts

1. Prepare the following solutions.
   1. Prepare the enzyme solution to contain 0.25% (w/v) macerozyme, 1.0% (w/v) cellulase, 400 mM D-mannitol, 8 mM calcium chloride (CaCl₂), 0.1% (w/v) bovine serum albumin (BSA), and 5 mM MES. Adjust the pH to 5.6 with KOH.
      1. Filter the enzyme solution using a cellulose acetate filter unit with a 0.45 μm pore size. Aliquot 25 mL of the enzyme solution into 50 mL conical tubes and keep at -20 °C. Thaw the enzyme solution at room temperature (RT) and mix well just before use.
   2. Prepare a 21% (w/v) sucrose solution by dissolving 21 g of sucrose in 100 mL of deionized water and autoclave the solution.
   3. Prepare W5 solution to contain 154 mM sodium chloride (NaCl), 125 mM CaCl₂, 5 mM potassium chloride (KCl), 5 mM glucose, and 1.5 mM MES. Adjust the pH to 5.6 with KOH and autoclave the solution.
   4. Prepare MaMg solution to contain 400 mM D-mannitol, 15 mM magnesium chloride (MgCl₂), and 5 mM MES. Adjust the pH to 5.6 with KOH and autoclave the solution.
   5. Prepare 1 M D-mannitol and 1 M calcium nitrate stock solutions to make the PEG solution. Autoclave these solutions.
2. Harvest intact leaf tissues from 2 weeks-old plants from ~ 1 – 2 B5 plates using a surgical knife and place the harvested leaves into a 50 mL conical tube containing 25 mL of enzyme solution. Place the conical tube containing the enzyme solution and leaf tissues horizontally on a rotary shaker with gentle agitation in the dark. It takes 8 – 12 h for full digestion of leaf tissues.
3. At 8 – 12 h after incubation, pour the enzyme solution (containing protoplasts released from leaf tissues) into a fresh Petri dish through a mesh with 140 µm pore size. Carefully layer the protoplast-containing enzyme solution on top of 15 mL of 21% (w/v) sucrose solution and centrifuge it at 98 x g for 10 min with the lowest acceleration and deceleration settings in a swinging-bucket rotor.
4. Using a Pasteur pipette, carefully transfer the protoplasts from the upper-most layer (enzyme solution) and at the interface between the enzyme solution and sucrose solution to a 50 mL conical tube containing 30 mL of W5 solution. Centrifuge this tube at 51 x g for 6 min. At this stage, protoplasts will be in the pellet at the bottom of the conical tube.
5. Discard the supernatant carefully using a pipette without disturbing the protoplasts pellet. Add 25 mL of W5 solution, and gently resuspend the protoplasts. Incubate the protoplast solution in a 4 °C refrigerator for at least 1 h for stabilization.

4. Protoplast Transformation using Polyethylene Glycol

1. Prepare a 40% PEG solution by adding 4 g of PEG 8000, 4 mL of 1 M mannitol solution, 1 mL of 1 M Ca(NO₃)₂, and 1.8 mL of distilled water into a 50 mL conical tube and mix well. Dissolve PEG by heating in a microwave oven for 20 – 30 s. Place the 40% PEG solution at RT for cooling down.
   NOTE: The 40% PEG solution should be prepared freshly every time. If protoplasts are not pelleted completely during the 4 °C incubation in the refrigerator, centrifuge the material at 46 x g for 2 min.
2. Remove the supernatant carefully but completely and add MaMg solution to the protoplast pellet to yield the concentration of 5 x 10⁶/mL.
   NOTE: The number of protoplast can be determined by a hemocytometer viewed under a microscope.
3. Place 10 µg of the plasmid DNA each empty 13 mL round-bottom tube and add 300 µL of protoplast solution using a pipette.
   NOTE: The end of the pipette tip should be cut off. Whenever protoplasts are sampled, the protoplast-containing solution should be resuspended right before pipetting so that the same number of protoplasts is added to each tube.
4. Mix the plasmid DNA with protoplasts by gently rotating the tubes and immediately add 300 µL of 40% PEG solution using a pipette. Mix gently but completely by rotating tubes and incubate for 30 min at room temperature; tilt the tube almost horizontally and rotate it several times.
5. Add 1 mL of W5 solution and mix gently but completely by rotating the tube by hand in a similar manner. Incubate the sample for 10 min at room temperature.
6. Repeat this step two times more using 1.5 mL and 2 mL of W5 solution, respectively. Incubate for 30 min after the final addition of W5 solution.
7. Centrifuge at 46 x g for 4 min. Discard the supernatant and add 2 mL of W5 solution. Mix gently but completely. Incubate at 22 °C in a dark chamber.

5. Analysis of the Protein Import into Chloroplasts

NOTE: After PEG-mediated transformation of protoplasts, incubation time ranges from 8 to 24 h.

1. Fluorescence Microscopy
   1. Place 10 µL of the protoplast solution on a slide glass using a pipette with a trimmed tip and carefully cover with a coverslip to avoid damaging the protoplasts.
   2. Place the slide on the stage of a fluorescence microscope equipped with excitation/emission filter sets for observing green fluorescent protein (GFP) and chlorophyll auto-fluorescence.
   3. Capture images with a cooled charge-coupled device (CCD) camera and process images using an image-editing software to produce pseudo-color images.
2. Total Protein Extraction and Immunoblotting
   1. Prepare denaturation buffer containing 2.5% (w/v) sodium dodecylsulfate (SDS), and 2% (v/v) 2-mercaptoethanol.
      NOTE: Protein import into chloroplasts can be quantified by measuring the degree of transit peptide processing via immunoblot analysis using anti-GFP antibody.
   2. Transfer protoplasts into a centrifuge tube and centrifuge at 46 x g for 4 min.
   3. Remove the supernatant and add 80 µL of denaturation buffer. Vigorously vortex for 5 s and add 5x SDS sample buffer (250 mM Tris-Cl (pH 6.8), 0.5 M DTT, 10% (w/v) SDS, 0.05% (w/v) Bromophenol blue, and 50% (v/v) glycerol). Mix well and boil for 10 min.
   4. Subject this protein sample to standard SDS-PAGE and immunoblotting with anti-GFP antibody.

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

Materials

List of materials used in this article

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