Arabidopsis organelle isolation and characterization

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Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 1062,DOI 10.1007/978-1-62703-580-4_29, © Springer Science+Business Media New York 2014

Chapter 29

Arabidopsis Organelle Isolation and Characterization

Nicolas L. Taylor , Elke Ströher , and A. Harvey Millar

Abstract

The subcellular energy organelles (chloroplast, mitochondria, and peroxisome) in plants are responsible for major metabolic processes including photosynthesis, photorespiration, oxidative phosphorylation, β-oxidation, and the tricarboxylic acid cycle. Arabidopsis thaliana provides a considerable challenge to organellar researchers that have traditionally focused their methods on the use of larger plants and storage organs from which organelles are relatively easy to isolate. In contrast, the small size and lack of abundant heterotrophic organs in Arabidopsis thaliana means that many traditional techniques have required signifi -cant modifi cation to yield enough isolated organelles for experimentation. However, these challenges are balanced by the advantages of working in an organism that has such a wide array of publically available genetic resources. Here we present methods for the isolation of chloroplasts, mitochondria and peroxi-somes from Arabidopsis thaliana plants and heterotrophic cell cultures as well as a number of commonly used assays to assess their functional integrity and purity.

Key words Arabidopsis , Mitochondrial isolation , Chloroplast isolation , Peroxisome isolation , Organellar proteomics , Subcellular proteomics

1 Introduction

The plant cell contains three distinct organelles that carry out the major energy metabolism of the cell: the chloroplast, mitochon-dria, and peroxisome. They house some of the most important cellular processes in plants including photosynthesis, respiration, oxidative phosphorylation, and lipid β-oxidation and coordinate their function for multicompartment metabolic processes such as photorespiration. While insight into the function of these organ-elles has been gained from whole plant investigations, the largest advances in understanding these organelles have stemmed from the ability to isolated them from other cellular components and study them in vitro. Arabidopsis thaliana is an important model system for the systematic study of plant genes and their expression. A number of features of this plant make it particularly attractive to plant scientists including high-quality sequence of its genomes, including those of chloroplasts and mitochondria, many available

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single-gene knockout lines, a short life cycle, and increasing data on its growth, development, and the phenotypes of many ecotypes and mutants. However this model plant is far from ideal for conducting biochemical investigations. The main problem is that Arabidopsis thaliana is small and thus only produces a minimal amount of biomass per plant. Traditionally, plant biochemists have focused their research on larger crop plants such as maize, pea, soybean, tobacco, or plants with large heterotrophic organs such as caulifl ower, potato, and beetroot. To overcome these limitations, it has become necessary to adapt existing organelle isolation and assay procedures. Separate methods have been developed and pub-lished for organelle isolations from mitochondria [ 1 – 4 ], chloro-plasts [ 5 – 7 ], and peroxisomes [ 8 – 10 ]. Here we present modifi ed step-by-step laboratory methods for the isolation of all three of these organelles from Arabidopsis thaliana for those unfamiliar with the process and pitfalls of intact organelle isolation. We also provide a number of assays that confi rm the functional integrity of organelles. In addition we provide a number of marker enzyme assays that can be used to determine cross organellar contamina-tion in isolated preparations.

2 Materials

1. Gradient buffer 1: 0.6 M mannitol, 20 mM TES-KOH (pH 7.5), 0.2 % (w/v) BSA.

2. Gradient buffer 2: 0.6 M sucrose, 20 mM TES-KOH (pH 7.5), 0.2 % (w/v) BSA.

3. Percoll. 4. Ice bucket with ice. 5. 25 mL syringe with a 19 gauge hypodermic needle. 6. Rack for the syringe. 7. Gradient maker (for isolation of mitochondria from seedlings

or seedling cultures). 8. Preparation:

(a) The different concentrations of Percoll are prepared in the appropriate gradient buffer. Gradients should be prepared in advance and stored on ice until required.

(b) For isolation of mitochondria or peroxisomes from seed-lings and heterotrophic cell suspension cultures, 3 different gradients are used.

(c) The fi rst is a step gradient consisting of 5 mL of 40 % (v/v) Percoll overlayered with 20 mL 23 % (v/v) Percoll and then 10 mL 18 % (v/v) Percoll, all made up using gradient buffer 1. The layers can be conveniently poured over one

2.1 Density Gradients for Mitochondrial Isolation

Nicolas L. Taylor et al.

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another by allowing the solution to run through a 19 gauge hypodermic needle placed against the inside of the centrifuge tube held at a 45° angle.

(d) The second is a single linear polyvinylchloride (PVP-40) gradient in 28 % (v/v) Percoll. Preparation of this gradient requires a gradient maker which consists of a Perspex block with two cylindrical chambers connected by a small pipe at their base (Fig. 1 ). One of the chambers has an outfl ow. Both the connecting pipe and outfl ow are metered by taps. Two solutions are prepared: a “heavy” solution (in this case 28 % (v/v) Percoll and 4.4 % (v/v) PVP-40 (from a 20 % w/v stock) made up in gradient buffer 2) and a “light” solution (28 % (v/v) Percoll made up in gradient buffer 2). The “light” solution (15 mL) is placed in the chamber with no outfl ow. The tap connecting the two chambers should be briefl y opened to displace air in the connecting pipe and ensure fl ow. The “heavy” solution (15 mL) is placed into the chamber with the outflow. A magnetic stir bar is placed in the chamber with outfl ow and the solutions rapidly stirred. The outfl ow pipe should be secured against the inside of a 40 mL centrifuge tube held at a 45° angle (above the fi nal level to which the gradient solution will reach in the tube). It is important that the end

Fig. 1 Preparation of linear gradients with a gradient maker. High density (“heavy solution”) and low density (“light solution”) are arranged in the chambers of the gradient maker as shown. Magnetic stir bar in the chamber containing the “heavy solution” should rotate at a speed suffi cient to ensure complete mixing. To pour the gradient, both taps should be opened simultaneously and the outfl ow should be allowed to run out by gravity and down the side of a centrifuge tube held below the apparatus

Organelle Isolation and Characterization

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of the outfl ow tube is held below the level of the gradient maker so that solution will fl ow by gravity (Fig. 1 ). The taps on both the connecting pipe and the outfl ow are then opened simultaneously. As the gradient pours, the level of solution in both chambers should drop at the same rate.

(e) The third is a step sucrose gradient for further purifi cation of leaf peroxisomes [ 10 ], following the fi rst gradients. Prepare solutions for the gradient, 3 mL of 40 % (w/w), 3 mL of 45 % (w/w), 3 mL of 50 % (w/w), 3 mL of 55 % (w/w), and 3 mL of 60 % (w/w) by addition of solid sucrose to gradient buffer 2. The layers can be conveniently poured over one another by allowing the solution to run through a 19 gauge hypodermic needle placed against the inside of the centrifuge tube held at a 45° angle.

1. 25 mL centrifugation tubes and adapters for the rotor. 2. 40 % (v/v) Percoll solution; 0.33 M sorbitol, 20 mM HEPES

(pH 7.9), 5 mM MgCl 2 , 2.5 mM EDTA (ethylenediamine-tetraacetic acid; disodium salt), 10 mM NaHCO 3 , 0.2 % (w/v) BSA (bovine serum albumin), 2 mM ascorbate in 40 % (v/v) Percoll.

3. 100 % (v/v) Percoll solution; 0.33 M sorbitol, 20 mM HEPES (pH 7.9), 5 mM MgCl 2 , 2.5 mM EDTA (ethylenediaminetet-raacetic acid; disodium salt), 10 mM NaHCO 3 , 0.2 % (w/v) BSA, 2 mM ascorbate in 100 % (v/v) Percoll.

4. Centrifuge with fi xed-angle rotor (Beckman Coulter Avanti J-26 XP or equivalent).

5. Ice bucket with ice. 6. 25 mL syringe with a 19 gauge hypodermic needle. 7. Rack for the syringe. 8. Preparation:

(a) For isolation of chloroplasts from leaf tissue, a step gradient consisting of 2.5 mL 100 % (v/v) Percoll solution and 10 mL 40 % (v/v) Percoll solution is used.

(b) The layers can be conveniently poured over one another by allowing the solution to run through a 25 mL syringe with a 19 gauge hypodermic needle placed against the inside of the 25 mL centrifuge tube held at a 45° angle on ice.

(c) A sigmoidal gradient self-forms during centrifugation due to the sedimentation of the Percoll polydispersed silica colloid.

(d) The gradient can be prepared the day before and stored in the cold room. Care must be taken to ensure that no movement occurs to disturb the gradient.

2.2 Density Gradients for Chloroplastic Isolation


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1. Gradient buffer 1: 0.6 M mannitol, 20 mM TES-KOH (pH 7.5), 0.2 % (w/v) BSA.

2. Gradient buffer 2: 0.6 M sucrose, 20 mM TES-KOH (pH 7.5), 0.2 % (w/v) BSA.

3. Percoll. 4. Ice bucket with ice. 5. 25 mL syringe with a 19 gauge hypodermic needle. 6. Rack for the syringe. 7. Preparation: See Subheading 2.1 .

1. Miracloth (Merck Biosciences). 2. Muslin. 3. Buchner funnel. 4. Waring blender. 5. Vacuum pump. 6. Cell extraction medium: 0.45 M mannitol, 50 mM sodium

pyrophosphate (Na 4 P 2 O 7 ), 0.5 % (w/v) BSA, 0.5 % (w/v) PVP-40, 2 mM EGTA (ethyleneglycoltetraacetic acid). Adjust pH to 8.0 with phosphoric acid. Add cysteine to 20 mM on day of use.

7. Enzyme buffer: 0.4 M mannitol, 3.5 mM MES, pH 5.7 with KOH, just before use add 0.4 % (w/v) cellulase RS, 0.05 % (w/v) pectolyase Y-23. Also prepare buffer without enzymes.

8. 250 mL centrifuge tubes. 9. Dounce homogenizer (No 4, 0,12 mm; or potter). 10. 50 mL centrifuge tubes. 11. Centrifuge with fi xed-angle rotor (Beckman Coulter Avanti

J-26 XP with JLA-10.500 and JA-25.50 or equivalent). 12. Mitochondria wash buffer: 0.3 M mannitol, 10 mM TES- KOH,

pH 7.5. 13. Soft-haired artist’s paintbrush. 14. 3 mL disposable pipette. 15. 40/23/18 % (v/v) Percoll step gradient ( see Subheading 2.1 ).

1. Buchner funnel. 2. Sterile distilled water. 3. Perspex vessel (45 × 60 × 220 mm). 4. Seedling extraction medium: 0.3 M sucrose, 25 mM sodium

pyrophosphate (Na 4 P 2 O 7 ), 2 mM EDTA (ethylenediaminetet-raacetic acid; disodium salt), 10 mM KH 2 PO 4 , 1 % (w/v) PVP-40, 1 % (w/v) BSA. Adjust pH to 7.5 with NaOH. Add ascorbic acid to 20 mM on day of use.

2.3 Density Gradients for Peroxisomal Isolation

2.4 Isolation of Mitochondria

2.4.1 From Heterotrophic Cell Suspension Cultures

2.4.2 From Seedlings and Hydroponic Seedling Cultures


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5. Polytron ® homogenizer equipped with a 200 mm long × 20 mm diameter dispersing head.

6. Miracloth (Merck Biosciences). 7. Muslin. 8. Centrifuge and centrifuge tubes as in Subheading 2.4.1 . 9. Soft-haired artist’s paintbrush. 10. 28 % (v/v) Percoll, 0–4.4 % (v/v) PVP-40 gradients ( see

Subheading 2.1 ). 11. Leaf mitochondria wash buffer: 0.3 M sucrose, 10 mM TES-

KOH, pH 7.5. 12. 3 mL disposable pipette.

1. Beaker for collecting tissue. 2. Balance. 3. Razor blade or scalpel. 4. Ice bucket with ice. 5. Grinding buffer: 0.33 M sorbitol, 20 mM tricine (pH 8.4), 5 mM

EGTA (ethyleneglycoltetraacetic acid), 5 mM EDTA (ethylenedi-aminetetraacetic acid; disodium salt), 10 mM NaHCO 3 , 0.1 % (w/v) BSA. Buffer can be stored in aliquots at −20 °C.

6. Wash buffer: 0.33 M sorbitol, 20 mM HEPES (pH 7.9), 5 mM MgCl 2 , 2.5 mM EDTA (ethylenediaminetetraacetic acid; disodium salt), 10 mM NaHCO 3 , 0.1 % (w/v) BSA. Buffer can be stored in aliquots at −20 °C.

7. 100 mM ascorbate stock solution prepared in grinding buffer and in wash buffer, respectively. Ascorbate has to be added to both buffers on the day of use. The stock solution offers a convenient way to prepare buffer as needed.

8. Waring blender. 9. Miracloth (Merck Biosciences). 10. Nylon mesh (20–50 μm pore size). 11. Funnel. 12. 100 mL measuring cylinder. 13. Parafi lm, pieces big enough to cover the opening of the

measuring cylinder. 14. 500 mL fl asks for collecting the suspension. 15. 50 and 25 mL centrifugation tubes with adapters for the rotor. 16. Wash buffer: 0.33 M sorbitol, 20 mM HEPES (pH 7.9),

5 mM MgCl 2 , 2.5 mM EDTA (ethylenediaminetetraacetic acid; disodium salt), 10 mM NaHCO 3 , 0.1 % (w/v) BSA, 2 mM ascorbate.

17. Soft-haired artist’s paintbrush. 18. Percoll gradients ( see Subheading 2.2 ).

2.5 Isolation of Chloroplasts

2.5.1 From Plant Leaves


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19. Centrifuge with fi xed-angle rotor (Beckman Coulter Avanti J-26 XP with JLA-10.500 and JA-25.50 or equivalent).

20. 3 mL disposable pipette. 21. 1.5 mL tubes for storing intact chloroplasts. 22. 4–5-week-old Arabidopsis plants grown under medium- day

conditions ( 12 h light/12 h dark, at 23 °C (day)/18 °C (night), light intensity of 120 μmol/m 2 /s and 60 % RH).

1. Miracloth (Merck Biosciences). 2. Muslin. 3. Buchner funnel. 4. Waring blender. 5. Vacuum pump. 6. Cell extraction medium: See item 6 in Subheading 2.4.1 . 7. 250 mL centrifuge tubes. 8. 50 mL centrifuge tubes. 9. Centrifuge with fi xed-angle rotor (Beckman Coulter Avanti

J-26 XP with JLA-10.500 and JA-25.50 or equivalent). 10. Centrifuge with swingout rotor (Beckman Coulter Optima

L-100 XP with SW40 rotor or equivalent or Beckman Coulter Avanti J-30I with JS-24.38 or equivalent).

11. Cell peroxisome wash buffer: 0.3 M mannitol, 10 mM TES- KOH, pH 7.5.

12. Soft-haired artist’s paintbrush. 13. 3 mL disposable pipette. 14. 40/23/18 % (v/v) Percoll step gradient ( see Subheading 2.3 ). 15. 40/45/50/55/60 % (w/w) sucrose step gradient ( see

Subheading 2.3 ).

1. Mitochondria isolated according to Subheading 3.1 . 2. Clark-type electrode, such as those manufactured by Hansatech

(Kings Lynn, UK) or Rank Brothers (Bottisham, UK). 3. Mitochondrial reaction medium: 0.3 M mannitol, 10 mM

TES- KOH (pH 7.5), 3 mM MgSO 4 ⋅7H 2 O, 10 mM NaCl, 5 mM KH 2 PO 4 , 0.1 % (w/v) BSA.

4. Stock solutions of respiratory substrates: 100 mM NADH, 1 M succinate, 50 mM ATP, 500 mM pyruvate, 50 mM malate, 30 mM NAD + , 10 mM thiamine pyrophosphate, 12 mM coen-zyme A. Coenzyme A should be made up in 1 % (w/v) cysteine and all other components should be made up in 500 mM Tes- KOH (pH 7.5).

5. 100 mM adenosine 5′-diphosphate (ADP) made up in 500 mM Tes-KOH (pH 7.5).

2.6 Isolation of Peroxisomes

2.6.1 From Heterotrophic Cell Suspension Cultures and Plant Leaves

2.7 Functional Assay of Mitochondria


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1. 5× sample buffer: 250 mM Tris–HCl, pH 6.8, 10 % (w/v) SDS, 50 % (w/v) glycerol, 0.01 % (w/v) bromophenol blue.

2. Reducing agent, dithiothreitol (DTT) or β-mercaptoethanol (β-Me).

3. Heating block. 4. Sample of broken and intact chloroplasts, isolated according to

Subheading 3.2 . 5. SDS gel (e.g., precast BioRad Criterion ® gels). 6. Running system: chamber (e.g., BioRad Criterion Cell ® ),

running buffer ( 25 mM Tris, 192 mM glycine, 0.1 % (w/v) SDS, do not adjust pH) and power supply.

7. Colloidal Coomassie staining solutions: Solution A, 2 % (w/v) orthophosphoric acid (85 % (v/v) H 3 PO 4 ) and 10 % (w/v) ammonium sulfate (NH 4 ) 2 SO 4 in H 2 O; Solution B, 5 % (w/v) Coomassie G250 in H 2 O; and fi xing solution, 40 % (v/v) methanol and 10 % (v/v) acetic acid ( see Note 1 ).

8. Staining container. 9. Platform shaker.

1. Sample of broken and intact chloroplasts, isolated according to Subheading 3.2 .

2. 1.5 mL reaction tubes. 3. Wash buffer (same as in Subheading 2.5 ). 4. Phase contrast microscope. 5. Slides and cover glass. 6. 3 mL disposable pipette.


2. Clark-type oxygen electrode, such as those manufactured by Hansatech (Kings Lynn, UK) or Rank Brothers (Bottisham, UK).

3. Slide projector, 150 W bulb or similar illuminator. 4. 2× assay medium: 660 mM sorbitol, 4 mM EDTA, 2 mM

MgCl 2 , 2 mM MnCl 2 , 100 mM HEPES-KOH (pH 7.6). 5. 50 mM potassium ferricyanide ( see Note 2 ). 6. 200 mM DL-glyceraldehyde.

1. Visible wavelength spectrophotometer. 2. 0.1 M KH 2 PO 4 –NaOH (pH 7.7). 3. 10 % (v/v) Triton X-100. 4. 1 M malate. 5. Mitochondria isolated according to Subheading 3.1 .

2.8 Assays of Chloroplast Integrity

2.8.1 SDS PAGE Analysis

2.8.2 Phase Contrast Microscopy

2.8.3 Oxygen Electrode and Ferricyanide Test for Intactness

2.9 Marker Enzyme Assays of Mitochondria

2.9.1 Fumarase


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6. Reaction master mix: 70 mM KH 2 PO 4 –NaOH, pH 7.7, 0.05 % (v/v) Triton X-100. 900 μL master mix per assay (i.e., to fi ll a 1 mL spectrophotometric cuvette after subsequent additions of sample and substrate) ( see Note 3 ).

1. Visible wavelength spectrophotometer. 2. 0.1 M HEPES-NaOH (pH 7.5). 3. 10 % (v/v) Triton X-100. 4. 20 mM nicotinamide adenine dinucleotide phosphate (NADP). 5. 0.1 M MnCl 2 . 6. 2,000 U/mL NADP-isocitrate dehydrogenase (ICDH)

(porcine heart). 7. 0.2 M aconitate. 8. Mitochondria isolated according to Subheading 3.1 . 9. Reaction master mix: 80 mM HEPES-NaOH, pH 7.5, 0.05 %

(v/v) Triton X-100, 0.5 mM NADP, 0.5 M MnCl 2 , 2 U NADP-ICDH. 900 μL master mix per assay (i.e., to fi ll a 1 mL spectrophotometric cuvette after subsequent additions of sample and substrate) ( see Note 3 ).


2. 2× reaction buffer: 200 mM Tris–HCl (pH 7.8), 20 mM MgCl 2 , 40 mM KCI, 20 mM DTT.

3. 100 mM adenosine 5′-triphosphate (ATP) disodium salt in 1× reaction buffer.

4. 100 mM phospho(enol)pyruvic acid monosodium salt hydrate in 1× reaction buffer.

5. 10 mM NADH in 1× reaction buffer, prepare only on day of use. 6. Pyruvate kinase (e.g., rabbit muscle), 400–800 units/mg protein. 7. Lactate dehydrogenase (e.g., Lactobacillus leichmannii ), 150–

300 units/mg protein. 8. Milli Q H 2 O. 9. Visible wavelength spectrophotometer.

1. Clark-type electrode, such as those manufactured by Hansatech (Kings Lynn, UK) or Rank Brothers (Bottisham, UK).

2. Reaction medium: 0.3 M sucrose, 5 mM KH 2 PO 4 , 10 mM TES-KOH, pH 7.2, 10 mM NaCl, 2 mM MgSO 4 , and 0.1 % (w/v) bovine serum albumin.

3. Sodium hydrosulfi te. 4. 1 % (v/v) H 2 O 2 solution. 5. Peroxisomes isolated according to Subheading 3.3 .

2.9.2 Aconitase

2.10 Marker Enzyme Assay of Chloroplasts

2.10.1 Phosphori-bulokinase

2.11 Marker Enzyme Assay of Peroxisomes

2.11.1 Catalase


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3 Methods

1. To preserve the integrity of isolated mitochondria, it is essential that all plasticware and glassware are free from traces of detergent ( see Note 4 ). All procedures should be done at 4 °C, solutions should be prechilled, and the work should be done in a cold room. Centrifugation should be done using a refrigerated centrifuge.

2. Separate cells from culture medium by fi ltration through a single layer of Miracloth using a Buchner funnel and a vacuum pump. Weigh cells.

3. Cells can be broken mechanically or enzymatically. The former is faster but the latter gives a higher yield. The choice really depends on the number of independent samples you wish to analyze.

4. Mechanical rupture. (a) Transfer up to 150 g FW of cells to a 500 mL Waring

blender vessel. Add at least 2 volumes of cell extraction medium per weight of cells ( see Note 5 ) and blend for 15 s at high speed and twice for 15 s at low speed. Leave 30 s intervals between bursts of blending to prevent excessive foaming and heating.

(b) Filter extract by wringing through one layer of Miracloth and two layers of muslin to remove cell debris and unbro-ken cells ( see Note 6 ).

(c) Transfer fi ltrate to 250 mL centrifuge tubes and centrifuge at 1,500 × g for 10 min. Gently pour resulting supernatant into fresh centrifuge tubes. Take care not to transfer any of the pellet which is quite loose (continues at step 6 ).

5. Enzymatic rupture. (a) Resuspend cells in enzyme buffer (ratio 50 g cells/100 mL

buffer). (b) Incubate the suspension for 3 h under low agitation at

25 °C in the dark (wrap the fl ask in foil) ( see Note 7 ). (c) Carefully pour the suspension in 500 mL centrifugation

tubes. (d) Centrifuge the suspension at 800 × g for 10 min with slow

brake. (e) Discard the supernatant; be careful not to discard the pellet

as it is loose. (f) Resuspend the pelleted protoplasts in enzyme buffer

without enzymes by swirling the tubes and centrifuge at 500 × g for 10 min.

(g) Remove the supernatant.

3.1 Isolation of Mitochondria

3.1.1 From Heterotrophic Cell Suspension Cultures


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(h) Resuspend the washed protoplasts in extraction buffer (ratio 50 g cells/100 mL buffer).

(i) Homogenize the protoplasts in a Dounce homogenizer (No 4, 0,12 mm; or potter) 3–4 times ( see Note 8 ).

(j) Centrifuge the homogenate at 2,500 × g for 5 min. 6. Centrifuge the supernatant from step 4 or 5 at 18,000 × g

for 15 min. Pour off and discard the resultant supernatant. 7. Add 1 mL of cell mitochondria wash medium to the pellet and

gently resuspend using a small artist’s paintbrush. Transfer all resuspended pellets to a single 50 mL centrifuge tube and add wash medium to 30 mL.

8. Repeat step 6 . Resuspend the fi nal pellet in 1 mL of cell mitochondria wash medium using an artist’s paintbrush as before. You now have an organelle suspension. It is important to move onto the density gradient centrifugation as quickly as possible.

9. Carefully pipette the organelle suspension onto the surface of the 40/23/18 % (v/v) step gradient. Centrifuge at 40,000 × g for 30 min. It is important that the centrifuge brake is disen-gaged at the end of the run because rapid deceleration can disturb the contents of the gradient.

10. Mitochondria appear as a diffuse white/pale brown band at the interface of the 40/23 % (v/v) Percoll layers ( see Note 9 ). Peroxisomes are just beneath but are not usually visible. Plastid membranes appear as a bright yellow band at the 23/18 % (v/v) Percoll interface. Aspirate the upper part of the gradient to waste. Using a 3 mL pipette, carefully transfer the mito-chondria band to a fresh 50 mL centrifuge tube. Generally, 7–10 mL is suffi cient to transfer all of the mitochondria. As much as possible, avoid the peroxisomal band below.

11. Add 20 mL of cell mitochondria wash medium to the mito-chondria and centrifuge at 18,000 × g for 10 min. Remove and discard the supernatant by aspiration. Take care not to disturb the pellet, which is very loose. It is not necessary to remove all the supernatant; leave 3–4 mL in the bottom of the tube.

12. Resuspend the mitochondrial pellet by gentle swirling and carefully pipette onto the 28 % (v/v) Percoll gradient. Centrifuge at 40,000 × g for 30 min with the brake off as before. Mitochondria will form a white/pale brown band in the upper part of the gradient and peroxisomes will band towards the bottom of the gradient. Carefully transfer the mitochondria to a fresh tube using a 5 mL pipette.

13. Add 20 mL of cell mitochondria wash medium to the mito-chondria and centrifuge at 18,000 × g for 10 min. Remove and discard the supernatant by aspiration.


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14. Repeat step 13 . Pour off and discard the supernatant (the pel-let is fi rmer at this stage) and gently resuspend the mitochon-dria in 0.5 mL of cell mitochondria wash medium using a small paintbrush.

1. To preserve the integrity of isolated mitochondria, it is essen-tial that all plasticware and glassware is free from traces of detergent ( see Note 4 ). All procedures should be done at 4 °C, solutions should be prechilled, and the work should be done in a cold room. Centrifugation should be done using a refriger-ated centrifuge.

2. Place leaves or seedlings up to 60 g in a Buchner funnel and wash with sterile distilled water. Leaves are best from 3-week- old plants, seedlings best at 10 days after germination.

3. Place washed leaves or seedlings in a Perspex vessel and cover with 300 mL seedling extraction medium. Homogenize tissue with a Polytron ® homogenizer equipped with a 200 mm long × 20 mm diameter dispersing head using three or four of 2 s bursts.

4. Filter extract by wringing through one layer of Miracloth and two layers of muslin to remove cell debris and unbroken cells ( see Note 6 ).

5. Transfer fi ltrate to 250 mL centrifuge tubes and centrifuge at 1,100 × g for 5 min. Gently pour resulting supernatant into fresh centrifuge tubes. Take care not to transfer any of the pellet which is quite loose.

6. Centrifuge the supernatant from step 5 at 18,000 × g for 20 min. Pour off and discard the resultant supernatant.

7. Add 1 mL of leaf mitochondria wash medium to the pellets and gently resuspend using a small paintbrush. Transfer resus-pended pellets to two 50 mL centrifuge tubes and add wash medium to each 30 mL.

8. Repeat step 6 . Resuspend the fi nal pellets in 1 mL of leaf mito-chondria wash medium using a paintbrush as before. You now have an organelle suspension. It is important to complete steps 1 – 8 and move onto the density gradient centrifugation as quickly as possible.

9. Carefully pipette the two organelle suspensions onto the surface of two 28 % (v/v) Percoll, 0–4.4 % (v/v) PVP-40 gradients. Centrifuge at 40,000 × g for 40 min. It is important that the centrifuge brake is disengaged at the end of the run because rapid deceleration can disturb the contents of the gradient.

10. Mitochondria will form a white/pale brown band towards the bottom of the gradient and thylakoids are apparent as a dark green band in the upper part of the gradient (Fig. 2 ).

3.1.2 From Plant Leaves and Seedling Cultures


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Aspirate the upper part of the gradient to waste. Using a 3 mL pipette, carefully transfer the mitochondria band to a fresh 50 mL centrifuge tube. Generally, 7–10 mL is suffi cient to transfer all of the mitochondria.

11. Add 20 mL of leaf mitochondria wash medium to the mito-chondria and centrifuge at 31,000 × g for 15 min. Remove and discard the supernatant by aspiration. Take care not to disturb the pellet, which is very loose. It is not necessary to remove all the supernatant—generally leave 3–4 mL in the bottom of the tube.

12. Repeat step 11 and pour off the resultant supernatant. Add 0.5 mL of leaf mitochondria wash medium and resuspend pellet gently with a small paintbrush.

1. To preserve the integrity of isolated chloroplasts, it is essential that all plasticware and glassware are free from traces of deter-gent ( see Note 4 ). All procedures should be done at 4 °C. All solutions, vessels, and consumables should be prechilled (best done by placing them in the cold room the day before the isolation) and the work should be done in a cold room. Centrifugation should be done using a refrigerated centrifuge

3.2 Isolation of Chloroplasts

Fig. 2 Appearance of organelle bands on different density gradients. ( a ) Organelles from heterotrophic Arabidopsis cell suspension cultures. ( b ) Organelles from Arabidopsis leaf material


564

at 4 °C. Once the tissue is harvested, work as quickly as possible, but with appropriate care.

2. Harvest approximately 20 g of leaf tissue, 5-week-old plants are best for intact chloroplast isolation ( see Note 10 ).

3. Measure 80 mL of grinding buffer using the measuring cylinder (ratio buffer to tissue is 4 mL/g). Add 1.6 mL of the 0.1 M ascorbate stock solution for a fi nal concentration of 2 mM ascorbate. Cover the measuring cylinder using parafi lm and mix quickly by inversion.

4. Place the leaf tissue in a blender vessel and cover with grinding buffer. Don’t overfi ll the blender vessel.

5. Blend the tissue 3–4 times for 5 s at high speed. Leave 30 s intervals between bursts of blending to prevent excessive foaming and heating.

6. Filter suspension through 3 layers of muslin. Gently squeeze the plant material to speed up the process ( see Note 11 ).

7. Filter suspension through nylon net ( see Note 11 ). 8. Transfer the fi ltrate to two 50 mL centrifugation tubes. 9. Centrifuge at 2,000 × g for 2 min with brake on. 10. Carefully aspirate the supernatant. Any froth adhering to the

sides of the tubes should be wiped off. 11. Gently resuspend the pellet in 2 mL wash buffer by gently

shaking the tube or using an artist’s paintbrush ( see Note 12 ). 12. Slowly lay the suspension on top of two 40/100 % (v/v) Percoll

gradients using a disposable pipette ( see Note 13 ). Avoid dis-turbances of the gradient. It is best if the tubes containing the gradient are slowly laid down on ice at a 45° angle as before.

13. Centrifuge at 3,500 × g for 30 min, brake off. It is important that the centrifuge brake is disengaged at the end of the run because rapid deceleration can disturb the contents of the gradient.

14. Intact chloroplasts are located at the interface of the 40 and 100 % (v/v) Percoll, whereas the broken chloroplasts are between the 0 and 40 % (v/v) Percoll interface (Fig. 3 ). Approximately 40–60 % (v/v) of the total chloroplasts are intact chloroplasts.

15. Optional: Take an aliquot of the broken chloroplasts for comparative analysis later on.

16. Aspirate the broken chloroplasts using a disposable tip con-nected to a vacuum line. Use slow circular movements alongside the inner wall of the tube.

17. Collect the intact chloroplasts (approximately 2 mL per gradient) using a disposable 3 mL pipette and transfer into a fresh 25 mL centrifugation tube.


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18. Add 2 mL wash buffer and resuspend the chloroplasts using a paintbrush, add further 8 mL wash buffer, and mix by swing-ing the tube.

19. Centrifuge at 2,000 × g for 2 min with brake on. 20. Aspirate the supernatant. 21. Resuspend the pellet in 250–500 μL of wash buffer.

1. To preserve the integrity of isolated peroxisomes, it is essential that all plasticware and glassware are free from traces of deter-gent ( see Note 4 ). All procedures should be done at 4 °C, solutions should be prechilled, and the work should be done in a cold room. Centrifugation should be done using a refriger-ated centrifuge.

2. Separate cells from culture medium by fi ltration through a single layer of Miracloth using a Buchner funnel and a vacuum pump. Weigh cells.

3. Transfer up to 150 g FW of heterotrophic cells or 100 g FW of 3–4-week-old leaves to a 500 mL Waring blender vessel. Add at least 2 volumes of cell extraction medium per weight of cells ( see Note 5 ) and blend for 15 s at high speed and twice for 15 s at low speed. Leave 30 s intervals between bursts of blending to prevent excessive foaming and heating.

4. Filter extract by wringing through one layer of Miracloth and two layers of muslin to remove starch, cell debris, and unbroken cells ( see Note 6 ).

3.3 Isolation of Peroxisomes

Fig. 3 Appearance of chloroplast bands on density gradient. 40/100 % (v/v) Percoll step gradient after centrifugation. The upper band represents broken chloroplasts; the lower band represents intact chloroplasts


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5. Transfer fi ltrate to 250 mL centrifuge tubes and centrifuge at 1,500 × g for 10 min. Gently pour resulting supernatant into fresh centrifuge tubes. Take care not to transfer any of the pellet which is quite loose.

6. Centrifuge the supernatant from step 5 at 18,000 × g for 15 min. Pour off and discard the resultant supernatant.

7. Add 1 mL of cell peroxisome wash medium to the pellet and gently resuspend using a small artist’s paintbrush. Transfer all resuspended pellets to a single 50 mL centrifuge tube and add wash medium to 30 mL.

8. Repeat step 6 . Resuspend the fi nal pellet in 1 mL of cell peroxi-some wash medium using an artist’s paintbrush as before. You now have an organelle suspension. It is important to complete steps 1 – 8 and move onto the density gradient centrifugation as quickly as possible.

9. Carefully pipette the organelle suspension onto the surface of the 40/23/18 % (v/v) step gradient. Centrifuge at 40,000 × g for 30 min. It is important that the centrifuge brake is disen-gaged at the end of the run because rapid deceleration can disturb the contents of the gradient.

10. Peroxisomes appear just below a diffuse white/pale brown band (mitochondria) at the interface of the 40/23 % (v/v) Percoll layers ( see Note 9 ). Plastid membranes appear as a bright yellow band in cell culture and as a green band in leaves at the 23/18 % (v/v) Percoll interface. Aspirate the upper part of the gradient to waste. Using a 3 mL pipette, carefully trans-fer the mitochondrial band and 40 % (v/v) Percoll layer to a fresh 50 mL centrifuge tube.

11. Fill the tube with cell peroxisome wash medium and centrifuge at 18,000 × g for 10 min. Remove and discard the supernatant by aspiration. Take care not to disturb the pellet, which is very loose. It is not necessary to remove all the supernatant; leave 3–4 mL in the bottom of the tube.

12. Resuspend the pellet by gentle swirling and carefully pipette onto the 28 % (v/v) Percoll gradient. Centrifuge at 40,000 × g for 30 min with the brake off as before. Peroxisomes will form a band towards the bottom of the gradient while mitochondria will form a white/pale brown band in the upper part of the gradient. Carefully remove the mitochondrial layers and trans-fer the peroxisomes to a fresh tube using a 5 mL pipette.

13. Add 20 mL of cell peroxisome wash medium to the mitochondria and centrifuge at 18,000 × g for 10 min. Remove and discard the supernatant by aspiration.

14. Repeat step 13 . Pour off and discard the supernatant (the pellet is fi rmer at this stage) and gently resuspend the peroxisomes in 0.5 mL of cell peroxisome wash medium using a small paintbrush.


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15. For leaf peroxisomes, preparations need to be further purifi ed by a third sucrose step gradient. The organelle preparation is laid on top of a 40–60 % (w/w) sucrose step gradient ( see Subheading 2.3 ) and centrifuged for 2 h, 125,000 × g in a swingout rotor. Peroxisomes are visible as a white band above the 55 % (w/w) sucrose layer.

1. Respiration of a variety of substrates can be measured as oxy-gen consumption using an oxygen electrode. The dependence of reaction rate on added ADP can be used to calculate the respiratory control ratio that gives an indication of the extent to which the mitochondria are coupled.

2. Set up the electrode according to the manufacturer’s instruc-tions using 50 % (w/v) saturated KCl as an electrolyte and cali-brate between air-saturated water (253 nmol O 2 /mL at 25 °C) and zero (established by adding a few crystals of sodium hydro-sulfi te to the water) ( see Note 14 ).

3. The assay should be conducted at 25 °C. To 1 mL of mito-chondrial reaction medium, add 5–20 μL of mitochondrial sus-pension ( see Note 15 ). Determine the background rate of oxygen consumption [rate (a)]. Add one of the following sub-strate combinations (all concentrations are fi nal), 1 mM NADH; 10 mM succinate, 0.5 mM ATP; and 5 mM pyruvate, 0.5 mM malate 0.3 mM NAD + , 0.1 mM thiamine pyrophosphate, 12 μM CoA, and determine the rate of oxygen consumption [rate (b)]. Add 1 μL of 100 mM ADP. The rate of oxygen con-sumption should increase: measure the initial linear rate [rate (c)]. After a few minutes the ADP will be depleted and the rate of oxygen consumption will be reduced to [rate (d)].

The respiratory control ratio is given by rate d rate arate b rate a

( ) ( )( ) ( )

−[ ]−[ ]

1. In contrast to broken chloroplasts, intact chloroplasts still contain all the soluble proteins from the stroma. This can easily be analyzed by separating the complex protein mixture of both fractions on 1-dimensional SDS PAGE [ 5 ].

2. Determine the appropriate protein amount to load based on the capacity of the gel and the detection method (e.g., 10 μg for a precast BioRad Criterion ® Gel with 18 wells and subse-quent colloidal Coomassie staining).

3. Prepare the protein sample by adding a quarter of 5× sample buffer and a reducing agent (DTT, fi nal concentration 100 mM, or β-Me, fi nal concentration 5 % (v/v)).

4. Prepare a sample of broken and intact chloroplasts, as well as a protein marker.

3.4 Functional Assay of Mitochondria

3.5 Assays of Chloroplast Integrity

3.5.1 SDS PAGE Analysis


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5. Heat the samples at 95 °C for 5 min. 6. Carefully load the samples into the wells. 7. Run the gel according to the manufactures’ instructions. 8. Prepare the colloidal Coomassie staining solution by combin-

ing 98 % (v/v) Solution A with 2 % (v/v) Solution B. Stir gently for 2 h.

9. Once the run is complete, transfer the gel into a clean container and add 50–100 mL fi xing solution.

10. Incubate on a platform shaker for at least 1 h. 11. Replace the fi xing solution with the same amount of colloidal

Coomassie staining solution and stain overnight. 12. This staining technique does not require a destaining of the

gel; therefore, it can be documented straight away. 13. The ratio between the major stromal protein, the large subunit

of Rubisco (ca. 52 kDa), and major thylakoid proteins, the light-harvesting complex proteins (multiple isoforms, around 30 kDa), is estimated. Stromal proteins should be enriched in the intact chloroplast fraction compared to the broken chloro-plast fraction.

1. Intact chloroplasts are refractile and surrounded by a bright halo compared with broken chloroplasts which are non- refractile [ 11 ] .

2. Dilute 2 μL of isolated chloroplasts (from Subheading 3.2 ) to 198 μL of wash buffer in a 1.5 mL tube and mix gently.

3. Pipette a drop onto a slide and place a glass cover on top. Take away excess solution with a tissue.

4. Check the chloroplasts using a phase contrast microscope. 5. Intact chloroplasts are round and green and under phase

contrast are surrounded by halo.

1. Ferricyanide is used as electron acceptor in the Hill reaction. As it can’t pass the chloroplast envelope, only broken chloro-plast can catalyze it. Total activity is measured after the osmotic rupture of the intact chloroplast [ 11 ]. The reduction can be measured using an oxygen electrode.

2. For intact chloroplasts: (a) Add 500 μL of assay medium, 20 μL of 50 mM potassium

ferricyanide, and 50 μL of 200 mM glyceraldehyde; add H 2 O up to 980 μL; and then add 20 μL chloroplasts.

(b) Illuminate for 2 min. (c) Add 20 μL 250 mM NH 4 Cl. (d) Monitor oxygen evolution rates.

3.5.2 Phase Contrast Microscopy

3.5.3 Oxygen Electrode and Ferricyanide Test for Intactness


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3. For total activity: (a) Add 20 μL chloroplasts to 410 μL H 2 O (at least tenfold

dilution). (b) Incubate for 2 min. (c) Add 500 μL of assay medium, 20 μL of 50 mM potassium

ferricyanide ( see Note 2 ), 50 μL of 200 mM glyceraldehyde. (d) Illuminate for 2 min. (e) Add 20 μL 250 mM NH 4 Cl. (f) Monitor oxygen evolution rates.

4. The percentage of intact chloroplasts is then calculated from the rates of the two measurements.

1. Add mitochondrial protein sample (10–100 μg protein) to 900 μL reaction master mix.

2. Add malate to start reaction to a fi nal concentration of 50 mM. 3. Progression of the reaction is measured directly at 340 nM

( ε = 2.55 mM −1 ).

1. Add mitochondrial protein sample (10–100 μg protein) to 900 μL reaction master mix.

2. Add aconitate to start reaction to a fi nal concentration of 8 mM ( see Note 16 ).

3. Progression of the reaction is measured as NADP reduction to NADPH at 340 nM ( ε = 6.22 mM −1 ).

1. Pipette 0.5 mL 2× reaction buffer and 5–20 μL of isolated intact chloroplasts in a 1 mL cuvette.

2. Add 2 mM ATP, 2 mM phosphoenol pyruvate, 0.2 mM NADH, 3.5 U/mL pyruvate kinase, 5 U/mL lactate dehydrogenase.

3. Add H 2 O to 975 μL and mix. 4. Monitor the A 340nm until constant. 5. Perform a baseline correction. 6. Add 25 μL 20 mM ribulose-5-phosphate (Ru5P) (fi nal conc.

0.5 mM) to initiate the reaction. 7. Activities are assayed by following the oxidation of NADH at

340 nm ( ε = 6.22 mM −1 ).

1. Catalase activity can be measured in an oxygen electrode by monitoring the production of oxygen after the addition of H 2 O 2 .

2. Set up the electrode according to the manufacturer’s instructions using 50 % (w/v) saturated KCl as an electrolyte and calibrate

3.6 Marker Enzyme Assays of Mitochondria

3.6.1 Fumarase

3.6.2 Aconitase

3.7 Marker Enzyme Assay of Chloroplasts

3.7.1 Phosphori-bulokinase

3.8 Marker Enzyme Assay of Peroxisomes

3.8.1 Catalase


570

between air-saturated water (253 nmol O 2 /mL at 25 °C) and zero (established by adding a few crystals of sodium hydrosulfi te to the water) ( see Note 14 ).

3. The assay should be conducted at 25 °C. To 1 mL of mitochondrial reaction medium, add 5–20 μL of mitochon-drial suspension ( see Note 15 ).

4. Add 4 μL of 1 % (v/v) H 2 O 2 and monitor oxygen evolution. Rates of oxygen evolution should be calculated as nmol O 2 per min per mg protein. Typical rates for highly purifi ed per-oxisomes ~14 μmol O 2 per min per mg protein [ 8 ].

4 Notes

1. These solutions can be prepared ahead of the experiment and stored at room temperature for 1 month.

2. Potassium ferricyanide itself has a low toxicity. But as it irritates skin and eyes, protective clothing should be worn when han-dling it. Avoid strongly acidic conditions as the highly toxic hydrogen cyanide gas is formed.

3. It is recommended to make up only about 10 mL of reaction master mix and use for a series of 8–10 assays immediately.

4. It is recommended that a dedicated stock of detergent-free glass- and plasticware is kept solely for organelle isolations. Buy new glass- and plasticware for this purpose and clean using hot water only.

5. One of the common causes of poor recovery of intact organ-elles is insuffi cient extraction medium in relation to the amount of tissue being extracted. Arabidopsis cells are highly vacuo-lated, and upon rupture, the cell volume considerably dilutes the osmoticum. If insuffi cient extraction medium is used, this dilution effect can lead to organelle rupture.

6. When wringing extracts through muslin/Miracloth, it is advis-able to apply steady, gentle pressure. Too much pressure and the cloth can rupture suddenly.

7. Do not digest more than 1 L suspension in each 3 L Erlenmeyer fl ask.

8. This step will disrupt most of the protoplasts but not the organelles.

9. A common reason for low yield of organelles is a failure to cor-rectly recognize the appropriate band on the gradient. Refer to Fig. 2 for guidance.

10. The quality of the plant tissue and the time of harvest are the keys to a good yield. The plants should be mature, but not old,


571

and healthy. This reduces the appearance of interfering substances, e.g., phenolics, considerably. When the tissue is collected in the morning, the amount of starch in the chloro-plasts is low. High amounts can rupture the chloroplasts prob-ably during centrifugation. The collected tissue should be kept in a beaker or on foil on ice.

11. Pre-wet the tissue with grinding buffer. This helps to reduce the loss of chloroplast due to absorption.

12. A complete resuspension of the chloroplasts in wash buffer is crucial for an optimal separation in the Percoll step gradient. The presence of a white ring in the pellet indicates starch. Too much starch in the chloroplasts reduces yield. Changing growth conditions of the plants can help to reduce the starch amount.

13. Another pipette can be used, but a 3 mL disposable pipette allows more careful application of the sample on top of the gradient.

14. The performance of oxygen electrodes deteriorates over time due to electrochemical deposition of chloride and oxide salts on the silver anode. It is therefore necessary to periodically clean the anode using aluminium oxide polishing paste.

15. It is important not to add too much mitochondrial suspension; otherwise the rate of oxygen consumption will exceed the response time of the oxygen electrode. It is therefore advisable to test several different concentrations of mitochondrial sus-pension and choose one in which the oxygen consumption rate in the presence of substrate gives an appropriate rate.

16. Aconitase activity can be determined through the measurement of isocitrate production from citrate. Isocitrate production rate is measured by activity of an isocitrate and NADP-dependent enzyme. Aconitase contains an Fe-S center that is readily dam-aged by H 2 O 2 -inhibiting activity of the protein, and the pro-tein itself has been shown to be degraded during prolonged oxidative stress.

Acknowledgements

This work was supported by the ARC Centre of Excellence for Plant Energy Biology (CE0561495) and AHM as an ARC Australian Professorial Fellow and ES as an ARC Australian Postdoctoral Fellow. The authors would like to thank Dr. Joshua Heazlewood (now at the Joint BioEnergy Institute (JBEI)) for the pictures of organelles from Arabidopsis cell suspension cultures separated by density gradient centrifugation.


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9. Reumann S, Babujee L, Ma C, Wienkoop S, Siemsen T, Antonicelli GE, Rasche N, Luder F, Weckwerth W, Jahn O (2007) Proteome analy-sis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms. Plant Cell 19:3170–3193

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