Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic Centrifugation

Daphné Seigneurin‐Berny1, Daniel Salvi1, Jacques Joyard1, Norbert Rolland1

1 Laboratoire de Physiologie Cellulaire Végétale, CNRS and Université Joseph Fourier, Grenoble, France
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.30
DOI:  10.1002/0471143030.cb0330s40
Online Posting Date:  September, 2008
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Abstract

Chloroplasts are plant‐specific organelles. They are the site of photosynthesis but also of many other essential metabolic pathways, such as syntheses of amino acids, vitamins, lipids, and pigments. This unit describes the isolation and purification of chloroplasts from Arabidopsis and spinach leaves. Differential centrifugation is first used to obtain a suspension enriched in chloroplasts (crude chloroplasts extract). In a second step, Percoll density gradient centrifugation is used to recover pure and intact chloroplasts. The Basic Protocol describes the purification of chloroplasts from Arabidopsis leaves. This small flowering plant is now widely used as a model organism in plant biology as it offers important advantages for basic research in genetics and molecular biology. The Alternate Protocol describes the purification of chloroplasts from spinach leaves. Spinach, easily available all through the year, remains a model of choice for the large‐scale preparation of pure chloroplasts with a high degree of intactness. Curr. Protoc. Cell Biol. 40:3.30.1‐3.30.14. © 2008 by John Wiley & Sons, Inc.

Keywords: chloroplast; Arabidopsis; spinach; purification; leaves

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Pure and Intact Chloroplasts from Arabidopsis Leaves
  • Alternate Protocol 1: Isolation of Pure and Intact Chloroplasts from Spinach Leaves
  • Support Protocol 1: Growing of Arabidopsis Plantlets
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Pure and Intact Chloroplasts from Arabidopsis Leaves

  Materials
  • Percoll gradients solution (see recipe)
  • Four to six boxes containing 3‐ to 4‐week‐old Arabidopsis plantlets (400 to 500 g of rosette material, see protocol 3)
  • Grinding medium (see recipe)
  • Washing medium (see recipe)
  • 80% (v/v) acetone
  • 50‐ml polypropylene tubes
  • Superspeed refrigerated centrifuge (e.g., Sorvall RC5), with the following rotors and corresponding tubes: fixed angle rotors GS‐3 with six 500‐ml plastic bottles and SS34 with eight 50‐ml polypropylene tubes; swinging bucket rotor HB‐6 with six 50‐ml polycarbonate tubes or equivalent
  • 1000‐ml and 5‐liter beakers
  • Ice and ice buckets
  • Motor‐driven blender, 3 speeds, 1 gallon (3.785 liter; e.g., Waring blender)
  • Muslin or cheesecloth, 80‐cm
  • Nylon blutex (50‐µm aperture; Tripette et Renaud)
  • Pasteur pipet
  • Vacuum aspirator with a flask as a liquid trap
  • Curved plastic spatula
  • 1‐ and 10‐ml pipets
  • 1‐ml microcentrifuge tube
  • Vortex
  • 1‐ml spectrophotometer glass cuvette
  • UV spectrophotometer
NOTE: All solutions, glassware, centrifuge tubes, and equipment should be precooled to 0° to 4°C and kept on ice throughout.NOTE: All operations are carried out at 0° to 5°C either by keeping samples on ice or by working in a cold room.

Alternate Protocol 1: Isolation of Pure and Intact Chloroplasts from Spinach Leaves

  Materials
  • 3 kg of spinach leaves from the market
  • Spinach leaf grinding medium (see recipe)
  • Ice
  • Spinach washing medium (see recipe)
  • 40% (v/v) Percoll and 80% (v/v) Percoll solutions (see recipes)
  • Motor‐driven blender, 3 speeds, 1 gallon (3.785 liter; Waring blender)
  • Muslin or cheesecloth, 80‐cm
  • Nylon blutex (50‐µm aperture; Tripette et Renaud)
  • 5‐liter beakers
  • Superspeed refrigerated centrifuge (Sorvall RC5), with the following rotors and corresponding tubes: fixed angle rotors GS‐3 with six 500‐ml plastic bottles and SS34 with eight 50‐ml polypropylene tubes; swinging bucket rotor HS‐4 with four 150‐ml polycarbonate tubes or equivalent
  • Pasteur pipet
  • Vacuum aspirator with a flask as a liquid trap
  • Ice buckets
  • Curved plastic spatula
  • 1‐ and 10‐ml pipets
  • 250‐ml cylinder
NOTE: All solutions, glassware, centrifuge tubes, and equipment should be precooled to 0° to 4°C and kept on ice throughout.NOTE: All operations are carried out at 0° to 5°C either by keeping samples on ice or by working in a cold room.

Support Protocol 1: Growing of Arabidopsis Plantlets

  Materials
  • Arabidopsis thaliana seeds
  • Compost
  • Large plastic cases (30‐cm × 45‐cm)
  • Growth rooms
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Figures

  •   FigureFigure 3.30.1 Outline of the strategy used to purify intact chloroplasts from Arabidopsis leaves.
  •   FigureFigure 3.30.2 SDS‐PAGE analysis of Percoll‐purified chloroplasts from Arabidopsis. Fractions (15 µg protein) were analyzed on a 12% SDS‐PAGE followed by Coomassie‐blue staining. In lanes B, the amount of protein was calculated in order to load the same amount of LHCP in the two lanes of the gel. The star and the arrow respectively indicate the major soluble protein from the stroma, RbcL, and the major membrane proteins from the thylakoids, the LHCPs. Note that the ratio of RbcL/LHCPs is strongly reduced in broken chloroplasts, as a consequence of breakage of the limiting envelope membranes and thus of loss of soluble proteins from the stroma. Abbreviations: CE: crude extract, BC: broken chloroplasts, IC: intact chloroplasts.
  •   FigureFigure 3.30.3 Schematic representation of the discontinuous Percoll‐gradient fractionation of the crude spinach chloroplasts preparation to yield intact and broken chloroplasts.

Literature Cited

Literature Cited
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