Isolation of Amyloplasts

Kay Denyer1, Marilyn Pike1

1 John Innes Centre, Norwich Research Park, Norwich, Norfolk
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.28
DOI:  10.1002/0471143030.cb0328s38
Online Posting Date:  March, 2008
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Abstract

Two different methods for the preparation of starch‐rich plastids are described together with protocols for the determination of plastid yield, purity, and intactness. The preparation of amyloplasts from maize endosperm and oilseed rape embryos are given as examples, but the protocols could be adapted for the isolation of starch‐rich plastids from other plant organs. A method for the determination of the quantitative distribution of an enzyme between the plastids and cytosol is given. Typical results and references for marker enzymes for a range of subcellular compartments are listed. Curr. Protoc. Cell Biol. 38:3.28.1‐3.28.15. © 2008 by John Wiley & Sons, Inc.

Keywords: amyloplast; plastid; starch

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Amyloplasts from Maize Endosperm
  • Alternate Protocol 1: Isolation of Amyloplasts from Oilseed Rape Embryos
  • Support Protocol 1: Determining the Yield and Purity of Amyloplasts
  • Support Protocol 2: Determination of Plastid Intactness by Latency
  • Support Protocol 3: Determination of Plasmid Intactness by Protection
  • Support Protocol 4: Determination of the Distribution of an Enzyme Between the Amyloplasts and Cytosol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Amyloplasts from Maize Endosperm

  Materials
  • Amyloplast isolation medium 1 (AIM 1; see recipe), ice cold
  • ∼10 g of developing endosperm harvested 11 to 22 days after pollination
  • Pasteur pipets and rubber pipet bulbs
  • Single‐edged razor blades
  • 90‐mm petri dish
  • Filtration cloth (e.g., Miracloth, pore size 22 to 25 µm; Calbiochem, or equivalent)
  • Small funnel
  • 30‐ml glass centrifuge tube
  • Refrigerated benchtop centrifuge (e.g., Beckman RT 6000D, or equivalent)
  • Measuring cylinder
  • Small (1‐ml) syringes and fine (23‐G) needles
NOTE: Carry out all procedures at 0° to 4°C using solutions and equipment prechilled to that temperature.

Alternate Protocol 1: Isolation of Amyloplasts from Oilseed Rape Embryos

  Materials
  • 1× and 2× amyloplast isolation medium 2 (AIM 2; see recipe), ice cold
  • Developing embryos of oil seed rape (Brassica oleracea variety Topas)
  • Percoll
  • Polytron homogenizer (e.g., model PT 3000, Kinematica, http://www.kinematica‐inc.com/; or equivalent)
  • 90‐mm petri dish
  • Filtration cloth (e.g., Miracloth, pore size 22 to 25 µm; Calbiochem, or equivalent)
  • 15‐ and 30‐ml glass centrifuge tubes
  • 50‐ml plastic graduated cylinder, prechilled
  • Small funnel
  • Wide‐ended pipet (see protocol 1, step 1) or small paintbrush
  • Refrigerated centrifuge (e.g., Sorvall RC5C with an HB4 rotor, or equivalent)
  • Small (1‐ml) syringes and fine (23‐G) needles
NOTE: Carry out all procedures at 0° to 4°C using solutions and equipment prechilled to that temperature.

Support Protocol 1: Determining the Yield and Purity of Amyloplasts

  Materials
  • Aliquots of homogenate, supernatant, and pellet fractions (see protocol 1 and protocol 2)
  • Additional reagents and equipment for assays of amyloplast marker enzymes (see references in Table 3.28.1)
NOTE: Carry out all procedures at 0° to 4°C using solutions and equipment prechilled to that temperature unless otherwise stated.

Support Protocol 2: Determination of Plastid Intactness by Latency

  Materials
  • 2 M sorbitol
  • Resuspended, intact plastids ( protocol 1 and protocol 2)
  • Cuvettes suitable for spectrophotometry
  • Additional reagents and equipment for assays of amyloplast marker enzymes (see references in Table 3.28.1)

Support Protocol 3: Determination of Plasmid Intactness by Protection

  Materials
  • Resuspended, intact plastids ( protocol 1 and protocol 2)
  • 400 mg/ml trypsin (Type IX, 6000 U/mg, from bovine pancreas) stock solution in H 2O (prepare immediately before use)
  • 600 mg/ml trypsin inhibitor (Glycine max, 12,000 U/mg) stock solution in H 2O (prepare immediately before use)
  • Small (1‐ml) syringes and fine (23‐G) needles
  • 25°C water bath
  • Cuvettes suitable for spectrophotometry
  • Additional reagents and equipment for assays of amyloplast marker enzymes (see references in Table 3.28.1)

Support Protocol 4: Determination of the Distribution of an Enzyme Between the Amyloplasts and Cytosol

  Materials
  • Aliquots of homogenate, supernatant, and pellet fractions (see protocol 1Basic and protocol 2Alternate Protocols)
  • Assay mix for enzyme X (the enzyme of interest; see Table 3.28.1)
  • Additional reagents and equipment for determining yield and purity of amyloplasts ( protocol 3) and assays of amyloplast marker enzymes (see references in Table 3.28.1)
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Figures

Videos

Literature Cited

Literature Cited
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   Denyer, K., Dunlap, F., Thorbjornsen, T., Keeling, P., and Smith, A.M. 1996. The major form of ADP‐glucose pyrophosphorylase in maize endosperm is extra‐ plastidial. Plant Physiol. 112: 779‐785.
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