Materials and Reagents

1. 50 ml Falcon tubes (Sigma-Aldrich, catalog number: Greiner227261 )
   
2. Muslin (MacCulloch & Wallis, catalog number: 4470 )
   
3. Plants
   1. Tomato (90 to 150 g of breaker +3 to 5 days fruit per condition)
      
   2. Sweet bell pepper (30 to 120 g of ripe fruit per condition; depending on fruit pigment content)
      
   3. Hot chilli pepper (~30 g of ripe fruit per condition)
   Note: Breaker implies the first visual appearance of yellow/orange colour on the fruit. It is usually difficult to get ~10 tomato fruits (for 90 to 150 g total) at the same ripening stage on the same day, so that is why we harvest fruit from breaker + 3 days to breaker + 5 days. Of course, if you can have enough fruit at the same ripening stage, it is even better.
4. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
   
5. Sucrose (Sigma-Aldrich, catalog number: S0389 )
   
6. Trizma base (Tris) (Sigma-Aldrich, catalog number: T1503 )
   
7. EDTA (Sigma-Aldrich, catalog number: E5134 )
   
8. Tricine (Sigma-Aldrich, catalog number: T0377 )
   
9. Sodium bisulphite (sodium metabisulfite) (Sigma-Aldrich, catalog number: S9000 )
   
10. Extraction buffer (see Recipes)
    
11. Gradient buffers (see Recipes)

Equipment

1. Cold room (4 °C)
   
2. -20 °C room
   
3. Centrifuge Sorvall RC-5C (Thermo Scientific)
   
4. Fixed angle rotor Sorvall GSA-3 (purple colour) (Thermo Scientific)
   
5. Fixed angle rotor Sorvall GSA-5 (green colour) (Thermo Scientific)
   
6. Ultracentrifuge Beckman L7 (Beckman Coulter)
   
7. Swing rotor SW28 (Beckman Coulter)
   
8. 500 ml centrifuge bottles (Thermo Fisher Scientific, Nalgene^TM, model: 3141-0500 )
   
9. 50 ml centrifuge tubes (Thick-wall UltraTubes) (Thermo Fisher Scientific, Nalgene^TM, model: 3110-0500 )
   
10. 38.5 ml centrifuge tubes (Beckman Coulter, Ultra-Clear^TM, model: 344058 )
    
11. Glass flask conical narrow neck 5 L (Fischer Scientific, Fisherbrand^TM, model: 11597422 )
    Note: This product has been withdrawn by the manufacturer. [Replacement item: Borosilicate glass narrow neck Erlenmeyer flasks (Fischer Scientific, Fisherbrand^TM, model: 15479103 )]
    
12. Waring blender (small laboratory blender 8010ES) (Scientific Labs, model: MIX1126 )
    
13. Potter-Elvehjem tissue grinder (VWR International, model: 14231-372 )
    
14. Fraction collector (Gilson, model: 203B )
    
15. Suitable ice containers
    
16. Funnel (glass or plastic, to fit 5 L conical flasks)

Procedure