Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels

Abstract

It has recently been reported that tomato fruit chromoplasts can synthesize ATP de novo using an ATP synthase complex harboring an atypical  -subunit which is also present in a variety of plant species. However many aspects related with the biochemical processes underlying this process remain largely unknown. Here we describe detailed protocols for the isolation of tomato fruit chromoplasts and the determination of ATP levels (end-point measurements) and ATP synthesis rates (kinetic measurements) in these organelles using bioluminescent luciferin/luciferase based assays. Materials and Reagents1. Red tomato fruits (MicroTom or Ailsa Craig, harvested 5-7 and 7-9 days after breaker stage, respectively) 2. Ultrapure water 3. NaCl (Sigma-Aldrich, catalog number: S7653) 4. Trizma ® base (Tris) (Sigma-Aldrich, catalog number: T6066) 5. HCl (Merck KGaA, catalog number: 100317) 6. Sorbitol (Sigma-Aldrich, catalog number: S1878) 7. MgCl2 (Sigma-Aldrich, catalog number: M2670) 8. KCl (Sigma-Aldrich, catalog number: P9541) 9. L-Ascorbic acid (Sigma-Aldrich, catalog number: 255564) 10. L-Cysteine (Sigma-Aldrich, catalog number: C7352) 11. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43819) 12. Sucrose (Sigma-Aldrich, catalog number: S8501) 13. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906) 14. MnCl2 (Sigma-Aldrich, catalog number: M3634) 15. KH2PO4 (Merck KGaA, catalog number: 104873)