Details

Autor(en) / Beteiligte

• Kim Hong, Hoang T.
• Bich Phuong, Truong T.
• Thu Thuy, Nguyen T.
• Wheatley, Matthew D.
• Cushman, John C.

Titel

Simultaneous chloroplast, mitochondria isolation and mitochondrial protein preparation for two-dimensional electrophoresis analysis of Ice plant leaves under well watered and water-deficit stressed treatments

Ist Teil von

• Protein expression and purification, 2019-03, Vol.155, p.86-94

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2019

Quelle

MEDLINE

Beschreibungen/Notizen

• This paper presents a simultaneous isolation of pure, intact chloroplasts and mitochondria from mature leaves of Ice plant (Mesembryanthemum crystallinum) and mitochondrial protein preparation for two-dimensional electrophoresis (2DE) analysis under well watered and water -deficit stressed treatments. The washed chloroplasts and mitochondria were purified with Percoll gradients prepared using a Master flex R pump. The chloroplast and mitochondrial proteins were extracted in lysis buffer containing a protease inhibitor mix supplemented with 1 μM Leupeptin and 1 μM E64, followed by precipitation with ice-cold acetone. The protein contents were determined by an EZQ protein quantitation kit. The results show that chloroplast and mitochondria isolated from Ice plant leaves via this protocol have pure and intact. The shape of chloroplast and mitochondria observed by microscopy were clear and sharp. This procedure was employed for assessing the significant differences in mitochondrial protein expression patterns from the well watered and water-deficit stressed treatment leaves collected at dawn (6 a.m.) and dusk (6 p.m.). The results showed 71 and 20 differentially abundant spots between control and CAM for 6 a.m. and 6 p.m., respectively. In addition, 32 protein spots were differentially abundant for 6 a.m. control compared with 6 p.m. control, and 45 protein spots were differentially abundant for 6 a.m. CAM compared with 6 p.m. CAM. Spots that displayed differential abundance for control compared with CAM likely included proteins involved in mitochondrial processes necessary for CAM function. Through further analysis, these proteins will be identified and characterized in the near future using mass-spectrometry-based techniques. •A protocol for simultaneous isolation of intact chloroplasts, mitochondria and their protein preparation for 2DE analysis from Ice plant.•Typical difference in mitochondrial proteins between well-watered (C3) and water-deficit stressed (CAM) treatment samples in the 2DE maps was firstly detected.•The drought stress treatment significantly effected on the protein expression of Ice plant mitochondria in which water-deficit stressed (CAM) samples showed more protein spots than those of well-watered (C3) samples at both time point of 6am and 6pm.