Abstract
Preparation of intact mitochondria requires the use of an osmoticum in high concentration that preserves the mitochondrial structure and prevents physical swelling and rupture of membranes. For this purpose mannitol is widely accepted as a component of the homogenization medium. The formation of sorbitol in plant mitochondria can be an important element of plant response to salinity and drought. The large difference in the concentration of possibly formed sorbitol and mannitol of the homogenisation medium makes the determination of sorbitol difficult. Gas-chromatography with flame ionization detection (GC-FID), gas-chromatography coupled to quadrupole mass spectrometry (GC-QPMS) and liquid chromatography/tandem mass spectrometry (LC/MS-MS) using hydrophilic interaction chromatography (HILIC) were tested to quantify this highly polar sugar alcohol: Although all three methods offered satisfactory results, the HPLC/MS-MS technique proved to be the best.
Highlights
Invertase activity and the corresponding transport of fructose, glucose and sucrose were described in mitochondria isolated from Jerusalem artichoke tubers [16]
On one hand the application of sucrose as osmoprotectant in the investigation of mitochondrial sorbitol formation is not possible, due to the mitochondrial invertase activity which cleaves sucrose into glucose and fructose, it would interfere with the observation of sorbitol formation
Three different techniques were compared: gas-chromatography with flame ionization detection (GC-FID), gas-chromatography coupled to quadrupole mass spectrometry (GC-QPMS), and hydrophilic interaction liquid chromatography coupled with electrospray ionisation and tandem mass spectrometry
Summary
Invertase activity and the corresponding transport of fructose, glucose and sucrose were described in mitochondria isolated from Jerusalem artichoke tubers [16]. Following this observation the enzyme was found in mitochondria from other plant species suggesting that it is a general feature of plant cells. A sucrose-glucose/ fructosesorbitol pathway has been described, as the central osmoregulation machinery, in the prokaryotic Zymomonas mobilis [9] This bacterial mechanism provides special importance to the observations that several plant species accumulate different sugar alcohols under drought and salt stress [3,5,13,15] and sorbitol can be one of the key components of the osmotic stress adaptation mechanism [12]. Three different techniques were compared: gas-chromatography with flame ionization detection (GC-FID), gas-chromatography coupled to quadrupole mass spectrometry (GC-QPMS), and hydrophilic interaction liquid chromatography coupled with electrospray ionisation and tandem mass spectrometry
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