Oxygen production and consumption by chloroplasts in situ and in vitro as studied with microscopic spin label probes

Abstract

A new spin-label oximetry approach able to measure the oxygen partial pressure in complex photosynthetic systems has been developed using bovine serum albumin (BSA)-coated light paraffin oil particles containing cholestane spin label (CSL). Paraffin oil particles protect the spin label against the action of chemically active metabolites. The amplitude of the electron paramagnetic resonance (EPR) signal from CSL measured at a saturating microwave power is sensitive to the concentration of oxygen. We demonstrate here the ability of this method to monitor the kinetics of light-induced oxygen production in situ, i.e., in the interior of a bean leaf. The oxygen release, observed during leaf illumination with continuous light, exhibits an overshoot that correlates with the well-known nonmonotonous behavior of the Photosystem I reaction center, P700. Short-term illumination of isolated bean chloroplasts, suspended in the presence of the electron mediator methylviologen, induces a reversible uptake of oxygen. However, after prolonged illumination, chloroplasts lose their ability to regenerate oxygen in the dark. The exhaustion of oxygen (and oxygen active forms) is accompanied by the loss of CSL paramagnetism and the capacity to photooxidize P700. Comparison of the kinetics of P700 redox transients with oximetric data demonstrates that oxygen concentration is the essential factor controlling electron transport in leaves and isolated chloroplasts.