Reversible heat-inactivation and photo-reactivation of oxygen evolving center (OEC) of spinach chloroplasts

Abstract

OEC of chloroplasts is very sensitive to irreversible heat-inactivation when heated at 50oC for 5 min. However, this study introduced a new method for the reversible heat-inactivation and the recovery of OEC activity by photo-reactivation. For the reversible heat-inactivation chloroplasts must be heated in the presence of reduced DCPIP (0.05-0.1 mM) by small amount (0.5 ml) in a tiny test tube in very short time (30 sec) by shaking very vigorously in a water bath at 50oC in the dark. By this heating the chloroplast OEC activity decreased to 15% but the 70% activity could be recovered by applying the photo-reactivation, that is the incubation of chloroplasts with Mn2+, Ca2+ and a reductant for 20 min under illumination of 300 lux white light at 20oC, the same conditions used for the reactivation of Tris-inactivated chloroplasts. The evidence of inactivation and reactivation in OEC or PSII reaction center was demonstrated by the observation of depression and restoration of actinic light-induced chlorophyll fluorescence rise. By photo-reactivation a part of low-potential form Cyt-b559 of heat-inactivated chloroplasts was brought back to the high-potential form