INTRODUCTION. It has been shown that mild trypsyn-treatment and heat-treatment of isola- ted chloroplasts above 350C led to the disruption of grana structures ...
MULTIPLE EFFECTS OF TRYPSIN- AND HEAT-TREATMENTS ON THE ULTRASTRUCTURE AND SURFACE CHARGE DENSITY OF PEA CHLOROPLAST MEMBRANES. INFLUENCE ON P700+ PARAMETERS.
Central Laboratory of Biophysics,Bulgarian Academy of Sciences, Acad. G. Bonchev Str. bl. 21, 1113 Sofia, Bulgaria. INTRODUCTION It has been shown that mild trypsyn-treatment and heat-treatment of isolated chloroplasts above 350C led to the disruption of grana structures resulting in the lack of thylakoid adhesion and formation of single nonappressed membranes(1,2,13).These changes in chloroplast organization are very similar to those observed by resuspending of thylakoids in low salt media(3).It is generally believed that the light-harvesting ChI a/b protein complex of photosystem II (LHCII) is required for the formation of grana(4,5) and this process might be achieved via electrostatic screening of the repulsion forces between negatively charged thylakoid membranes(6).Allowing that trypsinand heat-induced destacking processes are accompanied by the well established alterations in either,the composition(4,5) or the aggregation state of LHCII (2,7),it could be expected that electrostatic mechanisms are responsible for the observed structural changes in both cases. Trypsin- and heatinduced multiple effects on the chloroplast organization manifested in formation of destacked or concentric (mielin-like) structures are presented in this study. The formation of these structures strongly depend on the incubation conditions and are accompanied by reversible changes in surface charge density of thylakoid membranes. The influence of trypsin- and heat-induced dynamical rearrangements of the thylakoid protein components on the efficiency of P700 photooxidation is discussed in terms of redistribution of excitation energy between the photosystems in favour to PS I. MATERIAL A D METHODS Intact chloroplasts from pea leaves were isolated as in (8).Envelope-free chloroplasts were obtained by osmotic shock and were resuspended in 0.01 M Tricine /pH 8.0/ and 0.33 M sucrose. Stacked or unstacked thylakoids were obtained by the addition of 5 mM MgC1 or 10 mM KCl to the r~suspension bu2 ffer respectively.Glutaraldehyde (GA) treatment was performed as recomended in (9).Trypsin-treatments were carried out at different trypsin/ChI ratio. The reaction was stopped by the addition of 20-fold excess trypsin-inhibitor. 0 90 light-scattering measurements were performed as in (10).Thylakoid surface charge density (6 ) was investigated as described in (11).The relative amounts of P700+ (determined by the size of ESR signal I at g=2.0025 region) and dark reduction rate of P700+ were measured in an ERS 220 spectrometer. Samples for electron microscopy were fixed in 2.5% GA and postfixed in 1% Os04' The fixed material was dehydrated and embedded in Durcupan-ACM/Fluca/. StsJned ultrathin sections were observed with a JEM 100B electron microscope. RESULTS AND DISCUSSION light-scattering measurements are used for assessiny the degree of thylakoid stacking (10),under different incubation conditions /see Fig.1./.The observecJdecre
