Freeze-fracture studies on barley plastid membranes: VIII. In viridis- 115, a mutant completely lacking Photosystem II, oxygen evolution enhancer 1 (OEE1) and the α-subunit of cytochrome b-559 accumulate in appressed thylakoids

Abstract

The barley nuclear gene mutant viridis-115 completely and specifically lacks Photosystem II (PS Independent electron- transport activity. This was associated with the absence of variable fluorescence in room-temperature fluorescence induction, and a change in the 77 K fluorescence emission spectrum due to fluorescence from light-harvesting chlorophyll a / b-protein of Photosystem II (LHC II) unquenched by PS II reaction centres. The low-temperature absorption spectrum lacked chlorophyll a absorbing at 683 nm. Thin section electron microscopy showed that the grana were about 5 times larger in diameter than the wild type, with almost no inter-grana lamellae. Quantitation of the freeze-fracture ultrastructure of viridis-115 revealed a 96% reduction in the number of endoplasmic fracture face of thylakoid (EFs) particles, the presumed site of the PS II reaction centres. No change was seen in the protoplasmic fracture face of stacked (PFs) or unstacked (PFu)thylakoid particle densities, and the mutant contained 68% of the wild-type density of endoplasmic fracture face of unstacked thylakoid (EFu) particles, making it unlikely that they represent PS II located in the stroma lamellae (PS II β). Calculations based on chlorophyll: P-700 and chlorophyll a/b ratios revealed a loss of between 60 and 70 molecules of chlorophyll a per PS II reaction centre. A double mutant, clo-f22800 × vir115 contained granain the absence of PS II and LHC II, both of which have been implicated in the maintenance of thylakoid adhesion. Three of the ‘core’ PS II polypeptides (CP47, CP43 and Dl) were severely deficient, whereas the a-subunit of cytochrome b-559 was present in near normal levels, although the high potential form was not detectable by spectroscopy. The chlorophyll a/b-proteins CP29 and LHC I were present at normal levels, as was the extrinsic 33 kDa oxygen evolution enhancer 1 (OEE1).