INTEGRATION OF NUCLEAR-ENCODED PROTEINS INTO PEA THYLAKOIDS WITH DIFFERENT PIGMENT CONTENTS

The in vitro membrane integration of the light-harvesting protein of p hotosystem II (LHCP), the Rieske FeS protein of the cytochrome (Cyt) b lf-complex, and the NADPH:protochlorophyllide oxidoreductase (Pchlide reductase) into pea thylakoids with different pigment composition was studied. Pea plants (Pisum sativum L. cv. Kelvedon Wonder) with differ ent contents of chlorophyll (Chi) and carotenoids were obtained by gro wing the seedlings in a greenhouse or in weak red light with or withou t the herbicide Norflurazon, an inhibitor of carotenoid biosynthesis. Chloroplasts from untreated and Norflurazon-treated plants grown in we ak red light contained approximately 29 and 14% of Chi compared to chl oroplasts from unheated plants grown in the greenhouse. The correspond ing carotenoid contents were 66 and 5%. Following an integration react ion using LHCP precursor protein and chloroplast lysate, thylakoids fr om untreated and Norflurazon-treated plants grown in weak red light co ntained approximately 30 and 5% of protease-protected LHCP, respective ly, compared to thylakoids of untreated plants grown in a greenhouse. In contrast to LHCP, the in vitro assembly of the Pchlide reductase wa s only sligthly reduced in chloroplast lysates of plants grown in weak red light compared to greenhouse-grown plants. In chloroplast lysates of Norflurazon-treated plants, however, the amount of membrane associ ated, protease-protected Pchlide reductase was reduced to 32% of the a mount in untreated plants grown under the same light conditions. In co ntrast, the integration of the Rieske FeS protein occurred to almost s imilar levels irrespective of light conditions and herbicide treatment s. Reconstitution assays where stroma from Norflurazon-treated plants was added to thylakoids from untreated plants, showed that the herbici de did not affect any stromal component(s) vital for the insertion rea ction. Removal of samples during the integration reaction of LHCP show ed that no degradation of the protein occurred during the assay. Neith er was the assembled protein degraded up to 24 h after the termination of the assay. This indicates that growing plants in weak red light, w ith or without Norflurazon treatment, mainly affected the primary step in thylakoid assembly of LHCP, i.e. the insertion reaction into the m embrane. The results further indicate that proteins normally bound to pigments also require pigments for membrane recognition or integration .