Developmental and immunological studies on chlorophyll proteins
Creators: name: White, Michael J.
Publisher: name: University of British Columbia
Year: 2010

description: All the chlorophyll in higher plants is bound to specific proteins. These chlorophyll-proteins absorb light energy which is used by the electron transport processes of the photosynthetic apparatus. The synthesis and assembly of the photosynthetic apparatus and chlorophyll proteins is regulated by light and other factors which are incompletely understood. Antibodies were used to investigate the accumulation of chlorophyll proteins in plant systems which were completely lacking one or more of the factors involved in chlorophyll protein synthesis or degradation. Immunological cross-reactions among the chlorophyll a + b apoproteins were observed and characterized, and theoretical work was undertaken to predict the probability that immunological cross-reactions may arise due to chance arrangement of amino acids. Chlorophyll a+b protein complexes associated with photosystem I were isolated and characterized. These included a light-harvesting antenna complex (LHCI), which was shown to contain Chi a, some Chi b, and four polypeptides of 21-24 kDa. This complex was isolated from CP Ia complexes, which also contain the P700 chlorophyll a protein, CP I. Antibodies were raised against the chlorophyll a + b protein complexes of both the photosystem I antenna (LHCI) and the photosystem II antenna (CP II and CP 29), and against the chlorophyll a protein containing the photosystem I reaction center (CP I). Antibodies to LHCI were obtained by using highly purified CP Ia as an antigen. When antisera to Chi a + b proteins were reacted with the entire electrophoretic spectrum of denatured thylakoid proteins, they cross-reacted with the polypeptides belonging to other Chi a+b proteins. The strongest cross-reactions were observed between antibodies to CP Ia and the polypeptides of the photosystem II complexes, CP II and CP 29. These cross-reactions observed with anti-CP Ia were shown not to be due to antibodies to CP I, but rather to antibodies directed against the photosystem I antenna complex, LHCI. Affinity-purification of the anti-CP Ia antibodies did not eliminate these cross-reactions. The data suggest there are amino acid sequence similarities among the chlorophyll a/b-binding polypeptides. Theoretical work was undertaken to determine if these cross-reactions were expected due to chance alone or not. Attempts to address this question led to the synthesis of a mathematical model to predict the probability that antibodies will react with proteins other than the antigen they were raised against. This theoretical work was done as a collaboration with Peter Sibbald of our lab, with input from Dr. Michael Waterman, one of the mathematicians whose work this model is based on. The sequence similarities predicted to exist among the chlorophyll a+b proteins appear to be more extensive than those which would be expected to arise due to chance alone. It is postulated that the Chi 0+6 proteins may have arisen from a common ancestral gene. At least some polypeptides belonging to each of the three Chi a + b proteins, including those of CP 29 and LHCI, were found in the Chi Mess barley (Hordeum vulgare L.) mutant chlorina f2. Similar experiments were performed on barley grown under intermittent light, which also completely lacks Chi b. Only two polypeptides belonging to the Chi a+b proteins were found under these conditions. Taken together with published studies in the literature, these results provide evidence for the existence of differing posttranscriptional controls among the various chlorophyll proteins, and between these two systems. Antibodies were also used to determine the kinetics of synthesis of the chlorophyll-binding polypeptides during the light-induced greening of dark-grown barley. The chlorophyll a + b antenna polypeptides of both photosystems were absent in the dark. They were first detected after 1-2 hours of illumination, and then increased in amount exponentially. The kinetics of accumulation are almost identical for all the polypeptides belonging to the chlorophyll a+b protein complexes, suggesting their synthesis may be regulated by the same mechanism despite the existence of the differing posttranscriptional controls mentioned above. CP 43, a chlorophyll a antenna protein of photosystem II, appeared to follow the same time course. In contrast to some reports in the literature, small amounts of the photosystem I P700 chlorophyll o protein were detected in etiolated plants. This protein also shows a light-induced increase, showing that light plays an important role in its synthesis or degradation, even if it is not an absolute requirement for transcription and translation.