Immunological Evidence for the Binding of β-Carotene and Xanthophylls onto Peptides of Photosystem I from Nicotiana tabacum

Abstract

Photosystem I preparations were obtained from wild type tobacco Nicotiana tabacum var. John William’s Broadleaf (JWB) and from the two chlorophyll-deficient mutants N. tabacum Su/su and N. tabacum Su/su var. Aurea. The preparations were characterized with respect to the chlorophyll a/b ratio, their photosynthetic activity and their absorption spectroscopic properties. Peptides from these preparations were analyzed by SDS polyacrylamide gel electrophoresis and transferred for the detection of bound carotenoids according to the Western blot procedure to nitrocellulose or Immobilon membranes. The PS I preparation from the wild type JWB consisted of the core and the LHCP complex. The core complex contains the two core peptides with the same apparent MW of 66 kDa and several peptides with the lesser molecular masses of 22, 20, 19, 17, 16, 10 and 9 kDa. The light-harvesting protein complex consists of 4 subunits with the molecular masses 28, 26, 25 and 24 kDa. The PS I preparations of the yellow-green mutant Su/su and of the Aurea mutant Su/su var. Aurea contain as impurity traces of the D1 and D2 core peptides of photosystem II and also traces of the chlorophyll-binding photosystem II peptides with the molecular masses 42 and 47 kDa. The peptides of the photosystem I preparation were characterized by specific photosystem I antisera: An antiserum to the photosystem I complex reacts in the Western blot only with the homologous peptides of photosystem I. In comparative analyses with photosystem II preparations this antiserum (directed to photosystem I) reacts, as expected, only with the peptides of the light-harvesting complex. An antiserum to the CP 1 core peptides reacts only with the 66 kDa peptides of photosystem I and gives no cross reaction with heterodimer forms of the D1/D2 core peptides of photosystem II. In the Western blot procedure by means of polyclonal monospecific antisera to carotenoids it was demonstrated that β-carotene is bound in high concentration onto the core peptides CP 1 and to a lesser extent onto the two larger subunits of the LHCP complex, exhibiting the molecular masses of 28 and 26 kDa. Neoxanthin is bound onto the same peptides. In contrast to this, lutein was only identified on the core peptides CP 1 and violaxanthin only on the larger subunits of the LHCP complex. As the carotenoids are labelled with antibodies, even after SDS treatment in the electrophoresis, it is assumed, that the carotenoids are covalently bound via the ionon ring to the respective peptide