Light-dependent assembly of pigment-protein complexes in etiolated leaves of Phaseolus coccineus L monitored by seminative gel electrophoresis, fluorescence spectroscopy and pigment analysis

In primary leaves of dark grown bean seedlings (Phaseolus coccineus L.) the transformation of etioplasts to chloroplasts upon illumination (1-120 h continuous light) was studied by recording the alterations of the emission peaks of laser-induced chlorophyll fluorescence spectra and corresponding induction kinetics using a diode array spectrophotometer. In addition, pigment-protein complexes solubilized from isolated plastids of different developmental stages were separated by nondenaturating Deriphat polyacrylamide gel electrophoresis. Chlorophyll fluorescence spectra (77K) of the green pigment-protein complexes were measured directly on the gels. Subsequently, the complexes were extracted by electroelution and their pigment composition analyzed by HPLC. It was found that in leaves in the early light-dependent developmental phase (after about 1 h) a fluorescence maximum at 688 nm appeared that was followed (after several hours) by an additional peak emission at 742 nm. When analyzing 77K fluorescence spectra of leaves an increasing intensity of fluorescence between 735 nm and 739 nm was also found after a prolonged illumination period. In protein complexes, which were isolated from dark-grown leaves and separated on gels, no appreciable amounts of chlorophyll were visible. However, if 12-h illuminated leaves had been used, two green protein bands were detectable assigned to PS I and PS II by their 77K fluorescence. From fully developed leaves (120 h light) eight green bands were finally detected and classified as derivatives of PS I or PS II. PS I complexes exhibited a Chl a: Chl b ratio of about 10: 1, whereas PS II complexes had a ratio of 2 : 1. beta-carotene was mainly localized in PS I complexes, whereas lutein, neoxanthin and violaxanthin were especially found in PS II complexes. It is concluded that during the light-dependent arrangement of etioplasts to chloroplasts the assembly of PS II units and the insertion of pigments therein preceeds the assembly of PSI.