In vitro-reconstitution and developmental expression of LHPP, the light-harvesting NADPH:protochlorophyllide (Pchlide) oxidoreductase:Pchlide complex of etiolated plants

Abstract

NADPH:protochlorophyllide oxidoreductase (POR, EC 1.1.33.1) is a key enzyme for the light-induced greening of angiosperms. In barley, two POR proteins exist, termed PORA and PORB. These have previously been proposed to form higher molecular weight light-harvesting complexes in the prolamellar body of etioplasts (C. Reinbothe, N. Lebedev & S. Reinbothe (1999) Nature 397: 80-84). Here we report the in vitro-reconstitution of such complexes from chemically synthesized protochlorophyllides (Pchlides) a and b and galacto- and sulfolipids. Low temperature (77K) fluorescence measurements revealed that the reconstituted, lipid-containing complex displayed the same characteristics of photoactive Pchlide 650/657 as the presumed native complex in the prolamellar body. Moreover, Pchlide F650/657 was converted to chlorophyllide (Chlide) 684/690 upon illumination of the reconstituted complex with a 1-msec flash of white light. Identification and quantification of acetone-extractable pigments revealed that only the PORB-bound Pchlide a was photoactive and converted to Chlide a, whereas Pchlide b bound to the PORA remained photoinactive. Non-denaturing PAGE of the reconstituted Pchlide a/b-containing complex further demonstrated a size similar to that of the presumed native complex in vivo, suggesting that both complexes may be identical. LHPP expression is under developmental control and optimized to allow efficient seedling de-etiolation.