Chloroplast biogenesis: Biosynthesis and accumulation of Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins by greening cotyledons

Abstract

Etiolated excised cucumber cotyledons ( Cucumis sativus L. cv. Alpha Green), while greening in distilled water, synthesized and accumulated several metalloporphyrins in the absence of added substrates or inhibitors. The metalloporphyrins, undetectable by conventional spectrophotometry, exhibited distinct fluorescence emission and excitation properties in situ and in organic solvents. The metalloporphyrins were partially segregated on thin layers of silica gel H into three Chromatographic bands and the bands were eluted in methyl alcohol:acetone (4:1 v v ). The metalloporphyrins in the eluted bands were characterized by their soret excitation and short-wavelength emission maxima. One of the metalloporphyrins of band 3 ( R f , 0.4−0.56) was identified as Mg-protoporphyrin monoester. It was accompanied by traces of two other metalloporphyrins. Band 2 ( R f , 0.32−0.48) was made up of three metalloporphyrins and had the Chromatographic mobility of endogenous protochlorophyllide. Band 1 ( R f , 0.22−0.43) was made up of two metalloporphyrins; it moved with endogenous chlorophyllide. The metalloporphyrins of band 2 and 1 exhibited fluorescence emission and excitation maxima similar to Mg-protoporphyrin monoester but slightly shifted to longer wavelengths. The Chromatographic and spectral properties of these compounds suggested that they represent intermediates between Mg-protoporphyrin IX monomethyl ester and protochlorophyllide. The analytical techniques described in this work may prove useful in the elucidation of the enzymology between protoporphyrin IX and protochlorophyllide.