STUDIES ON THE BIOSYNTHESIS OF PROTOCHLOROPHYLLIDE ESTER IN ETIOLATED AND GREEN TISSUES OF HIGHER PLANTS

Cucumber cotyledons incubated in darkness with (alpha),(alpha)'-dipyridyl and (delta)-aminolevulinic acid (ALA) accumulated a pigment which had the spectroscopic properties of Mg-protoporphyrin monomethyl ether (MPE) but physical properties similar to those of protochlorophyllide (Pchlide) ester. When this compound was subjected to demetallation and acid hydrolysis the resulting porphyrin had the chromatographic and spectral properties of protoporphyrin (Proto). This new pigment appears to be a fully esterified MPE. Analysis of the MPE-like pigment preparation by HPLC revealed four peaks which were all spectrally identical to MPE. These pigments probably differed in mobility due to different esterifying alcohols. Analysis of the alcohols bound to cucumber MPE ester indicate that phytol is not present. Similar results were obtained when Pchlide ester preparations were analyzed.;The biosynthetic origin of Pchlide ester was investigated by analyzing the efficacy of various ('14)C-labeled porphyrins. A ration was taken of the relative incorporation of label from a putative precursor into Pchlide ester as compared with incorporationg into Pchlide from that precursor. This ratio was called the R value. The R values from Proto, MPE, and Pchlide were determined and compared with the reference vaue from ALA (R(,ALA)). If Pchlide ester was derived solely from the esterification of Pchlide the R(,X) value from any intermediate "X" should equal the R(,ALA) value. It was determined that R(,Proto) was slightly higher than R(,ALA), while R(,MPE) and R(,Pclide) were significantly lower than R(,ALA). Thus, Pchlide ester is apparently synthesized from precursors other than Pchlide. Several models suggesting routes for the biosynthesis of Pchlide ester were advanced.;An aqueous extract of cucumber cotyledons was found to inhibit the biosynthesis of Pchl in cucumber. The inhibition was observed both in vivo and in vitro. The extract did not cause any significant oxidation of C-4 or C-5 labelled ('14)C-ALA to CO(,2). (NH(,4))(,2)SO(,4) precipitated most of the activity in the 30 to 60% fraction, the inhibitory activity migrated into a sucrose graduent, and the activity was heat labile. These results indicate that the active component may be a macromolecule, possibly a protein. It is conjectured that this protein is an enzyme whose product actively inhibits the synthesis of Pchl.;The assay developed to analyze the biosynthesis of Pchlide ester was modified to localize the site of inhibitory activity. The inhibitor did not significantly affect the esterification of ('14)C-Pchlide to ('14)C-Pchlide ester. When etiochloroplasts were incubated with the inhibitor in th presence of ('14)C-Proto or ('14)C-MPE, the conversion of these substrates to Pchlide was significantly blocked. The relative proportions of monovinly to divinyl MPE, Pchlide and Pchlide ester, as monitored by spectrofluorimetry at 77(DEGREES)K, were not affected by incubation with the inhibitor. The inhibitor did not seem to affect the reduction of vinyl groups on the macrocycle nor the esterificatin of the propionic acid groups at positions 6 and 7. It, therefore, appears that the inhibitor is blocking reaction(s) resulting in the closure of ring V of the macrocycle.;Contamination of chlorophyll (Chl) preparations by Pchlide ester may complicate the analysis of minor Chl components, therefore a new chromatographic system was developed which allows the partial separation of Pchlide ester from Chl. Using this system, it became possible to examine mature photosynthetic tissues for thepresence of Pchlide ester. A number of diverse plant species were examined and found to contain traces of Pchlide ester. Such a universal occurrence of this pigment in mature tissues suggests that it may play a fundamental role inthylakoid repair.