8-vinyl reduction and chlorophyll a biosynthesis in higher plants

A technique involving solid-phase extractions and polyethylene h.p.l.c. suitable for the routine compositional analysis of the total protochlorophyllide pool of plants is described. The resynthesis kinetics of the individual components of the pool have been studied in briefly illuminated etiolated tissue of wheat (Triticum aestivum) and cucumber (Cucumis sativus) during subsequent redarkening. The data are interpreted in terms of a precursor-product relationship between the di- and mono-vinyl analogues of protochlorophyllide during their reaccumulation in darkness. The interconversion is assumed to be catalysed by an 8-vinyl reductase, which shows greater activity in wheat than in cucumber. Analyses of the redox state of the nicotinamide nucleotide of the pool during the process are compatible with NADPH as the cofactor of the putative reductase.

The Esterification of Chlorophyllide a in Greening Bean Leaves

The accumulation of esterfied chlorophyll a (Chl a) in etiolated bean leaves was determined during a dark period of 60 minutes after 1 minute irradiation with white light. The separation of the pigments esterified with geranylgeraniol (GG), dihydrogeranylgeraniol (DHGG), tetrahydrogeranylgeraniol (THGG) and phytol (P) was performed by HPLC. The last step for chlorophyll a biosynthesis is confirmed to be an esterification of chlorophyllide a to ChlGG followed by a stepwise hydrogenation of the pigment-bounded alcohols to ChlDHGG and ChlTHGG and finally to Chl a.

Chlorophyll a biosynthesis

Protoporphyrin IX is believed to be an intermediate common to both haem and chlorophyll biosynthesis. The pathway specific to chlorophyll starts with magnesium protoporphyrin and its monomethyl ester. Two routes have been proposed for conversion of the latter compound to protochlorophyllide: A, formation of the isocyclic ring followed by reduction of the 4-vinyl group, or B, reduction of the 4-vinyl group followed by formation of the isocyclic ring. Membranes prepared from isolated barley etioplasts are found to convert magnesium 2,4-divinylphaeoporphyrin a 5 monomethyl ester to chlorophyllide a at a rate equal to that of chlorophyll synthesis in intact leaves: this result supports route A. NADPH is necessary to maintain the two successive reductive steps: reduction of the 4-vinyl group and then the photoreduction of ring IV to yield chlorophyllide. The prohaem content of etiolated leaves does not increase during the phase of active chlorophyll synthesis although evidence is presented that suggests that the ALA synthesis reaction that regulates chlorophyll synthesis is common to both pathways. This and other regulatory aspects are discussed.