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A pool of chlorophyllide b has been detected in greening cucumber cotyledons. This pool exhibited the same spectrofluorometric properties as that of Chl b in diethyl ether at 298 and 77 K (Rebeiz, C.A., Belanger, F.C., Freyssinet, G. and Saab, D.G. (1980) Biochim. Biophys. Acta 590, 234–247) but had the chromatographic mobility and solubility of a monocarboxylic phorbin. The presence of a free carboxylic group and of a formyl group in this putative chlorophyllide b pool was demonstrated by methylation with diazomethane and by its conversion into chlorophyllide b oxime upon treatment with hydroxylamine. The concentration of this chlorophyllide b pool in greening tissues was in the same range as those of the protochlorophyllide and chlorophyllide a pools. Less than 15% of the chlorophyllide b pool could have arisen from chlorophyllase activity in vitro, as confirmed by the extent of hydrolysis of 14 C-labeled Chl b added to the tissue just before extraction.

The conversion of divinyl protochlorophyllide a (DV Pchlide a) (E443 F625), where E and F refer to fluorescence excitation and emission maxima respectively in ether at 77 K, into chlorophyllide a (Chlide a) (E459 F675) is reported. This reaction was demonstrated by first inducing etiolated cucumber cotyledons to accumulate DV Pchlide a (E443 F625) devoid of monovinyl (MV) contamination by a series of light/dark treatments. The accumulated DV Pchlide a (E443 F625) was then converted into Chlide a (E459 F675) by a 2.5-ms light pulse (L). The Chlide a (E459 F675) thus generated exhibited spectrofluorometric properties which differed from those of MV Chlide a (E447 F674) at 77 K and at room temperature before and after demetallation. The techniques described in this work permit the preparation of Chlide a (E459 F675) devoid of MV Chlide a contamination, for further chemical structural identification of this novel chlorophyll (Chl) precursor.

By subjecting etiolated cucumber cotyledons to three successive light/ dark treatments, the cotyledonary tissue was induced to accumulate divinyl (DV) protochlorophyllide (Pchlide). The DV Pchlide-enriched etiochloroplasts were isolated and the DV Pchlide was converted into DV chlorophyllide a (Chlide a) by irradiating the plastids with a 2.5 ms pulse of actinic white light. After a few minutes of incubation in the dark, most of the newly formed DV Chlidea was converted into monovinyl (MV) Chlide a in vitro. When etiolated cucumber cotykedons were induced to accumulate [14C]DV Pchlide and the latter was photoconverted into [14C]DV Chlide a by a 2.5 ms actinic light treatment, the [14C]DV Chlide a was converted into [14C]MV Chlide a, during a post-illumination 5-min-dark incubation period.