Throughout this paper, protochlorophyll (Pchl) will be used to designate the epiphasic pigment on petroleum ether-alkaline acetone partition, assumed to be an esterified form of Mg vinylpheoporphyrin a5 which moves on chromatography in a similar manner to chlorophyll a (Chl). Protochlorophyllide (Pchlide) designates the hypophasic pigment assumed to be unesterified at the propionic acid residue of ring IV, which moves in a similar manner to Pchlide extracted from etiolated barley leaves.Pchl(ide) will be used when the exact nature of the protopigment has not been determined or when a mixture of the two species is present. Chlide designates chlorophyllide a.It has recently been demonstrated that both Pchl and Pchlide are present in dark-grown cells of Euglena and that both protopigments are transformed to Chl-like pigments upon illumination (9, 10). Klein and Schiff (19) investigated proplastid development in 2-to 9-day-old etiolated bean leaves and observed that (a) the 3-day-old bean proplastids are about the same size as 'This research was supported by Grant GM14595 from the National Institutes of Health. Because of the marked similarities between plastid precursors in young dark-grown bean leaves and those of Euglena, we undertook a study of the forms of protopigments present and their phototransformation at various stages in the devef7opment of etiolated bean seedlings, particularly at times preceeding 7 to 9 days, the material commonly employed in such studies. In the usual material, Pchlide is the predominant protopigment which is phototransformed to Chlide a and then finally esterified to yield Chl a. Pchl photoconversion is extremely low and is not regarded as a significant route of Chl formation (8,12,16,31,32,35,37,39). In this work we show that Pchl conversion in young etiolated bean seedlings represents a highly significant fraction of the convertible protopigments, and results in the direct formation of Chl. A brief abstract of this work has appeared (22).
MATERIALS AND METHODSSeeds of Phaseolus vulgaris L. var. Red Kidney were germinated in wet vermiculite at 26 C in total darkness. The vermiculite was kept wet at all times by uniformly watering the trays, each containing 200 beans, with 1 liter of water each day. After time periods ranging from 2 to 17 days, a specified number of seeds, depending on the age, were removed, washed with water, and the cotyledons separated to expose the primary leaf pair. All manipulations were performed under dim green safelights (27) or in complete darkness. Roughly 1000 leaf pairs were required for each analysis of 3-day-old material. The number of leaf pairs required could be reduced to 35 at 5 days, 20 at 9 days, 15 at 13 days, and 10 by the 17th day.The methods for pigment extraction were modified from Shlyk (31). Before pigment extraction, the leaves were either (a) frozen in approximately 25 ml of liquid N2, (b) steamed for 10 min, or (c) extracted directly. All solvents used were reagent grade (Fisher).For experiments in which the leaves were heated, the spec...
