Our studlies wvith Euglena (2, 6, 14, 19, 20) have suggestedl that dark-grown cells containi approximately 30 proplastids whiclh develop into about 10 chloroplasts xvhen the cells are exposed to light. The existence of these proplastids, originally inferred from studlies on ultraviolet inactivation ain(l photoreactivation of chloroplast formiiation (14, 19). was confirimie(I by fluorescence ani(I electron miiicroscopy (2, 6). Other workers have stu(lie(l some of the initial steps invTolved wvhen dlark-groxvn cells are exposed to light, suclh as the conversion of protochlorophyll to chlorophyll (17). Other studies also exist of the related problemii of the physiology of chloroplast development in highler plants (3, 7, 23, 27). Our earlier vork (20) also showed that chloroplast development in Euglena could be separated experimentally into 2 phases: replication of the system which nmanufactures the chloroplast ancI development of the proplastid into the mature chloroplast. In this study x-e correlate the onset and kinetics of pigment formlation., O. evolution and COi. fixation with the developmilenit of the proplastid inito the mature chloroplast. Materials and Methods GroTl/t of Euglenia. Euigleina gracilis var. bacillaris Prinlgslheim (14) xv-as maintained aseptically in the (lark at 250, for 1 year in 250-ml Erlenmeyer flasks containiing 100 ml of Hutner's meclium pH 3.5 (10) xvith 5-ml transfers every 3 days. The starting culture wN-as a dark-grown stock maintainedl in our laboratory for over 2 years (14). The imiiportance of prolonged dark groxvtlh to entirely deplete chlorophyll and chloroplast structures has been repeatedly emlphasized (14, 17); our dark-grown cells contain protochlorophyll ancI proplastids and lack chloroplasts and chlorophyll completely. All manipulations xvere carried out under a green safelight in a (larkroolmi as described I)reviously (14).

The structure and physiology of the etioplast was investigated in developing primary leaves of 3-to 9-day-old dark-grown bean (Phaseolus rulgaris L. var. Red Kidney) seedlings. Increase in total protochlorophyll(ide) content followed that of leaf fresh weight. In 3-to 4-day-old bean leaves more than 50% of the protochlorophyll(ide) is in the form of protochlorophyll(ide) 628, which is nontransformable by light. Most of the transformable pigment is protochlorophyll(ide) 635, with smaller amounts of protochlorophyll (ide) 650. During leaf development from the 3rd to the 7th day phototransformable protochlorophyll(ide) with an absorbance maximum at 650 nm accumulates faster than nontransformable protochlorophyll(ide) or protochlorophvll(ide) 635. This increase in protochlorophyll(ide) 650 is correlated with the formation and enlargement of prolamellar bodies.The transformable protochlorophyll(ide) is converted by light directly to chlorophyll(ide) 672 in young leaves which do not yet have prolamellar bodies, and chlorophyll(ide) 672 may arise largely from the protochlorophyll(ide) 635. In older leaves the protochlorophyll(ide), largely protochlorophvll(ide) 650, is converted to chlorophyll(ide) 683, and a Shibata shift results in a change in the wavelength of absorption to 672 nm. The increase in protochlorophyll(ide) 650, the formation of prolamellar bodies, and the presence of the Shibata shift appear to be closely correlated. A model is briefly presented to provide a unified interpretation of these findings, including certain similarities between dark-grown Euglena cells and 3-to 4-day-old etiolated bean leaves.Etioplasts in the leaves of higher plants contain an elaborate internal membrane system composed of the prolamellar body and, attached to it, a number of perforated double membrane sheets (14, for references). The P'l in the etioplasts exists in at least three different forms, which can be characterized by their absorption maxima in the red region. Two of these forms, absorbing at 650 nm (Pa0) and 635 nm (P.), are con- The structural development of the etioplast in the leaves of the growing bean seedling has been studied repeatedly, most recently by Weier and Brown (24). The time course of protochlorophyll(ide) accumulation and regeneration in etiolated bean leaves has been studied by Akoyunoglou and Siegelman (1) as a function of seedling age. We shall report here details of etioplast development in 3-to 9-day-old bean seedlings; particularly the accumulation of convertible and nonconvertible protochlorophyll(ide), changes in the absorption spectra of the developing leaves in vivo, and structural changes in the etioplasts of the dark-grown leaves.MATERIALS AND METHODS Phaseolus vulgaris L. var. Red Kidney seeds were germinated in wet vermiculite in a dark chamber at 26 C for 2 to 9 days. One-gram samples of the primary leaves removed from the embryos or the seedlings after various times were then extracted with 80% (v/v) acetone in the presence of a small amount of MgCO3, and the total protochlor...

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