EVENTS SURROUNDING THE EARLY DEVELOPMENT OF EUGLENA CHLOROPLASTS—XI PROTOCHLOROPHYLL(IDE) AND ITS PHOTOCONVERSION*

Abstract

Abstract— The small amounts of chlorophyll precursors in dark‐grown cells of Euglena gracilis Klebs var. bacillaris Pringsheim and the presence of contaminants which interfere with their purification have made their isolation and characterization difficult. We now extract cells with acetone: 0.1 M NH4OH (9:1 v/v). Protochlorophyll is obtained by extracting this solution with petroleum ether (30–50° b.p.) and extracting this petroleum ether fraction with 80% acetone to remove substances which interfere with subsequent chromatography. Protochlorophyllide remaining in the original acetone: NH4OH fraction is extractable into diethyl ether after adjusting the pH to approximately 5.5. Both pigments are verified and further purified by chromatography on Whatmann 3 MM paper using benzene:petroleum ether:acetone (30:10:1) or cellulose MN 300 thin layers with methanol:methylene chloride:water (100:18:20). These pigments resemble their well‐described barley counterparts in solubility properties, spectral absorption maxima in ether (432, 530, 571 and 623 nm) and chromatographic behavior. Substantial amounts of protopheophytin and protopheophoribide are also found along with the Mg2+— containing pigments in cell extracts even when precautions are taken to avoid loss of Mg2+ during extractions and purification.Using heated cells before and chilled solvents after illumination to preclude enzymatic modification of pigments, chlorophyll and very small amounts of chlorophyllide are found as products of the photo‐conversion of the protopigments suggesting that both protochlorophyll and, to a much smaller extent, protochlorophyllide are photoconvertible in these organisms. These properties join several others which suggest that the Euglena chorophyll‐forming system more closely resembles that found in young bean leaves rather than the chlorophyll‐forming system of the older leaf material usually studied.

Occurrence of protochlorophyll and its phototransformation to chlorophyll in mutant C‐2A’ of Scenedesmus obliquus

“…The finding that PChl can be photoconverted to Chl in Scenedesmus is in accordance with the reports about Euglena gracilis (Cohen and Schiff 1976) and the Chlorella mutant YG-1 (Sasa and Sugahara 1976). The different photoconvertabilities of PChl in algae and higher plants and the physiological meaning are not yet understood and will be a topic of future research.…”

“…Besides the generally occurrring PChlide, also the occurrence of protochlorophyll (PChl), i.e. the already esterified PChlide, has been quite frequently reported (Singh 1953, Jones 1966, Cohen and Schiff 1976, Sasa and Sugahara 1976, Lancer et al 1976, Sundqvist et al 1980, Sundqvist and Ryberg 1983, Virgin 1984. However, its role in Chl biosynthesis and the possibility of its photoreduction to Chl are still not clear.…”

“…However, its role in Chl biosynthesis and the possibility of its photoreduction to Chl are still not clear. PChl, which was found in angiosperms, could not be photoreduced to Chl (Griffiths 1980, Shioi andSasa 1983), whereas in Euglena gracilis (Cohen and Schiff 1976) and in a mutant strain of Chlorella regularis (Sasa and Sugahara 1976) PChl could be photoreduced to Chl.…”

REGULATION OF PROTOCHLOROPHYLL(IDE) LEVELS IN DARK‐GROWN NON‐DIVIDING Euglena—II. INFLUENCE OF UTILIZABLE SUBSTRATES AND INHIBITORS OF PROTEIN SYNTHESIS*

“…the extraction, purification. and concentration of Pchl(ide) is a lengthy and delicate process (Cohen and Schiff. 1976).…”

“…this extra phototransformable pigment m u a t he formed from the conversion of non-PT-Pchl(ide) to PT-Pchl(ide). (In growingcells, only about 15-2O"i of the total Pchl(ide) is phototransformable) (Cohen and Schiff, 1976). There is a precedent for this conversion in earlier work using etiolated leaves of higher plants.…”