D. Stegwee

Changing cell-wall compositions in hypocotyls of dark-grown bean seedlings.

Hypocotyls of dark-grown 6-day-old seedlings of Phaseolus vulgaris L. proved to be sufficiently homogeneous to permit studies relating the rate of cell elongation to the composition of the primary cell walls. Whereas the levels of cellulose and uronic acids remained practically constant during and after cell extension, all other components showed major or minor changes. Cell-wall protein, as such, decreased by more than 50%, but indications are that hydroxyproline-rich glycoprotein increased with a decreasing rate of cell elongation, concomitant with a rise in the degree of arabinosylation of wall-bound hydroxyproline. As cell elongation slowed down, non-cellulosic glucose accumulated, presumably in the form of a β-(1–4)glucan closely associated with cellulose. These findings confirm the notion that the primary cell wall is a highly dynamic structure.

Aminolaevulinate dehydratase in greening leaves of Phaseolus vulgaris L.

Summary In greening leaves of Phaseolus vulgaris cv. Prelude a 4-fold increase of the activity of aminolaevulinate dehydratase (ALAD, E.C. 4.2.1.24) was observed. Some properties of this enzyme, which is involved in the biosynthesis of chlorophyll and heme compounds, were examined. Studies with the specific inhibitors of protein synthesis, chloramphenicol and cycloheximide, showed that the increase of enzyme activity was due to de novo synthesis on cytoplasmic ribosomes. Experiments with chloroplasts isolated in a non-aqueous medium indicated that at least part of the enzyme is localized within these organelles. These results are discussed with reference to the known inhibition of chlorophyll synthesis by cycloheximide and to possible factors limiting this synthesis.

Localization of arabinogalactan proteins in the membrane system of etiolated hypocotyls of Phaseolus vulgaris L.

The subcellular distribution of arabinogalactan protein (AGP) in etiolated bean hypocotyls was studied by isopycnic density centrifugation on sucrose gradients at different Mg2+ concentrations. The distribution of hydroxyproline (a major amino acid in AGP) in the membrane-containing fractions indicated that hydroxyproline-containing proteins were associated with rough endoplasmic reticulum, possibly with the Golgi apparatus, and with the plasma membrane. Non-specific binding of hydroxyproline-containing molecules to membranes could be excluded. To detect AGPs, fractions obtained after isopycnic density centrifugation were isoelectrofocused on polyacrylamide gels, and the gels were stained with β-Gal-Yariv reagent. Bands appeared only at low pH values, where also most hydroxyproline was found. In the fractions at low densities (presumably membranefree), several bands were visible supporting the idea of the heterogeneous character of soluble AGP. The distribution of AGP in the membranous fractions strongly indicated that AGP was associated with the plasma membrane. Specific agglutination of protoplasts in the presence of β-Gal-Yariv reagent indicated that AGP was exposed at the outside of the cell membrane.

Gametogenesis in Liquid Cultures of Chlamydomonas eugametos

Summary: At the end of the exponential phase of vegetative growth in liquid cultures, cells of Chlamydomonas eugametos develop into gametes. Gametogenesis can occur without nitrogen deficiency. In continuous light, mating competence lasts only for a few hours, resulting in a low percentage of gametes. Nevertheless, mating competence is considered to be a general property of newly born cells in late exponential phase, since cultivation of both mating-types in mixed cultures normally leads to a high yield of paired cells. No evidence was found for an influence of the presence of partner gametes on gametogenesis. During gametogenesis of the mt - mating-type, extractable biologically active agglutination factor appears in the cell body. Subsequently, after a gametogenic division, mating competence and agglutinability are expressed, both fluctuating synchronously with the light/dark regime.

Aminolaevulinate dehydratase in greening cells of Euglena gracilis

Summary Aminolaevulinate dehydratase (ALAD, E. C. 4.2.1.24) is studied in greening cells of Euglena gracilis . Exposure of dark-grown non dividing cells to light conditions results in a rapid increase in ALAD, whereas chlorophyll synthesis exhibits a lag period. The activity of ALAD increases more than fivefold. The increase of ALAD activity in greening cells was inhibited by cycloheximide but was not affected by chloramphenicol, indicating that ALAD is formed on cytoplasmic ribosomes. The intracellular distribution pattern of ALAD was investigated. The yield of intact chloroplasts in a crude cell homogenate was improved by a protease treatment before rupturing the cells in the French pressure cell. The distribution pattern of ALAD followed that of ribulosediphosphate carboxylase and NADP-linked glyceraldehyde-3-phosphate dehydrogenase, typical chloroplast enzymes.

Cell-cell adhesion in conjugatingChlamydomonas gametes: a self-enhancing process

SummaryThe flagellar adhesiveness of gametes ofChlamydomonas eugametos increases during conjugation such that the cell-cell contacts are intensified. The rise in adhesiveness is due to an increase in agglutinin exposure which can be visualized by immunolabeling. The adhesiveness in the one cell is stimulated by the agglutinins of the adherent partner cell, and vice versa. Thus, sexual cell-cell adhesion is a self-enhancing process. In addition, it is shown that the gametes are able to activate potential partners at distance via agglutinin-rich vesicles which they shed into their environment.

The activity of Triton X-100 soluble chlorophyllase in liposomes.

Chlorophyllase (chlorophyll-chlorophyllidohydrolase, EC 3.1.1.14) was isolated and purified from Phaseolus vulgaris L. chloroplasts and etioplasts dissolved in 1% Triton X-100 and 10% glycerol. A 100 and 40-fold purification, respectively, was achieved. Enzyme preparations from both sources had similar affinities for chlorophyll a when assayed in a Triton X-100 medium. When electrophoresed in sodium dodecyl sulphate polyacrylamide gels the major band in both preparations migrated as a peptide of 30,000 daltons. Chlorophyll containing liposomes were also used as a substrate for chlorophyllase. The rate of hydrolysis did not follow Michaelis-Menten kinetics. When chlorophyllide a or methyl chlorophyllide a was incorporated in the liposomes, then in the presence of phytol dissolved in methanol, methylchlorophyllide a and chlorophyll a were shown to be synthesized. Apparently the purified enzyme in the presence of lipids, is endowed with both synthetic and hydrolytic activity.

Effect of chloramphenicol on the development of proplastids in Euglena gracilis: I. The synthesis of ribulosediphosphate carboxylase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase and aminolaevulinate dehydratase

Summary In a crude homogenate of dark-grown Euglena ribulose-diphosphate carboxylase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase and aminolaevulinate dehydratase activities are found. These enzymes are localized exclusively or (as for aminolaevulinate dehydratase) partly in the proplastids. If the cells are being cultivated for a number of generations in the dark in the presence of chloramphenicol, then the synthesis of ribulosediphosphate carboxylase and NADP-linked glyceraldehyde-3-phosphate dehydrogenase is inhibited, but that of aminolaevulinate dehydratase is not changed. The results signify that in the dark proteins are synthesized on the ribosomes of the proplastids. The synthesis of ribulosediphosphate carboxylase and NADP-linked gly-ceraldehyde-3-phosphate dehydrogenase is dependent on the protein synthesis in the proplastid. The aminolaevulinate dehydratase in the proplastid is probably synthesized in the cytoplasm and subsequently incorporated in the proplastid. The proplastid appears to be equivalent to the chloroplast in relation to the synthesis of these enzymes.

Turnover and transport of agglutinins in conjugatingChlamydomonas gametes

SummaryDuring gamete-gamete adhesion in the unicellular green algaChlamydomonas eugametos, the sexual adhesion molecules or agglutinins that are located on the flagella are subject to tip-oriented migration and rapid inactivation. It is demonstrated that sexual adhesiveness is maintained by incorporation of additional agglutinins, recruited from a cellular pool. The location of this reservoir is unknown but, as indicated by its insensitivity to the chaotropic agent guanidine thiocyanate, it appears to be distinct from the large amount of agglutinins on the plasma membrane of the cell body. By viewing flagella of conjugating gametes in a confocal scanning laser microscope after immuno-labelling of the agglutinins, evidence was obtained for a linear arrangement of the agglutinins in two rows on the flagellar surface. This suggests that after insertion at the base of the flagellum, the agglutinins follow linear tracks to the tip and that the transport system is confined to two longitudinal domains. It is estimated that the half-life of flagellar agglutinins drops from 1–2 h in nonconjugating gametes to 1 min during conjugation, which suggests that after incorporation at the flagellar base, the agglutinins migrate to the tip with a velocity of 100 nm/s. Presumably after arrival at the tip, the molecules are inactivated. It is postulated that rapid turnover and transport of agglutinins are required for optimal signalling between partner gametes.

Ferredoxin and ferredoxin-NADP-oxidoreductase in leaves ofPhaseolus vulgaris L.

During light-induced greening of 10-dayold etiolated bean seedlings a strong increase is observed of ferredoxin (Fd) and of ferredoxin-NADP-oxidoreductase (FNR; E.C. 1.6.99.4) activity, both known to reside in chloroplasts. The production of Fd starts immediately upon illumination and proceeds almost linearly for at least the next 72 h. It is inhibited by chloramphenicol (CAP) but not by cycloheximide (CHI), beit that in the presence of the latter Fd synthesis was impaired after 48 h of illumination. We conclude that for the elaboration of functional Fd in greening chloroplasts protein synthesis on chloroplast ribosomes is required. The increase of FNR activity shows a lag of about 24 h and is blocked by both CAP and CHI. When CAP is applied at 24 h after the illumination started it has no effect. We suggest that the synthesis of FNR on cytoplasmic ribosomes requires prior synthesis of protein(s) on chloroplast ribosomes.The nature of possible interactions between chloroplasts and cytoplasm is discussed.