Cellular Interactions during the Mating Process in Chlamydomonas eugametos.

A method to determine the mating competence of Chlamydomonas eugametos was developed. The contribution of each mating type in the pair formation was investigated using asymmetric gamete mixtures. It was established that pair formation is not mediated by a pheromonal attraction mechanism between partner gametes, but depends on collision chances. On the other hand, it was demonstrated that during transient contacts between partner gametes the flagellar agglutinability of both partners is stimulated, evidently to prepare a successful mating. The plus mating type was generally less agglutinable than the minus mating type and was a rate-limiting factor in the mating process.

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 Mg(2+) 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 ß-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 ß-Gal-Yariv reagent indicated that AGP was exposed at the outside of the cell membrane.

Arabinogalactan Protein from a Crude Cell Organelle Fraction of Phaseolus vulgaris L.

Sonication of a crude cell organelle fraction from hypocotyl tissue of dark-grown bean seedlings, and from suspension-cultured cells released a hydroxyproline-containing protein. The purification of this protein is described. It was found to be an arabinogalactan protein composed of 90% carbohydrate and 10% protein. The major sugars are galactose, arabinose, and uronic acids, and the major amino acids are hydroxyproline, serine, and alanine. Its molecular weight was estimated at 1.4 x 10(5) daltons and the isoelectric point at pH 2.3. The molecule is soluble in 5% trichloroacetic acid and can be precipitated with beta-galactosyl Yariv antigen. Pulse-chase experiments indicated that it was a secretory protein. The biosynthesis of arabinogalactan proteins is discussed.

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 ß-(1-4)glucan closely associated with cellulose. These findings confirm the notion that the primary cell wall is a highly dynamic structure.

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.

The coding site of chloroplast ferredoxin.

Ferredoxins were isolated and purified from leaves of different species of Nicotiana and Petunia and from spinach leaves. Their spectral properties, degree of homogeneity, and molecular weights were determined. The preparations were further analyzed by polyacrylamide gel electrophoresis of tryptic hydrolysates. This allowed us to distinguish between not only ferredoxins of Nicotiana, Petunia, and spinach, but even ferredoxins of various Nicotiana species. We used the differences in tryptic peptide compositions as phenotypic markers to study the mode of inheritance of chloroplast ferredoxin to see whether the coding site is in the chloroplast or in the nucleus. Analysis of the tryptic peptide composition of ferredoxin from different interspecific hybrids of Nicotiana showed that the characteristics of both parental ferredoxins were present. The results indicate that the primary structure of at least the male ferredoxin is coded for in the nucleus. In some of the hybrids the relative contribution of the male parent appeared to be low, suggesting that the female genome (presumably that part located in the plastome) exerted a dominating influence.

Qualitative and quantitative immunofluorescence studies of chloroplast ferredoxin : Application to investigations of ferredoxin inheritance in Nicotiana hybrids.

Antibodies were raised in rabbits against 2Fe-2S ferredoxin from N. tabacum L. The antibodies showed partial cross-reactivity in the double diffusion test with ferredoxins from Spinacia oleracea L., Petunia inflata Fries., P. axillaris Lam., Phaseolus vulgaris L., Chlamydomonas remhardii Dang. A complete cross-reaction was observed with ferredoxins from five other Nicotiana species, thus with this test it was impossible to discriminate between these ferredoxins. Therefore the following test was performed. Heterologous ferredoxin (i.e., ferredoxin other than from N. tabacum) was coupled covalently to Sepharose beads. Rabbit anti-N. tabacum-serum was then pre-incubated with this ferredoxin which resulted in complete abolition of cross-reactivity with free heterologous ferredoxin. However, the serum retained antibody activity against specific antigenic determinants of N. tabacum ferredoxin. When this serum was tested against ferredoxin purified from the hybrid: N. tabacum (♀)xN. glutinosa (♂) it gave a positive reaction. The relative content of maternal N. tabacum ferredoxin in the hybrid was estimated by using a fluorescent derivative of this specific antibody and estimating the cross-reactivity compared with that of artificial mixtures of pure N. tabacum and N. glutinosa ferredoxins. The hybrid contained 50% of maternal ferredoxin. This technique was also applied to ferredoxins of other species of Nicotiana and to the ferredoxin from the hybrid N. clevelandii (♀)xN. glutinosa (♂). We conclude that it provides a good test system for the study of the expression of chloroplast ferredoxin in Nicotiana hybrids in general.

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.