Sabine Waffenschmidt

Algal Lipid Bodies: Stress Induction, Purification, and Biochemical Characterization in Wild-Type and Starchless Chlamydomonas reinhardtii

ABSTRACT When the unicellular green soil alga Chlamydomonas reinhardtii is deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in the sta6 mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid “popped-cell” microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are ∼50% saturated (C16 and C18) fatty acids and ∼50% unsaturated fatty acids, half of which are in the form of oleic acid (C18:1). The FFA are ∼50% C16 and ∼50% C18. The LB-derived TAG yield from a liter of sta6 cells at 107 cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production in C. reinhardtii has the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.

Tumour genes in plants: T-DNA encoded cytokinin biosynthesis.

Gene 4 from the T‐region of Ti plasmids is responsible for cytokinin effects in crown gall cells; we investigated whether it codes for an enzyme of hormone biosynthesis. In a first set of experiments, gene 4 from octopine plasmid pTiAch5 and nopaline plasmid pTiC58 was expressed in Escherichia coli, and the gene products were identified by reaction with antiserum raised against a decapeptide derived from the DNA sequence of the gene. Extracts from cells expressing the gene contained high isopentenyl‐transferase activity catalyzing the formation of N6‐(△2‐isopentenyl)adenosine from 5′‐AMP and △2‐isopentenylpyrophosphate. The cytokinin was identified by sequential h.p.l.c. chromatography and mass spectrometry. In a second set of experiments it was shown that crown gall cells contained isopentenyltransferase activity and a protein of mol. wt. 27 000 which was identified as the product of gene 4 by reaction with the antiserum. Isopentenyltransferase activity was specifically inhibited by the antiserum. No comparable enzyme activity or immunoreactive protein was detected in cytokinin‐autotrophic, T‐DNA free tobacco cells. The results establish that gene 4 from the T‐region of octopine and nopaline Ti plasmids codes for an enzyme of cytokinin biosynthesis.

Plus and Minus Sexual Agglutinins from Chlamydomonas reinhardtii

Gametes of the unicellular green alga Chlamydomonas reinhardtii undergo sexual adhesion via enormous chimeric Hyp-rich glycoproteins (HRGPs), the plus and minus sexual agglutinins, that are displayed on their flagellar membrane surfaces. We have previously purified the agglutinins and analyzed their structural organization using electron microscopy. We report here the cloning and sequencing of the Sag1 and Sad1 genes that encode the two agglutinins and relate their derived amino acid sequences and predicted secondary structure to the morphology of the purified proteins. Both agglutinin proteins are organized into three distinct domains: a head, a shaft in a polyproline II configuration, and an N-terminal domain. The plus and minus heads are related in overall organization but poorly conserved in sequence except for two regions of predicted hydrophobic α-helix. The shafts contain numerous repeats of the PPSPX motif previously identified in Gp1, a cell wall HRGP. We propose that the head domains engage in autolectin associations with the distal termini of their own shafts and suggest ways that adhesion may involve head–head interactions, exolectin interactions between the heads and shafts of opposite type, and antiparallel shaft–shaft interactions mediated by carbohydrates displayed in polyproline II configurations.

Identification of the Minus-Dominance Gene Ortholog in the Mating-Type Locus of Gonium pectorale

The evolution of anisogamy/oogamy in the colonial Volvocales might have occurred in an ancestral isogamous colonial organism like Gonium pectorale. The unicellular, close relative Chlamydomonas reinhardtii has a mating-type (MT) locus harboring several mating-type-specific genes, including one involved in mating-type determination and another involved in the function of the tubular mating structure in only one of the two isogametes. In this study, as the first step in identifying the G. pectorale MT locus, we isolated from G. pectorale the ortholog of the C. reinhardtii mating-type-determining minus-dominance (CrMID) gene, which is localized only in the MT− locus. 3′- and 5′-RACE RT–PCR using degenerate primers identified a CrMID-orthologous 164-amino-acid coding gene (GpMID) containing a leucine-zipper RWP-RK domain near the C-terminal, as is the case with CrMID. Genomic Southern blot analysis showed that GpMID was coded only in the minus strain of G. pectorale. RT–PCR revealed that GpMID expression increased during nitrogen starvation. Analysis of F1 progeny suggested that GpMID and isopropylmalate dehydratase LEU1S are tightly linked, suggesting that they are harbored in a chromosomal region under recombinational suppression that is comparable to the C. reinhardtii MT locus. However, two other genes present in the C. reinhardtii MT locus are not linked to the G. pectorale LEU1S/MID, suggesting that the gene content of the volvocalean MT loci is not static over time. Inheritance of chloroplast and mitochondria genomes in G. pectorale is uniparental from the plus and minus parents, respectively, as is also the case in C. reinhardtii.

Between-Species Analysis of Short-Repeat Modules in Cell Wall and Sex-Related Hydroxyproline-Rich Glycoproteins of Chlamydomonas

Protein diversification is commonly driven by single amino acid changes at random positions followed by selection, but, in some cases, the structure of the gene itself favors the occurrence of particular kinds of mutations. Genes encoding hydroxyproline-rich glycoproteins (HRGPs) in green organisms, key protein constituents of the cell wall, carry short-repeat modules that are posited to specify proline hydroxylation and/or glycosylation events. We show here, in a comparison of two closely related Chlamydomonas species—Chlamydomonas reinhardtii (CC-621) and Chlamydomonas incerta (CC-1870/3871)—that these modules are prone to misalignment and hence to both insertion/deletion and endoduplication events, and that the dynamics of the rearrangements are constrained by purifying selection on the repeat patterns themselves, considered either as helical or as longitudinal face modules. We suggest that such dynamics may contribute to evolutionary diversification in cell wall architecture and physiology. Two of the HRGP genes analyzed (SAG1 and SAD1) encode the mating-type plus and minus sexual agglutinins, displayed only by gametes, and we document that these have undergone far more extensive divergence than two HRGP genes (GP1 and VSP3) that encode cell wall components—an example of the rapid evolution that characterizes sex-related proteins in numerous lineages. Strikingly, the central regions of the agglutinins of both mating types have diverged completely, by selective endoduplication of repeated motifs, since the two species last shared a common ancestor, suggesting that these events may have participated in the speciation process.

A ZYGOTE-SPECIFIC PROTEIN WITH HYDROXYPROLINE-RICH GLYCOPROTEIN DOMAINS AND LECTIN-LIKE DOMAINS INVOLVED IN THE ASSEMBLY OF THE CELL WALL OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

The cell wall of Chlamydomonas reinhardtii zygotes, which forms rapidly after the fusion of wall‐free gametes, provides a tractable system for studying the properties and assembly of hydroxyproline‐rich glycoproteins, the major proteinaceous components of green algal and plant cell walls. We report the cloning of the zsp2 gene and the analysis of its ZSP‐2 product, a 58.9 kDa polypeptide that is synthesized exclusively by zygotes. The protein contains two (SP)x repeats, establishing it as a member of the cell wall hydroxyproline‐rich glycoproteins family. It also contains a 4‐fold iteration of an amino acid sequence centered around cysteine residues, a configuration found in both plant and animal lectins. Furthermore, we report four observations on pellicle composition and production. First, cell‐free preparations of the pellicle matrix are rich in hydroxyproline, arabinose, and galactose and contain bundles of very long fibrils. Second, glutathione blocks pellicle formation and results in the accumulation of long fibrils in the growth medium. Third, antibody to ZSP‐2 also blocks pellicle formation. Fourth, ZSP‐2 immunolocalizes to the boundary between the outer layers of the wall proper and the pellicle matrix. These observations are consistent with the possibility that the Cys‐rich (glutathione‐sensitive) lectin‐like domains of ZSP‐2 may bind to sugar residues on the long fibrils and anchor them to the cell wall, thereby initiating and maintaining pellicle formation.

Matrix‐lysis and release of daughter spheroids in volvox carteri ‐ a proteolytic process

In the genus Volvocales we find the first simple organisms in which stem and somatic cells are differentiated and which establish an organized so@1 hierarchy, Tlms, they are interesting objects to study differentiation and ageing processes. In these multicellular green algae a number of systematic differentiating cell divisions and subsequent re-organization (inversion) of the cellular aggregate [l ] produces: (i) A sheath (which in some cases is syncytiai) of numerous somatic c&Is which are fitted out with an eye-spot and two flagella for locomotion and protection. They excrete a matrix which consists of several outer layers of hydroxyproline-rich glycoprotein f2] and an inner gel, presumably a ~ly~ccba~de~ degradable by Iysozyme. Within this matrix are: (ii) Vegetative gonidia in a f&d and limited number or, after a phase shift caused by the sexuality inducing glycoprotein [3,4], eggs and spermbundles. At maturation the vegetative daughter spheroids are released at a defined term, whereas tie male spheroid disintegrates completely into sperm packets. For release each daughter spheroid dissolves the somatic sheath of the parental colony at a circumscribed area in their immediate nei~bourbood and penetrates into the medium, possibly extruded by the elastic pressure of the matrix and starts to rotate vividly. A lysis of cell walls is also described for zoospore liberation in Chlamydomonas [S]. In both cases an enzymatic reaction involving hydrolysis may be responsible. We were able to purify the enzyme from I&&ox carteri to almost homogeneity and to characterize its catalytic properties as a neutral serine (glyca)protease.

Oligosaccharide side chains of wall molecules are essential for cell-wall lysis in Chlamydomonas reinhardtii.

The glycoproteins of the cell walls of Chlamydomonas are lysed during the reproductive cycle by proteases (autolysins) which are specific for their substrates. The autolysin which digests the wall of sporangia to liberate the zoospore daughter cells in the vegetative life cycle is a collagenase-like enzyme which attacks only selected domains in its wall substrates containing (hydroxy)-proline clusters. Cell-wall fractions obtained by salt-extraction (NaClO4) and oxidizing agents (NaClO2) and the insoluble residue were tested as substrates. The most-crosslinked insoluble inner part of the wall is the best substrate for the sporangia autolysin. Oligosaccharides obtained from the insoluble cell-wall fraction of sporangia by hydrolysis with Ba(OH)2 inhibit autolysin action. We conclude that the oligosaccharide side chains of wall substrates are essential for forming the reactive enzyme-substrate complex.

Characterization of a sperm lysin of Volvox carteri

SummaryA cell-wall degrading enzyme has been isolated from mature sperm packets of the green flagellate Volvox carteri (Poona strain). This sperm lysin (S-lysin) is a Ca2+-dependent protease of 34 kDa with an essential serine group in its active centre. Neither SH group-blocking reagents nor transition metal chelators inhibit its action. S-lysin degrades the hydroxyproline-rich glycoprotein structures of the cell walls of sheath cells and gonidia (eggs) of vegetative and sexual spheroids in a characteristic manner. In asexual spheroids the somatic envelope is totally disintegrated, whereas in sexual spheroids pores are formed by local lysis at sites of adjacent eggs. Although S-lysin is very similar to the G-lysin of the closely related Chlamydomonads, it is species specific and does not attack the mother or daughter cell walls of Chlamydomonas reinhardtii. S-lysin resembles the aerosin of animal sperm cells in some aspects of its action.

Mass spectrometric analysis of hydroxyproline glycans

Mass spectrometric techniques are presented which allow one to analyze the sugar part bound to hydroxyproline in hydroxyproline-rich glycoproteins. The hydroxyproline (Hyp) glycans obtained by alkaline hydrolysis give abundant [M + Na](+) ions by electrospray ionization which after collision-induced dissociation (CID) yield inter alia [Hyp - H + Na](+). In mixtures a parent ion scan of this species will indicate the various molecular species which can then be analyzed by MS(n) after CID in an ion trap, where successive losses of the sugar units are observed. Methylation techniques allow one to distinguish between linear and branched isomeric structures.