Shaio-Lim Mau

Arabinogalactan Proteins Are Required for Apical Cell Extension in the Moss Physcomitrella patens

Cell biological, structural, and genetic approaches have demonstrated the presence of arabinogalactan proteins (AGPs) in the moss Physcomitrella patens and provided evidence for their function in cell expansion and specifically in the extension of apical tip-growing cells. Inhibitor studies indicated that apical cell expansion in P. patens is blocked by synthetic AGP binding β-glucosyl Yariv reagent (βGlcYR). The anti-(1→5)-α-l-arabinan monoclonal antibody LM6 binds to some AGPs in P. patens, to all plasma membranes, and to the cell wall surface at the most apical region of growing protonemal filaments. Moreover, LM6 labeling of cell walls at the tips of apical cells of P. patens was abolished in the presence of βGlcYR, suggesting that the localized movement of AGPs from the plasma membrane to the cell wall is a component of the mechanism of tip growth. Biochemical and bioinformatic analyses were used to identify seven P. patens ESTs encoding putative AGP core proteins from homology with Arabidopsis thaliana, Brassica napus, and Oryza sativa sequences and from peptide fragments isolated from βGlcYR-precipitated AGPs. Gene knockout by homologous recombination of one of these genes, P. patens AGP1, encoding a classical AGP core protein, resulted in reduced cell lengths in protonemal filaments, indicating a role for AGP1 in apical cell expansion in P. patens.

Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility

The identification, isolation and aminoterminal sequencing of two S-genotype-associated proteins from style extracts of Lycopersicon peruvianum Mill. is reported. There is a high level of homology between these two sequences and with the amino-terminal sequences of other S-allele-associated glycoproteins isolated from Nicotiana alata Link et Otto. These sequences were obtained by a new high-sensitivity method of selected twodimensional gel analysis followed by electroelution and purification of proteins by inverse-gradient high-performance liquid chromatography before sequencing.

Isolation and partial characterization of components of Prunus avium L. styles, including an antigenic glycoprotein associated with a self-incompatibility genotype.

Several components of buffer extracts of Prunus avium L. styles (cv. Lambert, S3S4) have been isolated and partially characterized: the major component is a glycoprotein (molecular weight approx. 90,000; 95% protein, 5.4% carbohydrate). A “sticky” uronic-acid-containing component and an arabinogalactan are also present. Two minor components are an antigenic glycoprotein associated with the self-incompatibility genotype (Antigen S) and a component found in styles of all Prunus species (Antigen P). The isolated glycoproteins have a substantial carbohydrate content (Antigen P 17.2%; Antigen S 16.3%), and have apparent molecular weights of 32,000 (Antigen P) and 37,000–39,000 (Antigen S). They are antigenically quite distinct. Material corresponding to Antigen S is secreted into the medium of suspension-cultured callus cells raised from both leaf and stem of P. avium.

Nucleotide sequence and style-specific expression of a novel proline-rich protein gene from Nicotiana alata

AbstractcDNA clones encoding a novel proline-rich protein (NaPRP4) have been isolated from a Nicotiana alata stylar cDNA library. The N-terminal part of the derived protein is highly rich in proline (32.2%) and contains several repeats such as Lys-Pro-Pro (7 times) and Pro-Thr-Lys-Pro-Pro-Thr-Tyr-Ser-Pro-Ser-Lys-Pro-Pro (twice); the C-terminal part, on the other hand, has a lower proline content (9.9%) and contains two potential N-glycosylation sites and all the six cysteine residues. Northern blot and in situ hybridisation analyses indicate that expression of the NaPRP4 gene is restricted to cells of the transmitting tract of the style.

The effect of isolated components of Prunus avium L. styles on in vitro growth of pollen tubes

A number of components isolated from styles of P. avium cv. Napoleon (S3S4) have been tested for their capacity to influence in vitro growth of pollen tubes from fresh and stored pollen (cv. Napoleon (S3S4)). An antigenic glycoprotein (Antigen S) is a potent inhibitor of in-vitro pollen tube growth, causing a 65% reduction in tube length at a concentration of 20 μg/ml. None of the other style components were effective inhibitors of pollen tube growth; neither were proteins of animal origin such as histone, serum albumin, cytochrome C, and the glycoproteins ovalbumin and thyroglobulin, effective inhibitors.

Molecular Basis of Cell Recognition During Fertilization in Higher Plants

SUMMARY The molecular basis of recognition between plant cells is incompletely understood. Some principles established for recognition between animal cells may well apply to plant cell recognition, although, in contrast to animal cells, plant cells are encased by cell walls that play an active role in plant cell–cell recognition. The interaction that controls fertilization in flowering plants involves recognition between pollen or pollen tubes and the female sexual tissues. In many flowering plant families, self-incompatibility (S) genes operate to prevent inbreeding. In plants that have gametophytically controlled self-incompatibility, recognition of common S alleles in pollen tube and style results in arrest of pollen tube growth within the style. Self-incompatibility therefore provides a model cell–cell recognition system that is genetically defined. We have taken two approaches to defining cell recognition involved in gametophytic self-incompatibility in Nicotiana alata. Firstly, we have established the major features of the pollen tube wall and the matrix of the style transmitting tissue that are in contact with the growing pollen tube. Secondly, we have established the nature of style glycoproteins that are associated with the S genotype and have initiated a program to clone the genes coding for the protein component of these glycoproteins. Analyses of the pollen tube are consistent with the major polymers being a (1→3)-β-d-glucan (callose) and a (1→5)-α-l-arabinan. The pollen tube has two distinct layers: gold immunocytochemistry using a monoclonal antibody directed to terminal α-l-arabinosyl residues shows the binding is confined to the outer layers. The major component of the extracellular matrix of the style transmitting tissue is a family of proteoglycans, the arabinogalactan-proteins. A major glycoprotein that segregates with the S2 allele is present in extracts of mature styles. This component has a high pI (>9.5) and an apparent molecular weight of 32 × 103. It is not present in extracts of immature styles of N. alata genotypes bearing the S2 allele, or in extracts from other organs of N. alata or styles of other members of the Solanaceae. The isolated glycoprotein is an effective inhibitor of in vitro pollen tube growth. This evidence suggests that the S2-associated glycoprotein is either the product of the S2 allele, or a gene closely associated with the S gene. We have prepared a cDNA library from styles of one genotype and are screening this library with mRNA from mature and immature styles. We have also prepared synthetic oligonucleotide probes to N-terminal sequences obtained from the isolated S2-associated glycoprotein for use in screening the library. This dual approach for establishing the detailed structures of the interacting components and the genetic basis of the interaction will give us a better understanding of the recognition events involved in self-incompatibility.

Structural Classes of Arabinogalactan-Proteins

Any consideration of the structural classes of arabinogalactan-proteins (AGPs) also raises the question, “What is an AGP?” The AGPs belong to the Hyp-rich glycoprotein (HRGP) family of molecules that also includes the extensins, Pro/Hyp-rich glycoproteins (P/HRGPs) and the solanaceous lectins (Showalter 1993, Kieliszewski and Lamport 1994, Du et al 1996a, Sommer-Knudsen et al 1998). In general, three criteria have defined AGPs: the presence of arabinogalactan chains, a Hyp-rich protein backbone and the ability to bind to a class of synthetic phenylazo dyes, the β-glycosyl Yariv et al 1962, Clarke et al 1979, Fincher et al 1983). It may now be necessary to reconsider our definitions. Arabinogalactan chains are found on proteins that do not bind the Yariv reagent [e.g., AG-peptide from wheat (Fincher et al 1974) and two glycoproteins from styles (Lind et al 1994, Sommer-Knudsen et al 1996)]. Some AGPs are Hyp-deficient, and others have short oligoarabinosides previously thought to be characteristic of the extensins and solanaceous lectins (Qi et al 1991, Baldwin et al 1993). This degree of variability is similar to that observed in glycosylation and protein backbones of the animal extracellular matrix (ECM) proteoglycans (Hardingham and Fosang 1992, Verma and Davidson 1994). These general criteria easily distinguish AGPs from the extensins and solanaceous lectins, but the boundaries between AGPs and P/HRGPs are less clearly defined. This variability raises several issues: (1) Are the criteria outlined above sufficient? (2) The AGPs are a family of molecules with different protein backbones, each existing as multiple glycoforms. Is there a distinct boundary between AGPs and P/HRGPs, or is the HRGP family a continuum of molecules? (3) Can the knowledge and experiences from the animal proteoglycan field provide additional criteria that would clarify our definitions?

Biotransformation of podophyllotoxin by Hordeum vulgare cell suspension cultures

Hordeum vulgare cell suspension cultures were used to modify podophyllotoxin (1) One major product (1a) and one minor product (1b) were detected in both the culture medium and cells. To optimize the yield of compound 1a, we showed that: (1) the optimal concentration of added podophyllotoxin (1) was 33 mg L−1; higher concentrations caused cell toxicity; (2) the stage of the cell cycle (lag/log/stationary) at which podophyllotoxin was added only marginally affected the yield of compound 1a; the optimal addition time was after lag phase, in which the yield of compound 1a reached ca. 76%, and (3) biotransformation of podophyllotoxin (1) was relatively slow; podophyllotoxin fed at 4 days after subculture resulted in yields of compound 1a of ca. 56, 64 and 76% after an additional 3, 6 and 10 days of incubation, respectively. Product 1a was purified and identified as isopicropodophyllone (1a) based on MS and NMR data.

Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B

Regioselective acylation of four polyhydroxylated natural compounds, deacetyl asperulosidic acid (1), asperulosidic acid (2), puerarin (3) and resveratrol (4) by Candida antarctica Lipase B in the presence of various acyl donors (vinyl acetate, vinyl decanoate or vinyl cinnamoate) was studied. Compounds 1, 2 and 4 were regioselectively acetylated with vinyl acetate to afford products, 3′-O-acetyl-10-O-deacetylasperulosidic acid (1a), 3′,6′-O-diacetyl-10-O-deacetylasperulosidic acid (1b), 3′-O-acetylasperulosidic acid (2a), 3′,6′-O-diacetylasperulosidic acid (2b), 4′-O-acetylresveratrol (4a), respectively, with yields of 22 to 50%, while reactions with vinyl decanoate and vinyl cinnamoate were slow with lower yields. Compound 3 was readily acylated with all three acyl donors and quantitatively converted to products 6″-O-acetylpuerarin (3a), 6″-O-decanoylpuerarin (3b), 6″-O-cinnamoylpuerarin (3c), respectively. The structures of these acylated products were determined by spectroscopic methods (MS and NMR).

Regioselective acylation of ginsenosides by Novozyme 435

Ginsenosides from Panax species were acylated regioselectively by Novozyme 435 with vinyl acetate as the acetyl donor in organic solvents to afford mono-acyl ginsenosides.