Stylar Matrix Research Articles

Exogenous free ubiquitin enhances lily pollen tube adhesion to an in vitro stylar matrix and may facilitate endocytosis of SCA.

Pollen tube adhesion and guidance on extracellular matrices within the pistil are essential processes that convey the pollen tube cell and the sperm cells to the ovule. In this study, we purified an additional molecule from the pistil that enhances pollen tube adhesion when combined with the SCA (stigma/stylar cysteine-rich adhesin)/pectin matrix in our in vitro assay. The enhancer of adhesion was identified as free ubiquitin (Ub). This was confirmed by use of bovine Ub as a substitute for lily (Lilium longiflorum Thunb.) stigma Ub. To study the interaction of SCA and Ub with the lily pollen tube, we labeled both proteins with biotin. We observed uptake of biotin-labeled SCA and Ub into the pollen tube cells in vitro using confocal microscopy. For SCA, a strong signal occurred first at the tip of the pollen tube, suggestive of an endocytosis event, and then progressively throughout the tube cytoplasm. SCA was also localized inside the in vivo pollen tube using immunogold electron microscopy and found to be present in endosomes, multivesicular bodies, and vacuoles, all known to be endocytic compartments. It was also confirmed that SCA is endocytosed in the in vitro adhesion assay. Internalization of SCA was increased in pollen tubes treated with exogenous Ub compared to those without Ub, suggesting that Ub may facilitate SCA endocytosis. These results show that Ub can act as an enhancer of pollen tube adhesion in vitro and that it is taken up into the pollen tube as is SCA. The Ub machinery may play a role in pollen tube adhesion and guidance in lily.

Lily ( Lilium longiflorum L.) pollen protoplast adhesion is increased in the presence of the peptide SCA

The style of lily produces a specialized extracellular matrix (ECM) in the transmitting tract epidermis that functions to guide pollen tubes to the ovary. This adhesive ECM contains low esterified pectins and a peptide, SCA (stigma/stylar cysteine-rich adhesin). Together they form a matrix to which pollen tubes adhere as they grow through the style. Pollen tubes also adhere to each other but only when grown in vivo, not in vitro. Pollen does not produce detectable SCA, but when SCA is added to an in vitro growth medium, it binds to pollen tubes that have esterified and low-esterified pectins in their walls. Since adhesion of the pollen tube to the stylar matrix requires tip growth, we hypothesized that the pectin wall at the pollen tube tip interacted with the SCA protein to initiate adhesion with the stylar pectin [Lord (2000) Trends Plant Sci 5:368–373]. Here, we use a pollen protoplast system to examine the effect of SCA on protoplast adhesion when it is added to the growth medium in the absence of the stylar pectin. We found that SCA induces a 2-fold increase in protoplast adhesion when it is added at the start of protoplast culture. This effect is less when SCA is added to the medium after the cell wall on the protoplast has begun to regenerate. We show that among the first components deposited in the new wall are arabinogalactan proteins (AGPs) and highly esterified pectins. We see no labeling for low esterified pectins even after 3 days of culture. In the pollen protoplast culture, adhesion occurs in the absence of the low esterified pectin. The newly formed wall on the protoplast mirrors that of the pollen tube tip in lily, which is rich in AGPs and highly esterified pectins. Thus, the protoplast system may be useful for isolating the pollen partner for SCA in this adhesion event.

The identification of a pollen-specific LEA-like protein inLilium longiflorum

A novel pollen-specific LEA-like protein, LP28, was detected in Lilium longiflorum using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Immunoblot analysis using antiserum raised against LP28 revealed that the protein was not found in somatic tissues or uninucleate microspores, but accumulated gradually in developing pollen following microspore mitosis. Furthermore, LP28 was abundant in germinated pollen after hydration. The cDNA clone corresponding to LP28 encoded a putative protein of 238 amino acids with a calculated molecular mass of 24·2 kDa and a pI of 4·7. The amino acid sequence is highly hydrophilic except for the N-terminal hydrophobic signal peptide. The sequence has similarities with group 3 LEA (late embryogenesis abundant) proteins. Immunocytochemical analyses demonstrated that LP28 was mainly found in cytoplasmic granules of the vegetative cell until pollen maturation, but after hydration it appeared in the elongating pollen tube wall. LP28 might be a unique pollen-specific protein that is transported to the pollen tube wall after germination. Therefore, it is assumed that LP28 plays a role not only in pollen maturation, but also in the growth of the pollen tube, which penetrates the stylar matrix.

Adhesion molecules in lily pollination

Adhesion occurs both between pollen tubes and between the pollen tube and transmitting tract epidermis (TTE) in lily. The stylar matrix secreted by the TTE can be isolated and used in an in vitro adhesion assay for pollen tubes. This bioassay was used to isolate two stigma/stylar adhesion molecules in lily: a pectic polysaccharide and a small cysteine-rich, basic protein we named SCA (stigma/stylar cysteine-rich adhesin). Both molecules were purified and used in an adhesion assay. Adhesion in the assay can be disrupted by treatment of the pectin with polygalacturonase and of SCA with proteinase K. The two molecules bind to each other in a pH-dependent fashion, and this binding is necessary for the adhesion assay to work. Antibodies to each of the molecules show their localization at the sites of pollen tube adhesion in the style. Pollen does not produce SCA but does bind this protein in vivo and in vitro. In vivo functional analyses are necessary to establish the roles of these molecules in lily pollination.

A lipid transfer-like protein is necessary for lily pollen tube adhesion to an in vitro stylar matrix.

Flowering plants possess specialized extracellular matrices in the female organs of the flower that support pollen tube growth and sperm cell transfer along the transmitting tract of the gynoecium. Transport of the pollen tube cell and the sperm cells involves a cell adhesion and migration event in species such as lily that possess a transmitting tract epidermis in the stigma, style, and ovary. A bioassay for adhesion was used to isolate from the lily stigma/stylar exudate the components that are responsible for in vivo pollen tube adhesion. At least two stylar components are necessary for adhesion: a large molecule and a small (9 kD) protein. In combination, the two molecules induced adhesion of pollen tubes to an artificial stylar matrix in vitro. The 9-kD protein was purified, and its corresponding cDNA was cloned. This molecule shares some similarity with plant lipid transfer proteins. Immunolocalization data support its role in facilitating adhesion of pollen tubes to the stylar transmitting tract epidermis.

A lipid transfer-like protein is necessary for Lily pollen tube adhesion to an in vitro stylar matrix: Park S, Jauh G, Mollet J, Eckard KJ, Nothnagel AN, Walling LL, Lord EM: Plant Cell 2000, 12:151–164

A lipid transfer-like protein is necessary for Lily pollen tube adhesion to an in vitro stylar matrix: Park S, Jauh G, Mollet J, Eckard KJ, Nothnagel AN, Walling LL, Lord EM: Plant Cell 2000, 12:151–164

High-pressure freezing improves the ultrastructural preservation of in vivo grown lily pollen tubes

We have used high-pressure freezing followed by freeze substitution (HPF/FS) to preserve in vivo grown lily pollen tubes isolated from the style. The results indicated that HPF/FS (i) allows excellent preservation of the pollen tubes, (ii) maintains in situ the stylar matrix secreted by the transmitting tract cells, and (iii) preserves the interactions that exist between pollen tubes. Particular attention has been given to the structure of the pollen tube cell wall and the zone of adhesion. The cell wall is composed of an outer fibrillar layer and an inner layer of material similar in texture and nature to the stylar matrix and that is not callose. The stylar matrix labels strongly for arabinogalactan proteins (AGPs) recognized by monoclonal antibody JIM13. The zone of adhesion between pollen tubes contains distinct matrix components that are not recognized by JIM13, and apparent cross-links between the two cell walls. This study indicates that HPF/FS can be used successfully to preserve in vivo grown pollen tubes for ultrastructural investigations as well as characterization of the interactions between pollen tubes and the stylar matrix.

A major stylar matrix polypeptide (sp41) is a member of the pathogenesis-related proteins superclass.

A novel stylar-specific glycosylated protein, sp41, was characterized. Sp41 constitutes greater than 12% of the transmitting tract tissue soluble proteins and is mainly localized in the extracellular matrix. Two cDNA clones corresponding to sp41 mRNA were isolated and sequenced. The decoded sequences are, respectively, 80% and 49% homologous to acidic and basic pathogen-induced (1-3)-beta-glucanases of the leaf. Thus a subfamily of (1-3)-beta-glucanase pathogenesis-related (PR) proteins constitutes one of the major stylar matrix proteins. The accumulation of sp41 transcripts in normally developing and elicitor-treated styles and leaves was followed using an RNase protection assay. During development sp41 transcript accumulation starts well after carpel differentiation. It is first detected in styles at 8 days before anthesis. The maximal level of accumulation is reached during anthesis. Elicitor-treated styles do not accumulate the leaf-type (1-3)-beta-glucanase transcript, although they retain the capacity to synthesize leaf-type pathogenesis-related proteins such as the pathogen-induced acidic chitinase. The developmental regulation of sp41 expression points to a role for them in the normal processes of flowering and reproductive physiology.