Two SCA (Stigma/Style Cysteine-rich Adhesin) Isoforms Show Structural Differences That Correlate with Their Levels of in Vitro Pollen Tube Adhesion Activity

Abstract

Lily pollen tubes grow adhering to an extracellular matrix produced by the transmitting tract epidermis in a hollow style. SCA, a small (ϳ9.4 kDa), basic protein plus low esterified pectin from this extracellular matrix are involved in the pollen tube adhesion event. The mode of action for this adhesion event is unknown. We partially separated three SCA isoforms from the lily stigma in serial size exclusion column fractions (SCA1, 9370 Da; SCA2, 9384 Da; SCA3, 9484 Da). Peptide sequencing analysis allowed us to determine two amino acid variations in SCA3, compared with SCA1. For SCA2, however, there are more sequence variations yet to be identified. Our structural homology and molecular dynamics modeling results show that SCA isoforms have the plant nonspecific lipid transfer protein-like structure: a globular shape of the orthogonal 4-helix bundle architecture, four disulfide bonds, an internal hydrophobic and solvent-inaccessible cavity, and a long C-terminal tail. The Ala 71 in SCA3, replacing the Gly 71 in SCA1, has no predictable effect on structure. The Arg 26 in SCA3, replacing the Gly 26 in SCA1, is predicted to cause structural changes that result in a significantly reduced volume for the internal hydrophobic cavity in SCA3. The volume of the internal cavity fluctuates slightly during the molecular dynamics simulation, but overall, SCA1 displays a larger cavity than SCA3. SCA1 displays higher activity than SCA3 in the in vitro pollen tube adhesion assay. No differences were found between the two SCAs in a binding assay with pectin. The larger size of the hydrophobic cavity in SCA1 correlates with its higher adhesion activity.During lily pollination, pollen tubes are guided to the ovary on an extracellular matrix (ECM) 2 produced by the specialized stylar transmitting tract epidermis (TTE). This guidance includes an adhesion event between the pollen tube and the TTE (1-4). For the purpose of identifying molecular factors in lily pollen tube adhesion, Jauh et al. (5) developed an in vitro bioassay using ECM molecules attached to a nitrocellulose membrane as an artificial style. Using this in vitro bioassay, two molecules from the style ECM, SCA (stigma/style cysteine-rich adhesin) and a low esterified pectic polysaccharide, were found to be responsible for pollen tube adhesion on the stylar matrix (6, 7). The level of SCA mRNA was high in stigma/style tissue, petal, and young leaf, but it was absent in pollen tubes (8). Localization of both SCA and the low esterified pectin on the surface of the stylar TTE supported their involvement in in vivo pollen tube adhesion. The mechanism of action for SCA and pectin in pollen tube adhesion is yet unknown. Neither of these molecules alone is active in the adhesion assay. Only when they were combined did SCA and pectin bind to each other in a pH-dependent manner and form the functional, adhesive matrix (6). The pH effect implies that a charge interaction occurs, rather than a tight cross-linking, between SCA and the low esterified pectin in lily pistils.SCA is a sm...

A Gain-of-Function Mutation ofArabidopsisLipid Transfer Protein 5 Disturbs Pollen Tube Tip Growth and Fertilization

“…SCA was also shown to be involved in pollen tube chemotropism by synergistically enhancing the activity of lily chemocyanin on the stigma (Kim et al, 2003). Further work revealed that lily SCA, a secreted pistil factor, is endocytosed through the pollen tube tip and may function in tip growth as a signal transducer (Kim et al, 2006;Chae et al, 2007). LTPs related to SCA in other plant species have a variety of proposed biological roles.…”

“…These features were also identified in SCA (Chae et al, 2007). Computational modeling indicates that SCA has an LTP-like structure with a globular shape of the orthogonal four-helix bundle architecture and a hydrophobic core (Chae et al, 2007). The hydrophobic cavity of several plant LTPs is known to interact with lipids and fatty acids in vitro without ligand specificity (Zachowski et al, 1998;Charvolin et al, 1999;Douliez et al, 2000aDouliez et al, , 2001Hamilton, 2004).…”

“…They contain four conserved disulfide bonds with eight Cys residues and two consensus pentapeptide motifs (Thr/Ser-X1-X2-Asp-Arg/Lys and Pro-Tyr-X-Ile-Ser) (Douliez et al, 2000b). These features were also identified in SCA (Chae et al, 2007). Computational modeling indicates that SCA has an LTP-like structure with a globular shape of the orthogonal four-helix bundle architecture and a hydrophobic core (Chae et al, 2007).…”

“…With elimination of the predicted N-terminal signal sequences, our sequence alignment showed that mature LTP5 protein has 49% amino acid identity to maize (Zea mays) LTP (Shin et al, 1995), 50% to lily SCA1 (Chae et al, 2007), and 97% to ltp5-1 ( Figure 6A). Conserved sequence motifs of the plant LTP family were also found in Arabidopsis LTP5: eight conserved Cys residues and two pentapeptide motifs (Thr/Ser-X1-X2-Asp-Arg/ Lys and Pro-Tyr-X-Ile-Ser) (Douliez et al, 2000b).…”

“…A previous study showed that SCA can facilitate the activity of chemocyanin for chemotrophic guidance of tip growth as well (Kim et al, 2003). SCA, as a pistil factor , may be involved in cell-cell communication both in pollen tube adhesion and tip growth (Chae et al, 2007).…”