Self-incompatibility (SI) is a mechanism that prevents self-fertilization in flowering plants. Rosaceous species such as Japanese pear and apple have gametophytic self-incompatibility (GSI) controlled by a single, multi-allelic locus, the S-locus.
When a pollen grain lands on a stigma, a discrimination process takes place as to whether the S-allele of the pollen matches one of the two S-alleles of the pistil. The pollen grain germinates on the stigma and grows toward the embryo; but, if its S-allele matches one of the S-alleles of the pistil, pollen tube growth is arrested in the style, and no fertilization take places. A pistil-specific protein encoded by the S-locus has been shown to be a ribonuclease (S-RNase) that recognizes the pollen S-allele. S-RNase-based GSI also operates in the Solanaceae and Scrophulariaceae. Two models for the S-allele-specific inhibition of pollen tube growth involving S-RNase (the S >-allele-specific uptake and RNase inhibitor models) have been proposed by researchers working on GSI, but at present there is not enough evidence to support either model. How S-RNase discriminates between self- and nonself-pollen, what the counterpart molecule interacting with S-RNase is, and how S-RNase
interacts with that molecule have yet to be clarified.

In order to elucidate the mechanism of the S-RNase-based GSI at molecular level, we have been studying the structure and function of Japanese pear S-RNases and searching unknown pollen S-product(s). To date, genomic and cDNA clones encoding seven S-RNase (S1- to S7-RNase) from pistils of Japanese pear have been isolated and sequenced. The posttranslational
modifications including S-S bonds and sugar chains have been analyzed by mass spectrometer. When their amino acid sequences were aligned with those of other rosaceous S-RNases sequenced so far, many conserved amino acids were stretched throughout the sequence, but were absent from the region from the 51st to 66th residue which was designated a hypervariable (HV) region.
Window analysis of the numbers of synonymous (dS) and nonsynonymous (dN) substitutions in rosaceous S-RNase genes detected four regions with an excess of dN over dS in which positive selection may ope! rate (PS regions).

The crystal structure of the S3-RNase has been determined at 1.5 ? resolution. It consists of eight helices and seven β-strands, and its folding topology is typical of RNase T2 family enzymes. A hypervariable (HV) region comprises a long loop and short α-helix. This region is far from the active site cleft, exposed on the molecule's surface, and positively
charged. Four positively selected (PS) regions are located on either side of the active site cleft, and accessible to solvent.
These structural features suggest that the HV or PS regions may interact with a pollen S-gene product(s) to recognize self and non-self pollen. Search for pollen S-product(s) is now in progress in our laboratory.

Pollen tube growth is one of the important processes for higher plant fertilization. When a pollen grain lands on a stigma, it adsorbs water and forms a pollen tube. The pollen tube invades the pistil, growing between the walls of the stigmatic cells, then traveling through an extracellular matrix in the transmitting tissue of the style, and finally arriving at the ovary, where it targets an ovule that contains an egg. The pollen tube extends by highly specialized and localized form of cell wall and membrane deposition called "tip growth". The pollen tube has a characteristic polarization of its cytoplasm and cytoskeletal componets, similar to other tip-growing cells such as root hairs, fungal hyphae, and axon of neuron.

Gradient of calcium ion, Rho GTPase, and change of actin cytoskeleton are believed to be responsible for the tip growth of the pollen tube, but its overall mechanism is still ambiguous. We have found about fifty kinds of proteins, which are expressed specifically during pollen tube growth, by two-dimensional polyacrylamide gel electrophoresis. In order to elucidate the mechanism of the pollen tube growth at molecular level, we are investigating their functions by analyzing the protein structures and the protein-protein interactions.