Sexual reproduction in many flowering plants involves self-incompatibility ( SI self-incompatibility ), which is one of the most important systems to prevent inbreeding. In many species, the self-/nonself-recognition of SI is controlled by a single polymorphic locus, the S-locus. Molecular dissection of the S-locus revealed that SI represents not one system, but a collection of divergent mechanisms. Here, we discuss recent advances in the understanding of three distinct SI mechanisms, each controlled by two separate determinant genes at the S-locus. In the Brassicaceae, the determinant genes encode a pollen ligand and its stigmatic receptor kinase; their interaction induces incompatible signaling(s) within the stigma papilla cells. In the Solanaceae-type SI, the determinants are a ribonuclease and an F-box protein, suggesting the involvement of RNA and protein degradation in the system. In the Papaveraceae, the only identified female determinant induces a Ca²⁺-dependent signaling network that ultimately results in the death of incompatible pollen.

Dissecting Self-Incompatibility: Although the pollen may be available for a flower to fertilize itself, molecular determinants on the pollen and the pistil prevent inbreeding in a process termed self-incompatibility. In the Petunia self-incompatibility, if male determinants (F-box proteins) on pollen are recognized by a female ribonuclease determinant on the pistil, the pollen tube is killed when its ribosomal RNA is digested. Outcrossed fertilizations can occur because of allelic diversity in the female that fails to recognize its male counterparts; however, the genetic diversity of the ribonuclease gene is greater than that of the known F-box gene. Kubo et al. (p. 796 ; see the Perspective by Indriolo and Goring ) have discovered that there are several related F-box genes in Petunia , each of which brings its own allelic diversity to bear—thus, increasing the variety of potential mating partners.

Pollen tube growth is crucial for the delivery of sperm cells to the ovule during flowering plant reproduction. Previous in vitro imaging of Lilium longiflorum and Nicotiana tabacum has shown that growing pollen tubes exhibit a tip-focused Ca²⁺ concentration ([Ca²⁺]) gradient and regular oscillations of the cytosolic [Ca²⁺] ([Ca²⁺]_cyt) in the tip region. Whether this [Ca²⁺] gradient and/or [Ca²⁺]_cyt oscillations are present as the tube grows through the stigma (in vivo condition), however, is still not clear. We monitored [Ca²⁺]_cyt dynamics in pollen tubes under various conditions using Arabidopsis (Arabidopsis thaliana) and N. tabacum expressing yellow cameleon 3.60, a fluorescent calcium indicator with a large dynamic range. The tip-focused [Ca²⁺]_cyt gradient was always observed in growing pollen tubes. Regular oscillations of the [Ca²⁺]_cyt, however, were rarely identified in Arabidopsis or N. tabacum pollen tubes grown under the in vivo condition or in those placed in germination medium just after they had grown through a style (semi-in vivo condition). On the other hand, regular oscillations were observed in vitro in both growing and nongrowing pollen tubes, although the oscillation amplitude was 5-fold greater in the nongrowing pollen tubes compared with growing pollen tubes. These results suggested that a submicromolar [Ca²⁺]_cyt in the tip region is essential for pollen tube growth, whereas a regular [Ca²⁺] oscillation is not. Next, we monitored [Ca²⁺] dynamics in the endoplasmic reticulum ([Ca²⁺]_ER) in relation to Arabidopsis pollen tube growth using yellow cameleon 4.60, which has a lower affinity for Ca²⁺ compared with yellow cameleon 3.60. The [Ca²⁺]_ER in pollen tubes grown under the semi-in vivo condition was between 100 and 500 μm. In addition, cyclopiazonic acid, an inhibitor of ER-type Ca²⁺-ATPases, inhibited growth and decreased the [Ca²⁺]_ER. Our observations suggest that the ER serves as one of the Ca²⁺ stores in the pollen tube and cyclopiazonic acid-sensitive Ca²⁺-ATPases in the ER are required for pollen tube growth.

Many flowering plants possess systems of self-incompatibility (SI) to prevent inbreeding. In Brassica, SI recognition is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus, which encodes both the male determinant S-locus protein 11 (SP11/SCR) and the female determinant S-receptor kinase (SRK). Upon self-pollination, the S-haplotype–specific interaction between the pollen-borne SP11 and the cognate stigmatic SRK receptor induces SI signaling in the stigmatic papilla cell and results in rejection of the self-pollen. Our genetic analysis of a self-compatible mutant revealed the involvement of a cytoplasmic protein kinase, M-locus protein kinase (MLPK), in the SI signaling, but its exact physiological function remains unknown. In this study, we identified two different MLPK transcripts, MLPKf1 and MLPKf2, which are produced using alternative transcriptional initiation sites and encode two isoforms that differ only at the N termini. While MLPKf1 and MLPKf2 exhibited distinct expression profiles, both were expressed in papilla cells. MLPKf1 localizes to the plasma membrane through its N-terminal myristoylation motif, while MLPKf2 localizes to the plasma membrane through its N-terminal hydrophobic region. Although both MLPKf1 and MLPKf2 could independently complement the mlpk/mlpk mutation, their mutant forms that lack the plasma membrane localization motifs failed to complement the mutation. Furthermore, a bimolecular fluorescence complementation assay revealed direct interactions between SRK and the MLPK isoforms in planta. These results suggest that MLPK isoforms localize to the papilla cell membrane and interact directly with SRK to transduce SI signaling.

The directional growth of the pollen tube from the stigma to the embryo sac in the ovules is regulated by pollen-pistil interactions based on intercellular communication. Although pollen tube growth is regulated by the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt), it is not known whether [Ca²⁺]_cyt is involved in pollen tube guidance and reception. Using Arabidopsis expressing the GFP-based Ca²⁺-sensor yellow cameleon 3.60 (YC3.60) in pollen tubes and synergid cells, we monitored Ca²⁺ dynamics in these cells during pollen tube guidance and reception under semi-in vivo fertilization conditions. In the pollen tube growing towards the micropyle, pollen tubes initiated turning within 150 μm of the micropylar opening; the [Ca²⁺]_cyt in these pollen tube tips was higher than in those not growing towards an ovule in assays with myb98 mutant ovules, in which pollen tube guidance is disrupted. These results suggest that attractants secreted from the ovules affect Ca²⁺ dynamics in the pollen tube. [Ca²⁺]_cyt in synergid cells did not change when the pollen tube grew towards the micropyle or entered the ovule. Upon pollen tube arrival at the synergid cell, however, [Ca²⁺]_cyt oscillation began at the micropylar pole of the synergid, spreading towards the chalazal pole. Finally, [Ca²⁺]_cyt in the synergid cell reached a maximum at pollen tube rupture. These results suggest that signals from the pollen tube induce Ca²⁺ oscillations in synergid cells, and that this Ca²⁺ oscillation is involved in the interaction between the pollen tube and synergid cell.