Background : Self-incompatibility (SI) in the Solanaceae, Rosaceae and Scrophulariaceae is gametophytically controlled by a single polymorphic locus, termed the S -locus. To date, the only known S -locus product is a polymorphic ribonuclease, termed SRNase, which is secreted by stylar tissue and thought to act as a cytotoxin that degrades the RNA of incompatible pollen tubes. However, understanding how S -RNase causes S -haplotype specific inhibition of pollen tubes has been hampered by the lack of a cloned pollen S -determinant gene.

Many flowering plants possess self-incompatibility (SI) systems that prevent inbreeding. In Brassica, SI is controlled by a single polymorphic locus, the S locus. Two highly polymorphic S locus genes, SLG (S locus glycoprotein) and SRK (S receptor kinase), have been identified, both of which are expressed predominantly in the stigmatic papillar cell. We have shown recently that SRK is the determinant of the S haplotype specificity of the stigma. SRK is thought to serve as a receptor for a pollen ligand, which presumably is encoded by another polymorphic gene at the S locus. We previously have identified an S locus gene, SP11 (S locus protein 11), of the S9 haplotype of Brassica campestris and proposed that it potentially encodes the pollen ligand. SP11 is a novel member of the PCP (pollen coat protein) family of proteins, some members of which have been shown to interact with SLG. In this work, we identified the SP11 gene from three additional S haplotypes and further characterized the gene. We found that (i) SP11 showed an S haplotype-specific sequence polymorphism; (ii) SP11 was located in the immediate flanking region of the SRK gene of the four S haplotypes examined; (iii) SP11 was expressed in the tapetum of the anther, a site consistent with sporophytic control of Brassica SI; and (iv) recombinant SP11 of the S9 haplotype applied to papillar cells of S9 stigmas, but not of S8 stigmas, elicited SI response, resulting in inhibition of hydration of cross-pollen. All these results taken together strongly suggest that SP11 is the pollen S determinant in SI.I n Brassica, SI is sporophytically controlled by a highly polymorphic locus, termed the S locus, with more than 100 haplotypes identified so far (1, 2). To date, two stigmatically expressed highly polymorphic genes have been identified at the S locus. One is the S locus glycoprotein (SLG) gene, which encodes a secreted glycoprotein abundantly present in the papillar cell of the stigma surface (3, 4), and the other is the S locus receptor kinase (SRK) gene, which encodes a putative receptor-like serine͞threonine protein kinase presumed to span the plasma membrane of the papillar cell (5). The predicted extracellular domain of SRK shares extensive sequence similarity with SLG. Results from our recent gain-of-function experiments have shown that SRK is the sole determinant of the S haplotype specificity of the stigma (6). SRK is thought to function as a receptor for the S determinant of pollen with the same S haplotype. Binding of SRK to the pollen S determinant then would elicit a signaling cascade in the papillar cell, leading to the rejection of self-pollen. Our recent study also has shown that the role of SLG is probably to enhance this recognition process, although how this is accomplished is not yet known (6).In contrast to the S determinant of the stigma, the pollen S determinant had long remained elusive. Doughty et al. (7,8) analyzed the pollen coat protein (PCP) of Brassica oleracea and identified a basic 7-kDa protein, termed PCP-A1 (protein 1 of cla...

Many higher plants have evolved self-incompatibility mechanisms to prevent self-fertilization. In Brassica self-incompatibility, recognition between pollen and the stigma is controlled by the S locus, which contains three highly polymorphic genes: S-receptor kinase (SRK), S-locus protein 11 (SP11) (also called S-locus cysteine-rich protein; SCR) and S-locus glycoprotein (SLG). SRK encodes a membrane-spanning serine/threonine kinase that determines the S-haplotype specificity of the stigma, and SP11 encodes a small cysteine-rich protein that determines the S-haplotype specificity of pollen. SP11 is localized in the pollen coat. It is thought that, during self-pollination, SP11 is secreted from the pollen coat and interacts with its cognate SRK in the papilla cell of the stigma to elicit the self-incompatibility response. SLG is a secreted stigma protein that is highly homologous to the SRK extracellular domain. Although it is not required for S-haplotype specificity of the stigma, SLG enhances the self-incompatibility response; however, how this is accomplished remains controversial. Here we show that a single form of SP11 of the S8 haplotype (S8-SP11) stabilized with four intramolecular disulphide bonds specifically binds the stigma membrane of the S8 haplotype to induce autophosphorylation of SRK8, and that SRK8 and SLG8 together form a high-affinity receptor complex for S8-SP11 on the stigma membrane.

Self-incompatibility (SI) response in Brassica is initiated by haplotype-specific interactions between the pollen-borne ligand S locus protein 11/SCR and its stigmatic S receptor kinase, SRK. This binding induces autophosphorylation of SRK, which is then thought to trigger a signaling cascade that leads to self-pollen rejection. A recessive mutation of the modifier (m) gene eliminates the SI response in stigma. Positional cloning of M has revealed that it encodes a membrane-anchored cytoplasmic serine/threonine protein kinase, designated M locus protein kinase (MLPK). Transient expression of MLPK restores the ability of mm papilla cells to reject self-pollen, suggesting that MLPK is a positive mediator of Brassica SI signaling.

Allyl sulfides are characteristic flavor components obtained from garlic. These sulfides are thought to be responsible for their epidemiologically proven anticancer effect on garlic eaters. This study was aimed at clarifying the molecular basis of this anticancer effect of garlic by using human colon cancer cell lines HCT-15 and DLD-1. The growth of the cells was significantly suppressed by diallyl trisulfide (DATS, HCT-15 IC 50 ‫؍‬ 11.5 M, DLD-1 IC 50 ‫؍‬ 13.3 M); however, neither diallyl monosulfide nor diallyl disulfide showed such an effect. The proportion of HCT-15 and that of DLD-1 cells residing at the G 1 and S phases were decreased by DATS, and their populations at the G 2 /M phase were markedly increased for up to 12 h. The cells with a sub-G 1 DNA content were increased thereafter. Caspase-3 activity was also dramatically increased by DATS. Fluorescence-activated cell sorter analysis performed on the cells arrested at the G 1 /S boundary revealed cell cycle-dependent induction of apoptosis through the transition of the G 2 /M phase to the G 1 phase by DATS. DATS inhibited tubulin polymerization in an in vitro cell-free system. DATS disrupted microtubule network formation of the cells, and microtubule fragments could be seen at the interphase. Peptide mass mapping by liquid chromatography-tandem mass spectrometry analysis for DATS-treated tubulin demonstrated that there was a specific oxidative modification of cysteine residues Cys-12␤ and Cys-354␤ to form S-allylmercaptocysteine with a peptide mass increase of 72.1 Da. The potent antitumor activity of DATS was also demonstrated in nude mice bearing HCT-15 xenografts. This is the first paper describing intracellular target molecules directly modified by garlic components.Allyl sulfides, e.g. diallyl monosulfide (DAS), 4 diallyl disulfide (DADS), and diallyl trisulfide (DATS), are characteristic flavor components of the essential oil prepared from garlic (Allium sativum L.). Garlic is widely served around the world, and it has been reported that allyl sulfides inhibit both the initiation and promotion stages of tumorigenesis in experimental carcinogenesis models for various types of cancer (1-5). Recently, several lines of investigation have shown that allyl sulfides suppress cell growth and induce apoptosis in multiple cancer cell lines (6 -12). We previously reported that the sulfur-containing volatile oils prepared from garlic and onion inhibit proliferation and induce differentiation of the human promyelocytic leukemia cell line HL-60 (13). However, the molecular mechanisms underlying the antitumorigenesis of allyl sulfides are still not fully understood.Microtubules are ubiquitous proteins present in eukaryotes as components of the cytoskeleton and play pivotal roles in a variety of cellular processes involving cell division, motility, and intracellular trafficking (14). The microtubules are dynamic polymers composed of ␣␤-tubulin heterodimers, and they form the mitotic spindles, which are known to introduce the replicated DNA molecules to the res...

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 μ  m. 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.

A diploid organism has two copies of each gene, one inherited from each parent. The expression of two inherited alleles is sometimes biased by the effects known as dominant/recessive relationships, which determine the final phenotype of the organism. To explore the mechanisms underlying these relationships, we have examined the monoallelic expression of S-locus protein 11 genes (SP11), which encode the male determinants of self-incompatibility in Brassica. We previously reported that SP11 expression was monoallelic in some S heterozygotes, and that the promoter regions of recessive SP11 alleles were specifically methylated in the anther tapetum. Here we show that this methylation is controlled by trans-acting small non-coding RNA (sRNA). We identified inverted genomic sequences that were similar to the recessive SP11 promoters in the flanking regions of dominant SP11 alleles. These sequences were specifically expressed in the anther tapetum and processed into 24-nucleotide sRNA, named SP11 methylation inducer (Smi). Introduction of the Smi genomic region into the recessive S homozygotes triggered the methylation of the promoter of recessive SP11 alleles and repressed their transcription. This is an example showing sRNA encoded in the flanking region of a dominant allele acts in trans to induce transcriptional silencing of the recessive allele. Our finding may provide new insights into the widespread monoallelic gene expression systems.

Self-incompatibility (SI) inBrassica is controlled sporophytically by the multiallelic S -locus. The SI phenotype of pollen in an S -heterozygote is determined by the relationship between the two S -haplotypes it carries, and dominant/recessive relationships often are observed between the two S -haplotypes. The S -locus protein 11 ( SP11 , also known as the S -locus cysteine-rich protein) gene has been cloned from many pollen-dominant S -haplotypes (class I) and shown to encode the pollen S -determinant. However, SP11 from pollen-recessive S -haplotypes (class II) has never been identified by homology-based cloning strategies, and how the dominant/recessive interactions between the two classes occur was not known. We report here the identification and molecular characterization of SP11 s from six class II S -haplotypes of B. rapa and B. oleracea . Phylogenetic analysis revealed that the class II SP11s form a distinct group separated from class I SP11s. The promoter sequences and expression patterns of SP11 s also were different between the two classes. The mRNA of class II SP11 , which was detected predominantly in the anther tapetum in homozygotes, was not detected in the heterozygotes of class I and class II S -haplotypes, suggesting that the dominant/recessive relationships of pollen are regulated at the mRNA level of SP11 s. INTRODUCTIONMany species of hermaphrodite plants have evolved mechanisms to prevent self-fertilization. Self-incompatibility (SI) is one physiological means of avoiding self-fertilization through recognition of self-pollen in or on the female pistil. Classic genetic analyses have revealed the presence of two major types of homomorphic SI systems, gametophytic and sporophytic (de Nettancourt, 1977). Although the recognition of self-pollen is controlled genetically by a single highly polymorphic locus called the S -locus in both of these systems, the SI phenotype of pollen (gametophyte) is determined by its own S -haplotype in the gametophytic system, whereas in the sporophytic system, the SI phenotype is controlled by the S -haplotypes of the diploid parent (sporophyte).The majority of the members of the cruciferous plant genus Brassica possess a strong sporophytic SI system. Thus, the SI phenotype of pollen as well as stigma is determined by relationships between the two S -haplotypes carried by its parent (Bateman, 1955). In other words, a codominant or a dominant/recessive relationship between the two S -haplotypes influences the ultimate SI phenotype of both pollen and stigma (Thompson and Taylor, 1966). The following observations have been made about dominance relationships among S -haplotypes: (1) codominance is common; (2) dominance/recessiveness is frequent in pollen; (3) dominance relationships among stigmas are different from those among pollen; and (4) dominance relationships are nonlinear (Thompson and Taylor, 1966;Ockendon, 1975;Visser et al., 1982; Hatakeyama et al., 1998a).Recent molecular studies have revealed that the S -locus of Brassica encodes three highly polymorphic ...