MLPK function is not required for self-incompatibility in the S29 haplotype of Brassica rapa L.

KEY MESSAGE: S29 haplotype does not require the MLPK function for self-incompatibility in Brassica rapa. Self-incompatibility (SI) in Brassicaceae is regulated by the self-recognition mechanism, which is based on the S-haplotype-specific direct interaction of the pollen-derived ligand, SP11/SCR, and the stigma-side receptor, SRK. M locus protein kinase (MLPK) is known to be one of the positive effectors of the SI response. MLPK directly interacts with SRK, and is phosphorylated by SRK in Brassica rapa. In Brassicaceae, MLPK was demonstrated to be essential for SI in B. rapa and Brassica napus, whereas it is not essential for SI in Arabidopsis thaliana (with introduced SRK and SP11/SCR from related SI species). Little is known about what determines the need for MLPK in SI of Brassicaceae. In this study, we investigated the relationship between S-haplotype diversity and MLPK function by analyzing the SI phenotypes of different S haplotypes in a mlpk/mlpk mutant background. The results have clarified that in B. rapa, all the S haplotypes except the S29 we tested need the MLPK function, but the S29 haplotype does not require MLPK for the SI. Comparative analysis of MLPK-dependent and MLPK-independent S haplotype might provide new insight into the evolution of S-haplotype diversity and the molecular mechanism of SI in Brassicaceae.

Genetic Diversity of Genes Controlling Unilateral Incompatibility in Japanese Cultivars of Chinese Cabbage.

In recent years, unilateral incompatibility (UI), which is an incompatibility system for recognizing and rejecting foreign pollen that operates in one direction, has been shown to be closely related to self-incompatibility (SI) in Brassica rapa. The stigma- and pollen-side recognition factors (SUI1 and PUI1, respectively) of this UI are similar to those of SI (stigma-side SRK and pollen-side SP11), indicating that SUI1 and PUI1 interact with each other and cause pollen-pistil incompatibility only when a specific genotype is pollinated. To clarify the genetic diversity of SUI1 and PUI1 in Japanese B. rapa, here we investigated the UI phenotype and the SUI1/PUI1 sequences in Japanese commercial varieties of Chinese cabbage. The present study showed that multiple copies of nonfunctional PUI1 were located within and in the vicinity of the UI locus region, and that the functional SUI1 was highly conserved in Chinese cabbage. In addition, we found a novel nonfunctional SUI1 allele with a dominant negative effect on the functional SUI1 allele in the heterozygote.

Spatiogenetic characterization of S receptor kinase (SRK) alleles in naturalized populations of Raphanus sativus L. var. raphanistroides on Yakushima island.

In various coastal areas of Japan, naturalized radish populations are observed. Radish is a cruciferous plant and exhibits self-incompatibility, involving a system controlled by a single locus with multiple S alleles. Although the S allele diversity of radish cultivars and wild radishes has been characterized, the S allele distribution in naturalized populations has not yet been analyzed in relation to the positions of the plants in situ. Here, we show the S allele distribution in naturalized radish populations of Yakushima, a small island in the East China Sea, with positions of the plants. Radish plants were sampled in coastal areas in Yakushima, and their S alleles were detected and characterized. Most of the S alleles had been previously identified in radish cultivars. However, four novel S alleles, which may be unique to Yakushima, were also found. Moreover, seeds in siliques from plants growing in the study areas were sampled, and S allele determination in DNA extracted from these seeds suggested that the plants had exchanged their pollen among their close neighbors. There was also a problem in that the PCR amplification of some SRK alleles was difficult because of their sequence diversity in the naturalized populations, as occurs in cultivars. Our results suggest that the exchange of S alleles between cultivars and naturalized populations occurs and that S alleles in naturalized populations are highly diverse. The methodology established in our study should be applicable to other self-incompatible species to dissect the diversity of S allele distribution in naturalized populations.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

Characterization of self-incompatible Brassica napus lines lacking SP11 expression.

Recognition of self-incompatibility (SI) is regulated by the SRK and SP11 genes in Brassicaceae. Brassica rapa and B. oleracea are self-incompatible, while most cultivated species of B. napus, which arose from hybridization between B. rapa and B. oleracea, are self-compatible. Various studies of the SRK and SP11 genes in self-compatible B. napus have been reported, but details of the mechanism in different B. napus lines are not fully understood. In this study, we confirmed the S haplotypes, SI phenotypes and SP11 expression in 10 representative lines of B. napus, and identified two SI lines (N110 and N343) lacking SP11 expression. In N343 (with BnS1 and BnS6 haplotypes), we confirmed that there is a 3.6-kb insertion in the promoter region of BnSP11-1, and that BnSP11-1 and BnSP11-6 are not expressed, as reported previously (expression of BnSP11-6 is suppressed by the BnS1 haplotype), although this line is self-incompatible. Similarly, in N110, with two novel S haplotypes (BnS8 and BnS9) in addition to BnS6, a 4.3-kb insertion was identified in the promoter region of BnSP11-9, and expression levels of BnSP11-6, BnSP11-8 and BnSP11-9 were all suppressed (BnSP11-6 and BnSP11-8 may be suppressed by BnS8 and BnS9, respectively), although the phenotype was self-incompatible. This observation of an SI phenotype without SP11 expression suggests the existence of unknown factor(s) that induce pollen-stigma incompatibility in B. napus.

Genetic and tissue-specific RNA-sequencing analysis of self-compatible mutant TSC28 in Brassica rapa L. toward identification of a novel self-incompatibility factor.

Self-incompatibility (SI) is a sophisticated system for pollen selectivity to prevent pollination by genetically identical pollen. In Brassica, it is genetically controlled by a single, highly polymorphic S-locus, and the male and female S-determinant factors have been identified as S-locus protein 11 (SP11)/S-locus cysteine-rich protein (SCR) and S-locus receptor kinase (SRK), respectively. However, the overall molecular system and identity of factors in the downstream cascade of the SI reaction remain unclear. Previously, we identified a self-compatible B. rapa mutant line, TSC28, which has a disruption in an unidentified novel factor of the SI signaling cascade. Here, in a genetic analysis of TSC28, using an F2 population from a cross with the reference B. rapa SI line Chiifu-401, the causal gene was mapped to a genetic region of DNA containing markers BrSA64 and ACMP297 in B. rapa chromosome A1. By fine mapping using an F2 population of 1,034 plants, it was narrowed down to a genetic region between DNA markers ACMP297 and BrgMS4028, with physical length approximately 1.01 Mbp. In this genomic region, 113 genes are known to be located and, among these, we identified 55 genes that were expressed in the papilla cells. These are candidates for the gene responsible for the disruption of SI in TSC28. This list of candidate genes will contribute to the discovery of a novel downstream factor in the SP11-SRK signaling cascade in the Brassica SI system.

Duplicated pollen-pistil recognition loci control intraspecific unilateral incompatibility in Brassica rapa.

In plants, cell-cell recognition is a crucial step in the selection of optimal pairs of gametes to achieve successful propagation of progeny. Flowering plants have evolved various genetic mechanisms, mediated by cell-cell recognition, to enable their pistils to reject self-pollen, thus preventing inbreeding and the consequent reduced fitness of progeny (self-incompatibility, SI), and to reject foreign pollen from other species, thus maintaining species identity (interspecific incompatibility)1. In the genus Brassica, the SI system is regulated by an S-haplotype-specific interaction between a stigma-expressed female receptor (S receptor kinase, SRK) and a tapetum cell-expressed male ligand (S locus protein 11, SP11), encoded by their respective polymorphic genes at the S locus2-6. However, the molecular mechanism for recognition of foreign pollen, leading to reproductive incompatibility, has not yet been identified. Here, we show that recognition between a novel pair of proteins, a pistil receptor SUI1 (STIGMATIC UNILATERAL INCOMPATIBILITY 1) and a pollen ligand PUI1 (POLLEN UNILATERAL INCOMPATIBILITY 1), triggers unilateral reproductive incompatibility between plants of two geographically distant self-incompatible Brassica rapa lines, even though crosses would be predicted to be compatible based on the S haplotypes of pollen and stigma. Interestingly, SUI1 and PUI1 are similar to the SI genes, SRK and SP11, respectively, and are maintained as cryptic incompatibility genes in these two populations. The duplication of the SRK and SP11 followed by reciprocal loss in different populations would provide a molecular mechanism of the emergence of a reproductive barrier in allopatry.

A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets.

In diploid organisms, phenotypic traits are often biased by effects known as Mendelian dominant-recessive interactions between inherited alleles. Phenotypic expression of SP11 alleles, which encodes the male determinants of self-incompatibility in Brassica rapa, is governed by a complex dominance hierarchy1-3. Here, we show that a single polymorphic 24 nucleotide small RNA, named SP11 methylation inducer 2 (Smi2), controls the linear dominance hierarchy of the four SP11 alleles (S44 > S60 > S40 > S29). In all dominant-recessive interactions, small RNA variants derived from the linked region of dominant SP11 alleles exhibited high sequence similarity to the promoter regions of recessive SP11 alleles and acted in trans to epigenetically silence their expression. Together with our previous study4, we propose a new model: sequence similarity between polymorphic small RNAs and their target regulates mono-allelic gene expression, which explains the entire five-phased linear dominance hierarchy of the SP11 phenotypic expression in Brassica.

Involvement of MLPK Pathway in Intraspecies Unilateral Incompatibility Regulated by a Single Locus With Stigma and Pollen Factors.

Plants have evolved many systems to prevent undesirable fertilization. Among these, incompatibility is a well-organized system in which pollen germination or pollen tube growth is inhibited in pistils. We previously found that a novel one-way pollen-stigma incompatibility response [unilateral incompatibility (UI)] occurred between two self-incompatible Brassica rapa plants, a Turkish line, and a Japanese cultivated hybrid variety, "Osome." Pollen from the Turkish line is rejected on the stigma of the Osome line, but the reverse cross is compatible; such a UI phenotype closely resembles self-incompatibility (SI). The pollen factor of this UI has been genetically explained by a single locus which is different from the S-locus. In this study, we performed further genetic analyses of this intraspecies UI and showed that the stigma factor was also controlled by a single locus, and we named the loci corresponding to the stigma and pollen factors of the intraspecies UI, stigmatic unilateral incompatibility (SUI), and pollen unilateral incompatibility (PUI) loci, respectively. Interestingly, segregation analyses of SUI and PUI indicated that they are closely linked to each other and behave as a single unit. To investigate the effect of an SI-related gene, MLPK in this UI, we produced segregation lines for SUI and mlpk A distorted segregation ratio of SUI phenotype in an mlpk background indicated involvement of MLPK in SUI, suggesting the existence of an MLPK-dependent novel pollen-stigma recognition mechanism.

Novel self-compatible lines of Brassica rapa L. isolated from the Japanese bulk-populations.

Self-incompatibility (SI) in Brassicaceae is sporophytically controlled by a single S-locus with multi allelic variety. The male S determinant, SP11/SCR (S-locus protein 11/S-locus cysteine-rich protein), is a small cysteine-rich protein, and the female S determinant, SRK (S-locus receptor kinase), functions as a receptor for SP11 at the surface of stigma papilla cells. Although a few of the following downstream factors in the SP11-SRK signaling cascade have been identified, a comprehensive understanding of the SI mechanism still remains unexplained in Brassicaceae. Analysis of self-compatible (SC) mutants is significant for understanding the molecular mechanism in SI reactions, thus we screened SC lines from a variety of Japanese bulk-populations of B. rapa vegetables. Two lines, TSC4 and TSC28, seem to have disruptions in the SI signaling cascade, while the other line, TSC2, seems to have a deficiency in a female S determinant, SRK. In TSC4 and TSC28, known SI-related factors, i.e. SRK, SP11, MLPK (M-locus protein kinase), THL (thioredoxin-h-like), and ARC1 (arm repeat containing 1), were expressed normally, and their expression levels were comparable with those in SI lines. On a B. rapa genetic linkage map, potential SC genes in TSC4 and TSC28 were mapped on linkage groups A3 and A1, respectively, whereas MLPK, ARC1, and THL were mapped on A3, A4, and A6, respectively. Although potential SC genes of TSC4 and MLPK were on the same linkage group, their positions were apparently independent. These results indicate that the SC genes of TSC4 and TSC28 are independent from the S-locus or known SI-related genes. Thus, the SC lines selected here have mutations in novel factors of the SI signaling cascade, and they will contribute to fill pieces in a signal transduction pathway of the SI system in Brassicaceae.

Expression and developmental function of the 3-ketoacyl-ACP synthase2 gene in Arabidopsis thaliana.

The 3-ketoacyl-ACP synthase (KAS) II is a fatty-acid-related enzyme which catalyzes the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. The fatty acid biosynthesis 1-1 (fab1-1) mutant of Arabidopsis thaliana is partially deficient in its activity of Arabidopsis thaliana 3-ketoacyl-ACP synthase 2 (AtKAS2), and its phenotype has been intensively studied in connection with the chilling resistance and fatty acid composition. In this study, we used the T-DNA insertion mutant of AtKAS2 to examine its possible role in plant development. Reverse transcription (RT)-PCR showed that the AtKAS2 gene was expressed in various plant organs, except for roots, and was highly expressed in siliques. The fusion of beta-glucuronidase (GUS) to the AtKAS2 promoter demonstrated that the promoter was active in various tissues such as embryos, stomatal guard cells, inflorescences and pollen grains. We were not able to identify atkas2 homozygous mutant adult plants in heterozygous mutant progeny. Phenotypic and genetic analyses showed that disruption of the AtKAS2 by T-DNA insertion caused embryo lethality, and the development of the embryos was arrested at the globular stage. Taken together, our results suggest that AtKAS2 is required for embryo development in Arabidopsis during the transition from the globular to the heart stage.

Molecular characterization of two anther-specific genes encoding putative RNA-binding proteins, AtRBP45s, in Arabidopsis thaliana.

RRM (RNA-recognition motif) domain is important for the post-transcriptional regulation of gene expression including RNA processing. In our previous study, we found one anther- and/or pollen-specific gene (LjRRM1, previously named as LjMfb-U93) in model legume, Lotus japonicus. Because of the richness of genomic information of another model plant, Arabidopsis thaliana, for functional analysis, we identified and characterized the orthologous genes in A. thaliana. By comparison of the partial nucleotide sequence of LjRRM1 to the public database, we identified three homologous genes (AtRBP45a, AtRBP45b, and AtRBP45c) in A. thaliana genome. Based on promoter analysis, both AtRBP45a and AtRBP45c were specifically expressed in immature anther tissues (tapetum cells) and mature pollen grains of transgenic plants. This expression pattern of AtRBP45a and AtRBP45c is quite similar to that of LjRRM1, indicating that AtRBP45a and AtRBP45c would be orthologous to LjRRM1. Because in another previous experiment, it was shown that proteins having RRM domains were related to pre-mRNA maturation, and as a conclusion, it is possible that LjRRM1, AtRBP45a, and AtRBP45c genes encoding RNA-binding proteins are functionally involved in the repression of translation in mature pollen grains in L. japonicus and A. thaliana.

Anther-specific genes, which expressed through microsporogenesis, are temporally and spatially regulated in model legume, Lotus japonicus.

Pollen germination and pollen tube elongation are important for pollination and fertilization in higher plants. To date, several pollen-specific genes have been isolated and characterized. However, there is little information about the precise spatial and temporal expression pattern of pollen-specific genes in higher plants. In our previous study, we identified 132 anther-specific genes in the model legume Lotus japonicus by using cDNA microarray analysis, though their precise expression sites in the anther tissues were not determined. In this study, by using in situ hybridization experiments, we determined the spatial and temporal expression sites of 46 anther-specific genes (ca. 35%), which were derived from two groups, cluster I-a and cluster II-a, according to flower developmental stages. In the case of the genes grouped into cluster I-a, thirteen clones were characterized. The specific hybridized signals were varied among the clones, and were observed in tapetum cells, microspores, and anther walls at the early developmental stage of anther tissues. In the case of the genes classified into cluster II-a, we used thirty three different cDNA clones encoding primary and secondary metabolism-related proteins, cell wall reconstruction-related proteins, actin reorganization-related proteins, and sugar transport-related proteins, etc., as a probe. Interestingly, all genes in these thirty three clones examined were specifically expressed in the bicellular pollen grains, though the signal intensity was varied among clones. From the data of the cluster II-a genes, the mRNAs related to pollen germination and pollen tube elongation were specifically transcribed and preserved in mature pollen grains.

Comparative analysis of the S-intergenic region in class-II S haplotypes of self-incompatible Brassica rapa (syn. campestris).

In the Brassica self-incompatibility (SI) system, a pollen determinant, SP11, is involved in dominance/recessive relationships in pollen SI phenotypes. In order to gain some insights into the genomic structure around the SP11 and the mechanisms that give dominance/recessive relationships, we characterized the genomic region containing SP11 and SRK genes in three pollen recessive class-II S haplotypes. The direction of transcription of S genes was completely conserved among class-II S haplotypes. However, the region between SP11 and SRK (S-intergenic region) was highly polymorphic without short repetitive sequences. In addition, we found a sequence similarity between the short repetitive sequence and 5'-upstream region of SP11. This sequence similarity was found to be potentially related to the expression of dominance relationships through the change of chromatin structure.

Molecular characterization of mature pollen-specific genes encoding novel small cysteine-rich proteins in rice (Oryza sativa L.).

In our previous cDNA microarray analysis, we identified 53 mature anther-specific genes, whose function was unknown, in rice. We reanalyzed these genes from the viewpoint of the specific amino acid motif. Out of 53 genes, three genes, Os-26, Os-32, and Os-169 (renamed as OsSCP1, OsSCP2, and OsSCP3), encoded cysteine-rich motif (Cys-X3-Cys-X13-Cys-X3-Cys), indicating that they were novel small cysteine-rich proteins. From the search of specific elements in promoter regions, several pollen-specific elements were found. In order to determine whether three promoters were functional in pollen or not, the gene constructs with promoter regions fused to the beta-glucuronidase gene were transformed into tobacco. Histochemical analysis showed that these promoters were active in the mature pollen grains and pollen tubes. Furthermore, OsSCP1 and OsSCP3 formed a multigene family tandemly in the rice genome. From the results, OsSCPs might have important roles in mature pollen development and pollen tube growth.

Linear dominance relationship among four class-II S haplotypes in pollen is determined by the expression of SP11 in Brassica self-incompatibility.

Self-incompatibility (SI) prevents self-fertilization by rejecting pollen from plants with the same S phenotype. The Brassica SI system is controlled sporophytically by multiple alleles at the single locus, S, and dominance relationships among S haplotypes are observed in both stigma and pollen. We have identified previously five different class-II S haplotypes in Brassica campestris. Here, we performed test-crosses between S heterozygotes and their respective parental S homozygotes for four of these class-II S haplotypes, and observed a linear dominance relationship on the pollen side. To determine how this relationship is controlled, we performed RNA gel blot analyses for six S heterozygotes and their respective parental S homozygotes using the corresponding SP11 clone as a probe. In all six S heterozygotes, SP11 derived from a dominant haplotype was predominantly expressed, and SP11 derived from a recessive haplotype was repressed. Thus, the linear dominance relationship of the SI phenotype on the pollen side is regulated by the expression of SP11.

The dominance of alleles controlling self-incompatibility in Brassica pollen is regulated at the RNA level.

Self-incompatibility (SI) in Brassica 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 SP11s 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 SP11s 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 SP11s.