Dominance in self-compatibility between subgenomes of allopolyploid Arabidopsis kamchatica shown by transgenic restoration of self-incompatibility.

The evolutionary transition to self-compatibility facilitates polyploid speciation. In Arabidopsis relatives, the self-incompatibility system is characterized by epigenetic dominance modifiers, among which small RNAs suppress the expression of a recessive SCR/SP11 haplogroup. Although the contribution of dominance to polyploid self-compatibility is speculated, little functional evidence has been reported. Here we employ transgenic techniques to the allotetraploid plant A. kamchatica. We find that when the dominant SCR-B is repaired by removing a transposable element insertion, self-incompatibility is restored. This suggests that SCR was responsible for the evolution of self-compatibility. By contrast, the reconstruction of recessive SCR-D cannot restore self-incompatibility. These data indicate that the insertion in SCR-B conferred dominant self-compatibility to A. kamchatica. Dominant self-compatibility supports the prediction that dominant mutations increasing selfing rate can pass through Haldane's sieve against recessive mutations. The dominance regulation between subgenomes inherited from progenitors contrasts with previous studies on novel epigenetic mutations at polyploidization termed genome shock.

Double-Locking Mechanism of Self-Compatibility in Arabidopsis thaliana: The Synergistic Effect of Transcriptional Depression and Disruption of Coding Region in the Male Specificity Gene.

Self-compatibility in Arabidopsis thaliana represents the relatively recent disruption of ancestral obligate cross pollination, recognized as one of the prevalent evolutionary pathways in flowering plants, as noted by Darwin. Our previous study found that inversion of the male specificity gene (SP11/SCR) disrupted self-incompatibility, which was restored by overexpressing the SCR with the reversed inversion. However, SCR in A. thaliana has other mutations aside from the pivotal inversion, in both promoter and coding regions, with probable effects on transcriptional regulation. To examine the functional consequences of these mutations, we conducted reciprocal introductions of native promoters and downstream sequences from orthologous loci of self-compatible A. thaliana and self-incompatible A. halleri. Use of this inter-species pair enabled us to expand the scope of the analysis to transcriptional regulation and deletion in the intron, in addition to inversion in the native genomic background. Initial analysis revealed that A. thaliana has a significantly lower basal expression level of SCR transcripts in the critical reproductive stage compared to that of A. halleri, suggesting that the promoter was attenuated in inducing transcription in A. thaliana. However, in reciprocal transgenic experiments, this A. thaliana promoter was able to restore partial function if coupled with the functional A. halleri coding sequence, despite extensive alterations due to the self-compatible mode of reproduction in A. thaliana. This represents a synergistic effect of the promoter and the inversion resulting in fixation of self-compatibility, primarily enforced by disruption of SCR. Our findings elucidate the functional and evolutionary context of the historical transition in A. thaliana thus contributing to the understanding of the molecular events leading to development of self-compatibility.

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.

Demonstration in vivo of the role of Arabidopsis PLIM2 actin-binding proteins during pollination.

In plant reproduction, pollination is the initial key process in bringing together the male and female gametophytes. When a pollen grain lands on the surface of the stigma, information is exchanged between the pollen and stigmatic cell to determine whether the pollen grain will be accepted or rejected. If it is accepted, the stigmatic papilla cell supplies water and other resources to the pollen for germination and pollen tube elongation. Cellular processes involving actin are essential for pollen germination and tube growth, and actin-binding proteins regulate these processes by interacting with actin filaments to assemble cytoskeletal structures and actin networks. LIM proteins, which belong to a subfamily of cysteine-rich proteins, are a family of actin-binding proteins in plants, and are considered to be important for formation of the actin cytoskeleton and maintenance of its dynamics. Although the physiological and biochemical characteristics of LIMs have been elucidated in vitro in a variety of cell types, their exact role in pollen germination and pollen tube growth during pollination remained unclear. In this manuscript, we focus on the pollen-specific LIM proteins, AtPLIM2a and AtPLIM2c, and define their biological function during pollination in Arabidopsis thaliana. The atplim2a/atplim2c double knockdown RNAi plants showed a reduced pollen germination, approximately one-fifth of wild type, and slower pollen tube growth in the pistil, that is 80.4 µm/hr compared to 140.8 µm/hr in wild type. These defects led to an occasional unfertilized ovule at the bottom of the silique in RNAi plants. Our data provide direct evidence of the biological function of LIM proteins during pollination as actin-binding proteins, modulating cytoskeletal structures and actin networks, and their consequent importance in seed production.

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.

Evolution of self-compatibility in Arabidopsis by a mutation in the male specificity gene.

Ever since Darwin's pioneering research, the evolution of self-fertilisation (selfing) has been regarded as one of the most prevalent evolutionary transitions in flowering plants. A major mechanism to prevent selfing is the self-incompatibility (SI) recognition system, which consists of male and female specificity genes at the S-locus and SI modifier genes. Under conditions that favour selfing, mutations disabling the male recognition component are predicted to enjoy a relative advantage over those disabling the female component, because male mutations would increase through both pollen and seeds whereas female mutations would increase only through seeds. Despite many studies on the genetic basis of loss of SI in the predominantly selfing plant Arabidopsis thaliana, it remains unknown whether selfing arose through mutations in the female specificity gene (S-receptor kinase, SRK), male specificity gene (S-locus cysteine-rich protein, SCR; also known as S-locus protein 11, SP11) or modifier genes, and whether any of them rose to high frequency across large geographic regions. Here we report that a disruptive 213-base-pair (bp) inversion in the SCR gene (or its derivative haplotypes with deletions encompassing the entire SCR-A and a large portion of SRK-A) is found in 95% of European accessions, which contrasts with the genome-wide pattern of polymorphism in European A. thaliana. Importantly, interspecific crossings using Arabidopsis halleri as a pollen donor reveal that some A. thaliana accessions, including Wei-1, retain the female SI reaction, suggesting that all female components including SRK are still functional. Moreover, when the 213-bp inversion in SCR was inverted and expressed in transgenic Wei-1 plants, the functional SCR restored the SI reaction. The inversion within SCR is the first mutation disrupting SI shown to be nearly fixed in geographically wide samples, and its prevalence is consistent with theoretical predictions regarding the evolutionary advantage of mutations in male components.

Integration of Brassica A genome genetic linkage map between Brassica napus and B. rapa.

An integrated linkage map between B. napus and B. rapa was constructed based on a total of 44 common markers comprising 41 SSR (33 BRMS, 6 Saskatoon, and 2 BBSRC) and 3 SNP/indel markers. Between 3 and 7 common markers were mapped onto each of the linkage groups A1 to A10. The position and order of most common markers revealed a high level of colinearity between species, although two small regions on A4, A5, and A10 revealed apparent local inversions between them. These results indicate that the A genome of Brassica has retained a high degree of colinearity between species, despite each species having evolved independently after the integration of the A and C genomes in the amphidiploid state. Our results provide a genetic integration of the Brassica A genome between B. napus and B. rapa. As the analysis employed sequence-based molecular markers, the information will accelerate the exploitation of the B. rapa genome sequence for the improvement of oilseed rape.