Electrostatic potentials of the S-locus F-box proteins contribute to the pollen S specificity in self-incompatibility in Petunia hybrida.

Self-incompatibility (SI) is a self/non-self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S-locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S-locus encodes a single S-RNase and a cluster of S-locus F-box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of 'like charges repel and unlike charges attract' between SLFs and S-RNases in Petunia hybrida. Strikingly, the alteration of a single C-terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S-RNases, providing a mechanistic insight into the self/non-self discrimination between cytosolic proteins in angiosperms.

Genome-wide transcription analyses in rice using tiling microarrays.

Sequencing and computational annotation revealed several features, including high gene numbers, unusual composition of the predicted genes and a large number of genes lacking homology to known genes, that distinguish the rice (Oryza sativa) genome from that of other fully sequenced model species. We report here a full-genome transcription analysis of the indica rice subspecies using high-density oligonucleotide tiling microarrays. Our results provided expression data support for the existence of 35,970 (81.9%) annotated gene models and identified 5,464 unique transcribed intergenic regions that share similar compositional properties with the annotated exons and have significant homology to other plant proteins. Elucidating and mapping of all transcribed regions revealed an association between global transcription and cytological chromosome features, and an overall similarity of transcriptional activity between duplicated segments of the genome. Collectively, our results provide the first whole-genome transcription map useful for further understanding the rice genome.

Cytological characterization of the tandem repetitive sequences and their methylation status in the Antirrhinum majus genome.

Tandem repetitive sequences are DNA motifs common in the genomes of eukaryotic species and are often embedded in heterochromatic regions. In most eukaryotes, ribosomal genes, as well as centromeres and telomeres or subtelomeres, are associated with abundant tandem arrays of repetitive sequences and typically represent the final barriers to completion of whole-genome sequencing. The nature of these repeats makes it difficult to estimate their actual sizes. In this study, combining the two cytological techniques DNA fiber-FISH and pachytene chromosome FISH allowed us to characterize the tandem repeats distributed genome wide in Antirrhinum majus and identify four types of tandem repeats, 45S rDNA, 5S rDNA, CentA1, and CentA2, representing the major tandem repetitive components, which were estimated to have a total length of 18.50 Mb and account for 3.59% of the A. majus genome. FISH examination revealed that all the tandem repeats correspond to heterochromatic knobs along the pachytene chromosomes. Moreover, the methylation status of the tandem repeats was investigated in both somatic cells and pollen mother cells from anther tissues using an antibody against 5-methylcytosine combined with sequential FISH analyses. Our results showed that these repeats were hypomethylated in anther tissues, especially in the pollen mother cells at pachytene stage.

Genome structure and evolution of Antirrhinum majus L.

Snapdragon (Antirrhinum majus L.), a member of the Plantaginaceae family, is an important model for plant genetics and molecular studies on plant growth and development, transposon biology and self-incompatibility. Here we report a near-complete genome assembly of A. majus cultivar JI7 (A. majus cv.JI7) comprising 510 Megabases (Mb) of genomic sequence and containing 37,714 annotated protein-coding genes. Scaffolds covering 97.12% of the assembled genome were anchored on eight chromosomes. Comparative and evolutionary analyses revealed that a whole-genome duplication event occurred in the Plantaginaceae around 46-49 million years ago (Ma). We also uncovered the genetic architectures associated with complex traits such as flower asymmetry and self-incompatibility, identifying a unique duplication of TCP family genes dated to around 46-49 Ma and reconstructing a near-complete ψS-locus of roughly 2 Mb. The genome sequence obtained in this study not only provides a representative genome sequenced from the Plantaginaceae but also brings the popular plant model system of Antirrhinum into the genomic age.

BUD2, encoding an S-adenosylmethionine decarboxylase, is required for Arabidopsis growth and development.

Polyamines are implicated in regulating various developmental processes in plants, but their exact roles and how they govern these processes still remain elusive. We report here an Arabidopsis bushy and dwarf mutant, bud2, which results from the complete deletion of one member of the small gene family that encodes S-adenosylmethionine decarboxylases (SAMDCs) necessary for the formation of the indispensable intermediate in the polyamine biosynthetic pathway. The bud2 plant has enlarged vascular systems in inflorescences, roots, and petioles, and an altered homeostasis of polyamines. The double mutant of bud2 and samdc1, a knockdown mutant of another SAMDC member, is embryo lethal, demonstrating that SAMDCs are essential for plant embryogenesis. Our results suggest that polyamines are required for the normal growth and development of higher plants.

Molecular cytogenetic characterization of the Antirrhinum majus genome.

As a model system in classical plant genetics, the genus Antirrhinum has been well studied, especially in gametophytic self-incompatibility, flower development biology, and transposon-induced mutation. In contrast to the advances in genetic and molecular studies, little is known about Antirrhinum cytogenetics. In this study, we isolated two tandem repetitive sequences, CentA1 and CentA2, from the centromeric regions of Antirrhinum chromosomes. A standard karyotype has been established by anchoring these centromeric repeats on meiotic pachytene chromosome using FISH. An ideogram based on the DAPI-staining pattern of pachytene chromosomes was developed to depict the distribution of heterochromatin in the Antirrhinum majus genome. To integrate the genetic and chromosomal maps, we selected one or two molecular markers from each linkage group to screen an Antirrhinum transformation-competent artificial chromosome (TAC) library. These genetically anchored TAC clones were labeled as FISH probes to hybridize to pachytene chromosomes of A. majus. As a result, the relationship between chromosomes and the linkage groups (LGs) in Antirrhinum has been established.

Integration of cytological features with molecular and epigenetic properties of rice chromosome 4.

It has been reported that rice chromosome 4 has eight major heterochromatic knobs within the heterochromatic half and that this organization correlates with chromosomal-level transcriptional activity. To better understand this chromosomal organization, we created a model based on the statistical distribution of various types of gene models to divide chromosome 4 into 17 euchromatic and heterochromatic regions that correspond with the cytological staining. Fluorescence in-situ hybridization (FISH) experiments using a set of bacterial artificial chromosome (BAC) clones from chromosome 4 placed all 18 clones in the region predicted by the model. Elevated levels of H3K4 di- and tri-methylation detected by chromatin-immunoprecipitation (ChIP) on chip were correlated with euchromatic regions whereas lower levels of these two modifications were detected in heterochromatic regions. Small RNAs were more abundant in the heterochromatic regions. To validate these findings, H3K4 trimethylation, H3K9 acetylation, H4K12 acetylation, and H3K9 di- and tri-methylation of 19 individual genes were measured by ChIP-PCR. Genes in heterochromatic regions had elevated H3K9 di- and tri-methylation while genes in euchromatic regions had elevated levels of the other three modifications. We also assayed cytosine methylation of these genes using the restriction enzymes McrBC, HapII, and Msp I. This analysis indicated that cytosines of transposable elements and some genes located in heterochromatic regions were methylated while cytosines of the other genes were unmethylated. These results suggest that local transcriptional activity may reflect the organization of the corresponding part of the chromosome. They also indicate that epigenetic regulation plays an important role in correlating chromosomal organization with transcriptional activity.

A snapshot of the Chinese SOL Project.

In 2003, the International Solanaceae Project (SOL) was initiated by an international consortium of ten countries including Korea, China, the United Kingdom, India, the Netherlands, France, Japan, Spain, Italy and the United States. The first major effort of the SOL aimed to produce a DNA sequence map for euchromatin regions of 12 chromosomes of tomato (Solanum lycopersicum) before 2010. Here we present an update on Chinese effort for sequencing the euchromatin region of chromosome 3.

Heterochromatic and genetic features are consistent with recombination suppression of the self-incompatibility locus in Antirrhinum.

Self-incompatibility (SI) is a genetic mechanism to prevent self-fertilization that is found in many species of flowering plants. Molecular studies have demonstrated that the S-RNase and SLF/SFB genes encoded by the single polymorphic S locus, which control the pollen and pistil functions of SI in three distantly related families, the Solanaceae, Scrophulariaceae and Rosaceae, are organized in a haplotype-specific manner. Previous work suggested that the haplotype structure of the two genes is probably maintained by recombination suppression at the S locus. To examine features associated with this suppression, we first mapped the S locus of Antirrhinum hispanicum, a member of the Scrophulariaceae, to a highly heterochromatic region close to the distal end of the short arm of chromosome 8. Both leptotene chromosome and DNA fiber fluorescence in situ hybridization analyses showed an obvious haplotype specificity of the Antirrhinum S locus that is consistent with its haplotype structure. A chromosome inversion was also detected around this region between A. majus and A. hispanicum. These results revealed that DNA sequence polymorphism and a heterochromatic location are associated with the S locus. Possible roles of these features in maintenance of the haplotype specificity involved in both self and non-self recognition are discussed.

SDG714, a histone H3K9 methyltransferase, is involved in Tos17 DNA methylation and transposition in rice.

Although the role of H3K9 methylation in rice (Oryza sativa) is unclear, in Arabidopsis thaliana the loss of histone H3K9 methylation by mutation of Kryptonite [also known as SU(VAR)3-9 homolog] reduces genome-wide DNA methylation and increases the transcription of transposable elements. Here, we report that rice SDG714 (for SET Domain Group Protein714) encodes a histone H3K9-specific methyltransferase. The C terminus of SDG714 confers enzymatic activity and substrate specificity, whereas the N terminus localizes it in the nucleus. Loss-of-function mutants of SDG714 (SDG714IR transformants) generated by RNA interference display a mostly glabrous phenotype as a result of the lack of macro trichomes in glumes, leaves, and culms compared with control plants. These mutants also show decreased levels of CpG and CNG cytosine methylation as well as H3K9 methylation at the Tos17 locus, a copia-like retrotransposon widely used for the generation of rice mutants. Most interestingly, loss of function of SDG714 can enhance transcription and cause the transposition of Tos17. Together, these results suggest that histone H3K9 methylation mediated by SDG714 is involved in DNA methylation, the transposition of transposable elements, and genome stability in rice.

Structure, divergence, and distribution of the CRR centromeric retrotransposon family in rice.

The centromeric retrotransposon (CR) family in the grass species is one of few Ty3-gypsy groups of retroelements that preferentially transpose into highly specialized chromosomal domains. It has been demonstrated in both rice and maize that CRR (CR of rice) and CRM (CR of maize) elements are intermingled with centromeric satellite DNA and are highly concentrated within cytologically defined centromeres. We collected all of the CRR elements from rice chromosomes 1, 4, 8, and 10 that have been sequenced to high quality. Phylogenetic analysis revealed that the CRR elements are structurally diverged into four subfamilies, including two autonomous subfamilies (CRR1 and CRR2) and two nonautonomous subfamilies (noaCRR1 and noaCRR2). The CRR1/CRR2 elements contain all characteristic protein domains required for retrotransposition. In contrast, the noaCRR elements have different structures, containing only a gag or gag-pro domain or no open reading frames. The CRR and noaCRR elements share substantial sequence similarity in regions required for DNA replication and for recognition by integrase during retrotransposition. These data, coupled with the presence of young noaCRR elements in the rice genome and similar chromosomal distribution patterns between noaCRR1 and CRR1/CRR2 elements, suggest that the noaCRR elements were likely mobilized through the retrotransposition machinery from the autonomous CRR elements. Mechanisms of the targeting specificity of the CRR elements, as well as their role in centromere function, are discussed.

A tiling microarray expression analysis of rice chromosome 4 suggests a chromosome-level regulation of transcription.

The complete genome sequence of cultivated rice (Oryza sativa) provides an unprecedented opportunity to understand the biology of this model cereal. An essential and necessary step in this effort is the determination of the coding information and expression patterns of each sequenced chromosome. Here, we report an analysis of the transcriptional activity of rice chromosome 4 using a tiling path microarray based on PCR-generated genomic DNA fragments. Six representative rice organ types were examined using this microarray to catalog the transcribed regions of rice chromosome 4 and to reveal organ- and developmental stage-specific transcription patterns. This analysis provided expression support for 82% of the gene models in the chromosome. Transcriptional activities in 1643 nonannotated regions were also detected. Comparison with cytologically defined chromatin features indicated that in juvenile-stage rice the euchromatic region is more actively transcribed than is the transposon-rich heterochromatic portion of the chromosome. Interestingly, increased transcription of transposon-related gene models in certain heterochromatic regions was observed in mature-stage rice organs and in suspension-cultured cells. These results suggest a close correlation between transcriptional activity and chromosome organization and the developmental regulation of transcription activity at the chromosome level.