The transcriptional response to salicylic acid plays a role in Fusarium yellows resistance in Brassica rapa L.

KEY MESSAGE: Fusarium yellows resistant and susceptible lines in Brassica rapa showed different salicylic acid responses; the resistant line showed a similar response to previous reports, but the susceptible line differed. Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans (Foc) is an important disease. Previous studies showed that genes related to salicylic acid (SA) response were more highly induced following Foc infection in Brassica rapa Fusarium yellows resistant lines than susceptible lines. However, SA-induced genes have not been identified at the whole genome level and it was unclear whether they were up-regulated by Foc inoculation. Transcriptome analysis with and without SA treatment in the B. rapa Fusarium yellows susceptible line 'Misugi' and the resistant line 'Nanane' was performed to obtain insights into the relationship between SA sensitivity/response and Fusarium yellows resistance. 'Nanane's up-regulated genes were related to SA response and down-regulated genes were related to jasmonic acid (JA) or ethylene (ET) response, but differentially expressed genes in 'Misugi' were not. This result suggests that Fusarium yellows resistant and susceptible lines have a different SA response and that an antagonistic transcription between SA and JA/ET responses was found only in a Fusarium yellows resistant line. SA-responsive genes were induced by Foc inoculation in Fusarium yellows resistant (RJKB-T23) and susceptible lines (RJKB-T24). By contrast, 39 SA-induced genes specific to RJKB-T23 might function in the defense response to Foc. In this study, SA-induced genes were identified at the whole genome level, and the possibility, the defense response to Foc observed in a resistant line could be mediated by SA-induced genes, is suggested. These results will be useful for future research concerning the SA importance in Foc or other diseases resistance in B. rapa.

The role of FRIGIDA and FLOWERING LOCUS C genes in flowering time of Brassica rapa leafy vegetables.

There is a wide variation of flowering time among lines of Brassica rapa L. Most B. rapa leafy (Chinese cabbage etc.) or root (turnip) vegetables require prolonged cold exposure for flowering, known as vernalization. Premature bolting caused by low temperature leads to a reduction in the yield/quality of these B. rapa vegetables. Therefore, high bolting resistance is an important breeding trait, and understanding the molecular mechanism of vernalization is necessary to achieve this goal. In this study, we demonstrated that BrFRIb functions as an activator of BrFLC in B. rapa. We showed a positive correlation between the steady state expression levels of the sum of the BrFLC paralogs and the days to flowering after four weeks of cold treatment, suggesting that this is an indicator of the vernalization requirement. We indicate that BrFLCs are repressed by the accumulation of H3K27me3 and that the spreading of H3K27me3 promotes stable FLC repression. However, there was no clear relationship between the level of H3K27me3 in the BrFLC and the vernalization requirement. We also showed that if there was a high vernalization requirement, the rate of repression of BrFLC1 expression following prolonged cold treatments was lower.

Genome-wide characterization of DNA methylation, small RNA expression, and histone H3 lysine nine di-methylation in Brassica rapa L.

Epigenetic gene regulation is crucial to plant life and can involve dynamic interactions between various histone modifications, DNA methylation, and small RNAs. Detailed analysis of epigenome information is anticipated to reveal how the DNA sequence of the genome is translated into the plant's phenotype. The aim of this study was to map the DNA methylation state at the whole genome level and to clarify the relationship between DNA methylation and transcription, small RNA expression, and histone H3 lysine 9 di-methylation (H3K9me2) in Brassica rapa. We performed whole genome bisulfite sequencing, small RNA sequencing, and chromatin immunoprecipitation sequencing using H3K9me2 antibody in a Chinese cabbage inbred line, RJKB-T24, and examined the impact of epigenetic states on transcription. Cytosine methylation in DNA was analysed in different sequence contexts (CG, CHG, and CHH) (where H could be A, C, or T) and position (promoter, exon, intron, terminator, interspersed repeat regions), and the H3K9me2 and 24 nucleotide small interfering RNAs (24 nt-siRNA) were overlaid onto the B. rapa reference genome. The epigenome was compared with that of Arabidopsis thaliana and the relationship between the position of DNA methylation and gene expression, and the involvement of 24 nt siRNAs and H3K9me2 are discussed.

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.

Role of DNA methylation in hybrid vigor in Arabidopsis thaliana.

Hybrid vigor or heterosis refers to the superior performance of F1 hybrid plants over their parents. Heterosis is particularly important in the production systems of major crops. Recent studies have suggested that epigenetic regulation such as DNA methylation is involved in heterosis, but the molecular mechanism of heterosis is still unclear. To address the epigenetic contribution to heterosis in Arabidopsis thaliana, we used mutant genes that have roles in DNA methylation. Hybrids between C24 and Columbia-0 (Col) without RNA polymerase IV (Pol IV) or methyltransferase I (MET1) function did not reduce the level of biomass heterosis (as evaluated by rosette diameter). Hybrids with a mutation in decrease in dna methylation 1 (ddm1) showed a decreased heterosis level. Vegetative heterosis in the ddm1 mutant hybrid was reduced but not eliminated; a complete reduction could result if there was a change in methylation at all loci critical for generating the level of heterosis, whereas if only a proportion of the loci have methylation changes there may only be a partial reduction in heterosis.

Related polymorphic F-box protein genes between haplotypes clustering in the BAC contig sequences around the S-RNase of Japanese pear.

Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB(-α-γ)) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S(4)-RNase and 378 kb around S(2)-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB(4-u1-u4, 4-d1-d2) and PpSFBB(2-u1-u5,) (2-d1-d5), respectively) were found, but PpSFBB(4-α-γ) and PpSFBB(2-γ) were absent. The PpSFBB(4) genes showed 66.2-93.1% amino acid identity with the PpSFBB(2) genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3-94.9% identity, while group II consisted of gene pairs with higher identities (>92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.

Deletion of a 236 kb region around S 4-RNase in a stylar-part mutant S 4sm-haplotype of Japanese pear.

Japanese pear (Pyrus pyrifolia Nakai) has a gametophytic self-incompatibility (GSI) mechanism controlled by a single S-locus with multiple S-haplotypes, each of which contains separate genes that determine the allelic identity of pistil and pollen. The pistil S gene is the S-ribonuclease (S-RNase) gene, whereas good candidates for the pollen S gene are the F-box protein genes. A self-compatible (SC) cultivar, 'Osa-Nijisseiki', which is a bud mutant of 'Nijisseiki' (S (2) S (4)), has a stylar-part mutant S(4)sm-haplotype, which lacks the S (4)-RNase gene but retains the pollen S gene. To delineate the deletion breakpoint in the S(4)sm-haplotype, we constructed a bacterial artificial chromosome (BAC) library from an S (4)-homozygote, and assembled a BAC contig of 570 kb around the S (4)-RNase. Genomic PCR of DNA from S (4)- and S(4)sm-homozygotes and the DNA sequence of the BAC contig allowed the identification of a deletion of 236 kb spanning from 48 kb upstream to 188 kb downstream of S (4)-RNase. The S(4)sm-haplotype lacks 34 predicted open reading frames (ORFs) including the S (4)-RNase and a pollen-specific F-box protein gene (termed as S (4) F-box0). Genomic PCR with a primer pair designed from the deletion junctions yielded a product specific for the S(4)sm-haplotype. The product could be useful as a maker for early selection of SC cultivars harboring the S(4)sm-haplotype.