Characterization of FLOWERING LOCUS C 5 in Brassica rapa L.

Brassica rapa L., which includes Chinese cabbage, turnip, and pak choi, has more complex flowering time regulation than does Arabidopsis thaliana due to the presence of multiple paralogous flowering time genes. FLOWERING LOCUS C (FLC) is one of the key genes regulating the flowering time, and B. rapa has four FLC paralogs. BrFLC5 on the reference genome is deemed a pseudogene because of a mutation (from G to A) in the splice site of the third intron, but there are some accessions with a G nucleotide in the splice site. In this study, we genotyped 310 B. rapa accessions and found that 19 had homozygous and 81 had heterozygous putative functional BrFLC5 alleles. Accessions of turnip showed the highest proportion with a functional BrFLC5 allele. BrFLC5 acts as a floral repressor when overexpressed in A. thaliana. The BrFLC5 expression level varied in pre-vernalized plants, and this transcriptional variation was not associated with the G/A polymorphism in the third intron. Three accessions having a higher BrFLC5 expression in pre-vernalized plants had a 584-bp insertion in the promoter region. Many regions homologous to this 584-bp sequence are present in the B. rapa genome, and this 584-bp inserted region has tandem duplications of an AT-rich sequence in its central region. The possibility that a high expression of a functional BrFLC5 could contribute to producing premature bolting-resistant lines in B. rapa vegetables is discussed. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01405-0.

Large insertion in radish GRS1 enhances glucoraphanin content in intergeneric hybrids, Raphanobrassica (Raphanus sativus L. x Brassica oleracea var. acephala).

Glucosinolates (GSLs), precursors of isothiocyanates (ITCs), are present in Brassicaceae plants have been found to have health benefits. Sulforaphane (4-(methylsulfinyl)butyl ITC) is an ITC stored in the form of 4-(methylsulfinyl)butyl GSL (glucoraphanin, 4MSOB) in Brassica vegetables, such as broccoli and kale. Sulforaphane activates Nrf2 expression, a transcription factor responsible for inducing physiological activities such as detoxification in the human body, and it represents a functional component unique to cruciferous vegetables. Raphanobrassica is an inter-generic hybrid between radish and kale, and it contains a high amount of 4MSOB. However, Raphanobrassica contains as much 4-methylsulfinyl-3-butenyl GSL (glucoraphenin, 4MSO3B) as it does 4MSOB. GLUCORAPHASATIN SYNTHASE 1 (GRS1) is an enzyme present in radish that synthesizes 4-methylthio-3-butenyl GSL (glucoraphasatin, 4MT3B), a precursor of 4MSO3B, using 4-(methylthio)butyl GSL (glucoerucin, 4MTB) as a substrate. Since the precursor of 4MSOB is also 4MTB, it was considered that both 4MSOB and 4MSO3B accumulate owing to competition in Raphanobrassica. We hypothesized that owing to the impaired function of GRS1 in Raphanobrassica, it may be possible to breed Raphanobrassica cultivars containing a high 4MSOB content. In this study, we generated Raphanobrassica populations with functional and defective GRS1 and compared the GSL composition in the two populations using high-performance liquid chromatography. The mean 4MSOB content in leaves of the defective-type populations was higher than that in the functional-type population, and the defective/functional ratio ranged from 2.02 to 2.51-fold, supporting this hypothesis. Furthermore, leaves, flower buds, stems, and roots contained higher amounts of 4MSOB in the defective population than in the functional population. The leaf 4MSOB content of defective Raphanobrassica grown in this study was comparable to that of previously studied vegetables (such as broccoli sprouts) with high 4MSOB content. Raphanobrassica with defective GRS1 represents a new leafy vegetable with high 4MSOB content which exhibits anti-cancerous and anti-inflammatory potentials.

Genome Triplication Leads to Transcriptional Divergence of FLOWERING LOCUS C Genes During Vernalization in the Genus Brassica.

The genus Brassica includes oil crops, vegetables, condiments, fodder crops, and ornamental plants. Brassica species underwent a whole genome triplication event after speciation between ancestral species of Brassica and closely related genera including Arabidopsis thaliana. Diploid species such as Brassica rapa and Brassica oleracea have three copies of genes orthologous to each A. thaliana gene, although deletion in one or two of the three homologs has occurred in some genes. The floral transition is one of the crucial events in a plant's life history, and time of flowering is an important agricultural trait. There is a variation in flowering time within species of the genus Brassica, and this variation is largely dependent on a difference in vernalization requirements. In Brassica, like in A. thaliana, the key gene of vernalization is FLOWERING LOCUS C (FLC). In Brassica species, the vernalization response including the repression of FLC expression by cold treatment and the enrichment of the repressive histone modification tri-methylated histone H3 lysine 27 (H3K27me3) at the FLC locus is similar to A. thaliana. B. rapa and B. oleracea each have four paralogs of FLC, and the allotetraploid species, Brassica napus, has nine paralogs. The increased number of paralogs makes the role of FLC in vernalization more complicated; in a single plant, paralogs vary in the expression level of FLC before and after vernalization. There is also variation in FLC expression levels between accessions. In this review, we focus on the regulatory circuits of the vernalization response of FLC expression in the genus Brassica.

The histone modification H3 lysine 27 tri-methylation has conserved gene regulatory roles in the triplicated genome of Brassica rapa L.

Brassica rapa L. is an important vegetable and oilseed crop. We investigated the distribution of the histone mark tri-methylation of H3K27 (H3K27me3) in B. rapa and its role in the control of gene expression at two stages of development (2-day cotyledons and 14-day leaves) and among paralogs in the triplicated genome. H3K27me3 has a similar distribution in two inbred lines, while there was variation of H3K27me3 sites between tissues. Sites that are specific to 2-day cotyledons have increased transcriptional activity, and low levels of H3K27me3 in the gene body region. In 14-day leaves, levels of H3K27me3 were associated with decreased gene expression. In the triplicated genome, H3K27me3 is associated with paralogs that have tissue-specific expression. Even though B. rapa and Arabidopsis thaliana are not closely related within the Brassicaceae, there is conservation of H3K27me3-marked sites in the two species. Both B. rapa and A. thaliana require vernalization for floral initiation with FLC being the major controlling locus. In all four BrFLC paralogs, low-temperature treatment increases H3K27me3 at the proximal nucleation site reducing BrFLC expression. Following return to normal temperature growth conditions, H3K27me3 spreads along all four BrFLC paralogs providing stable repression of the gene.

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.

Long noncoding RNAs in Brassica rapa L. following vernalization.

Brassica rapa L. is an important agricultural crop that requires a period of prolonged cold for flowering. This process is known as vernalization. Studies have shown that long noncoding RNAs (lncRNAs) play important roles in abiotic stress responses and several cold-responsive noncoding RNAs have been suggested to be involved in vernalization. We examined the transcriptome of the Chinese cabbage inbred line (B. rapa L. var. pekinensis) RJKB-T24, and identified 1,444 long intergenic noncoding RNAs (lincRNAs), 551 natural antisense transcripts (NATs), and 93 intronic noncoding RNAs (incRNAs); 549 of the 2,088 lncRNAs significantly altered their expression in response to four weeks of cold treatment. Most differentially expressed lncRNAs did not lead to a change of expression levels in mRNAs covering or near lncRNAs, suggesting that the transcriptional responses to four weeks of cold treatment in lncRNA and mRNA are independent. However, some differentially expressed mRNAs had NATs with expression altered in the same direction. These genes were categorized as having an abiotic stress response, suggesting that the paired-expression may play a role in the transcriptional response to vernalization or cold treatment. We also identified short-term cold treatment induced NATs in BrFLC and BrMAF genes, which are involved in vernalization. The lncRNAs we identified differed from those reported in Arabidopsis thaliana, suggesting the role of lncRNAs in vernalization differ between these two species.

The production and characterization of a BoFLC2 introgressed Brassica rapa by repeated backcrossing to an F1.

Flowering time is an important agronomic trait for Brassica rapa crops, and previous breeding work in Brassica has successfully transmitted other important agronomic traits from donor species. However, there has been no previous attempts to produce hybrids replacing the original Brassica FLC alleles with alien FLC alleles. In this paper, we introduce the creation of a chromosome substitution line (CSSL) containing a homozygous introgression of Flowering Locus C from Brassica oleracea (BoFLC2) into a B. rapa genomic background, and characterize the CSSL line with respect to the parental cultivars. The preferential transmission of alien chromosome inheritance and the pattern of transmission observed during the production of the CSSLs are also discussed.

Genome re-sequencing, SNP analysis, and genetic mapping of the parental lines of a commercial F1 hybrid cultivar of Chinese cabbage.

The genome-wide characterization of single nucleotide polymorphism (SNP) between cultivars or between inbred lines contributes to the creation of genetic markers that are important for plant breeding. Functional markers derived from polymorphisms within genes that affect phenotypic variation are especially valuable in plant breeding. Here, we report on the genome re-sequencing and analysis of the two parental inbred lines of the commercial F1 hybrid Chinese cabbage cultivar "W77". Through the genome-wide identification and classification of the SNPs and indels present in each parental line, we identified about 1,500 putative non-functional genes in each parent. We designed cleaved amplified polymorphic sequence (CAPS) markers using specific mutations found at Eco RI restriction sites in the parental lines and confirmed their Mendelian segregation by constructing a linkage map using 96 F2 plants derived from the F1 hybrid cultivar, "W77". Our results and data will be a useful genomic resource for future studies of gene function and metagenomic studies in Chinese cabbage.

Epigenetic regulation of agronomical traits in Brassicaceae.

Epigenetic regulation, covalent modification of DNA and changes in histone proteins are closely linked to plant development and stress response through flexibly altering the chromatin structure to regulate gene expression. In this review, we will illustrate the importance of epigenetic influences by discussing three agriculturally important traits of Brassicaceae. (1) Vernalization, an acceleration of flowering by prolonged cold exposure regulated through epigenetic silencing of a central floral repressor, FLOWERING LOCUS C. This is associated with cold-dependent repressive histone mark accumulation, which confers competency of consequence vegetative-to-reproductive phase transition. (2) Hybrid vigor, in which an F1 hybrid shows superior performance to the parental lines. Combination of distinct epigenomes with different DNA methylation states between parental lines is important for increase in growth rate in a hybrid progeny. This is independent of siRNA-directed DNA methylation but dependent on the chromatin remodeler DDM1. (3) Self-incompatibility, a reproductive mating system to prevent self-fertilization. This is controlled by the S-locus consisting of SP11 and SRK which are responsible for self/non-self recognition. Because self-incompatibility in Brassicaceae is sporophytically controlled, there are dominance relationships between S haplotypes in the stigma and pollen. The dominance relationships in the pollen rely on de novo DNA methylation at the promoter region of a recessive allele, which is triggered by siRNA production from a flanking region of a dominant allele.

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.

Development of primer sets that can verify the enrichment of histone modifications, and their application to examining vernalization-mediated chromatin changes in Brassica rapa L.

Epigenetic regulation is crucial for the development of plants and for adaptation to a changing environment. Recently, genome-wide profiles of histone modifications have been determined by a combination of chromatin immunoprecipitation (ChIP) and genomic tiling arrays (ChIP on chip) or ChIP and high-throughput sequencing (ChIP-seq) in species including Arabidopsis thaliana, rice and maize. Validation of ChIP analysis by PCR or qPCR using positive and negative regions of histone modification is necessary. In contrast, information about histone modifications is limited in Chinese cabbage, Brassica rapa. The aim of this study was to develop positive and negative control primer sets for H3K4me3 (trimethylation of the 4(th) lysine of H3), H3K9me2, H3K27me3 and H3K36me3 in B. rapa. The expression and histone modification of four FLC paralogs in B. rapa, before and after vernalization, were examined using the method developed here. After vernalization, expression of all four BrFLC genes was reduced, and accumulation of H3K27me3 was observed in three of them. As with A. thaliana, the vernalization response and stability of FLC repression correlated with the accumulation of H3K27me3. These results suggest that the epigenetic state during vernalization is important for high bolting resistance in B. rapa. The positive and negative control primer sets developed here revealed positive and negative histone modifications in B. rapa that can be used as a control for future studies.

Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice.

Cytoplasmic male sterility (CMS) is an important agricultural trait characterized by lack of functional pollen, and caused by ectopic and defective mitochondrial gene expression. The pollen function in CMS plants is restored by the presence of nuclear-encoded restorer of fertility (Rf) genes. Previously, we cloned Rf2, which restores the fertility of Lead Rice (LD)-type CMS rice. However, neither the function of Rf2 nor the identity of the mitochondrial gene causing CMS has been determined in LD-CMS rice. Here, we show that the mitochondrial gene orf79 acts as a CMS-associated gene in LD-CMS rice, similar to its role in BT-CMS rice originating from Chinsurah Boro II, and Rf2 weakly restores fertility in BT-CMS rice. We also show that RF2 promotes degradation of atp6-orf79 RNA in a different manner from that of RF1, which is the Rf gene product in BT-CMS rice. The amount of ORF79 protein in LD-CMS rice was one-twentieth of the amount in BT-CMS rice. The difference in ORF79 protein levels probably accounts for the mild and severe pollen defects in LD-CMS and BT-CMS rice, respectively. In the presence of Rf2, accumulation of ORF79 was reduced to almost zero and 25% in LD-CMS and BT-CMS rice, respectively, which probably accounts for the complete and weak fertility restoration abilities of Rf2 in LD-CMS and BT-CMS rice, respectively. These observations indicate that the amount of ORF79 influences the pollen fertility in two strains of rice in which CMS is induced by orf79.

The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes a mitochondrial glycine-rich protein.

Cytoplasmic male sterility (CMS) is associated with a mitochondrial mutation that causes an inability to produce fertile pollen. The fertility of CMS plants is restored in the presence of a nuclear-encoded fertility restorer (Rf) gene. In Lead Rice-type CMS, discovered in the indica variety 'Lead Rice', fertility of the CMS plant is restored by the single nuclear-encoded gene Rf2 in a gametophytic manner. We performed map-based cloning of Rf2, and proved that it encodes a protein consisting of 152 amino acids with a glycine-rich domain. Expression of Rf2 mRNA was detected in developing and mature anthers. An RF2-GFP fusion was shown to be targeted to mitochondria. Replacement of isoleucine by threonine at amino acid 78 of the RF2 protein was considered to be the cause of functional loss in the rf2 allele. As Rf2 does not encode a pentatricopeptide repeat protein, unlike a majority of previously identified Rf genes, the data from this study provide new insights into the mechanism for restoring fertility in CMS.

Cytoplasmic-nuclear genomic barriers in rice pollen development revealed by comparison of global gene expression profiles among five independent cytoplasmic male sterile lines.

Cytoplasmic male sterility (CMS) is one of the most ideal phenomena known in higher plants to describe the incompatibilities between mitochondrial-nuclear genomic interactions. To elucidate the dependency of pollen development on mitochondrial genotypes and cytoplasmic-nuclear genomic barriers, we employed five CMS isogenic lines of rice, CW-, W11-, LD-, BT- and WA-type CMS lines, that exhibit distinct pollen-defective phenotypes, and we characterized the CMS phenotypes and the nuclear gene expression patterns in conjunction with their mitochondrial genomic structures. These five CMS lines carried independent mitotypes, and W11, LD and BT mitochondrial genomes were relatively close with respect to their phylogeny. In anthers at the uninucleate microspore and bicellular pollen stages, 8,199 genes significantly changed their expression in at least one of the CMS lines. Common expression patterns were observed in BT, LD and W11 after k-means clustering. Among the genes encoding putative mitochondrial proteins, ALTERNATIVE OXIDASE 1A, a gene for the well-known mitochondrial stress marker, was included in the group ectopically up-regulated in anthers at the bicellular pollen stage of BT, LD and W11. Several other clusters were also regulated in a cytoplasm-specific manner during pollen development. These clear similarities in gene regulatory networks of BT-, LD- and W11-CMS lines indicate that the phylogenetic relationships of the mitochondrial genotypes are strongly correlated with nuclear gene expression patterns and pollen abortion phenotypes, providing evidence of the mitochondrial epistacy over the nuclear genome during pollen development.

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm.

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of 'Lead Rice' and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6-orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of 'Chinsurah Boro II'. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6-orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.