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.

Comparison of transcriptome profiles by Fusarium oxysporum inoculation between Fusarium yellows resistant and susceptible lines in Brassica rapa L.

KEY MESSAGE: Resistant and susceptible lines in Brassica rapa have different immune responses against Fusarium oxysporum inoculation. Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans (Foc) is an important disease of Brassicaceae; however, the mechanism of how host plants respond to Foc is still unknown. By comparing with and without Foc inoculation in both resistant and susceptible lines of Chinese cabbage (Brassica rapa var. pekinensis), we identified differentially expressed genes (DEGs) between the bulked inoculated (6, 12, 24, and 72 h after inoculation (HAI)) and non-inoculated samples. Most of the DEGs were up-regulated by Foc inoculation. Quantitative real-time RT-PCR showed that most up-regulated genes increased their expression levels from 24 HAI. An independent transcriptome analysis at 24 and 72 HAI was performed in resistant and susceptible lines. GO analysis using up-regulated genes at 24 HAI indicated that Foc inoculation activated systemic acquired resistance (SAR) in resistant lines and tryptophan biosynthetic process and responses to chitin and ethylene in susceptible lines. By contrast, GO analysis using up-regulated genes at 72 HAI showed the overrepresentation of some categories for the defense response in susceptible lines but not in the resistant lines. We also compared DEGs between B. rapa and Arabidopsis thaliana after F. oxysporum inoculation at the same time point, and identified genes related to defense response that were up-regulated in the resistant lines of Chinese cabbage and A. thaliana. Particular genes that changed expression levels overlapped between the two species, suggesting that they are candidates for genes involved in the resistance mechanisms against F. oxysporum.

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.

Capsicum ethanol extracts and capsaicin enhance interleukin-2 and interferon-gamma production in cultured murine Peyer's patch cells ex vivo.

We investigated the effects of red pepper (Capsicum annuum Lin.) extracts (capsicum extract) and its main pungent capsaicin on T helper 1 (Th1) and 2 (Th2) cytokine production in cultured murine Peyer's patch (PP) cells in vitro and ex vivo. Direct administration of capsicum extract (1 and 10 mug/ml) and capsaicin (3 and 30 muM) resulted in suppression of interleukin (IL)-2, interferon (IFN)-gamma, IL-4 and IL-5 production. In an ex vivo experiment using PP cells removed from the mice after oral administration of capsicum extract (10 mg/kg/day for 4 consecutive days), IL-2, IFN-gamma and IL-5 increased in response to concanavalin A (Con A). Oral administration of 3 mg/kg/day capsaicin, one active constituent of the extract, also enhanced IL-2, INF-gamma and IL-4 production in response to Con A stimulation but did not influence the production of IL-5. Orally administered capsazepine (3 mg/kg/day), a selective transient receptor potential vanilloid 1 (TRPV1) antagonist, slightly enhanced IL-2 production also irrespective of Con A stimulation. The capsaicin-induced enhancement of both IL-2 and IFN-gamma production was not reduced by oral administration of capsazepine (3 mg/kg/day), suggesting a TRPV1 receptor-independent mechanism. Flow cytometric analysis revealed that the population of CD3(+) cells in the PP cells was significantly reduced while CD19(+) cells increased after oral administration of capsicum extract (1 and 10 mg/kg/day) and capsaicin (0.3 and 3 mg/kg/day). Capsazepine (3 mg/kg/day) weakly but significantly reversed these effects. Orally administered capsicum extract and capsaicin did not change the T cell subset (CD4(+) and CD8(+)), Th1 (IFN-gamma(+)) and T2 (IL-4(+)) ratio. These findings indicate that capsicum extract and capsaicin modulate T cell-immune responses, and their immunomodulatory effects on murine PP cells are partly due to both TRPV1-dependent and -independent pathway.

The liquid culture filtrates of Paecilomyces tenuipes (Peck) Samson (=Isaria japonica Yasuda) and Paecilomyces cicadae (Miquel) Samson (=Isaria sinclairii (Berk.) Llond) regulate Th1 and Th2 cytokine response in murine Peyer's patch cells in vitro and ex vivo.

The effects of liquid culture filtrates of medicinal entomogenous fungi, Paecilomyces tenuipes (Peck) Samson (=Isaria japonica Yasuda or Isaria tenuipes) (PTCF) and Paecilomyces cicadae (Miquel) Samson (=Isaria sinclairii (Berk.) Llond) (PCCF), on cytokine productions in cultured Peyer's patches (PP) from C57BL/6J mice were investigated in vitro and ex vivo. In an in vitro experiment, PTCF (100 and 10 microg/ml) enhanced the production of T helper 1 (Th1) cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, in cultured PP cells stimulated with 5 microg/ml concanavalin A (Con A) but did not influence on the production of T helper 2 (Th2) cytokines, IL-4 and IL-5. PTCF also enhanced the production of granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-10 in the cultured PP cells. While, PCCF enhanced the production of IFN-gamma but did not alter the level of IL-2 in the PP cells. In an ex vivo experiment using PP cells removed from the mice after oral treatment of PTCF (10 and 100 mg/kg daily for 7 consecutive days), the production of IL-2 and IFN-gamma were increased in response to Con A. On the other hand, orally treated PCCF (10 mg/kg/day) suppressed IL-2 production but did not change the levels of IFN-gamma and IL-10 in the isolated PP cells. The flow cytometric analysis revealed that the population of CD3(+) cells in the PP cells slightly but significantly increased after oral administration of PCCF. Orally administered PTCF did not change the population of T (CD3(+)), B (CD19(+)), T cell subset (CD4(+)and CD8(+)) and Th1 (IFN-gamma(+)) and Th2 (IL-4(+)). From PTCF, the fraction rich in proteoglycans was separated as active fraction that stimulates Th1 immune response. These results indicate that the mode of action of PTCF and PCCF on mucosal immune response is different and this is contributed to their metabolites. Taken together, there is a possibility of PTCF and PCCF being therapeutic or preventive agents for immune diseases such as cancer, allergy and parasitic disease through activation of mucosal immune response.

Differential expression of three oxidosqualene cyclase mRNAs in Glycyrrhiza glabra.

The cultured cells and intact plants of Glycyrrhiza glabra (Fabaceae) produce betulinic acid and oleanane-type triterpene saponins (soyasaponins and glycyrrhizin). To elucidate the regulation of triterpenoid biosynthesis in G. glabra, the cDNA of lupeol synthase, an oxidosqualene cyclase (OSC) responsible for betulinic acid biosynthesis, was cloned, and expression patterns of lupeol synthase and two additional OSCs, beta-amyrin synthase and cycloartenol synthase, were compared. The mRNA expression levels of lupeol synthase and beta-amyrin synthase were consistent with the accumulation of betulinic acid and oleanane-type triterpene saponins, respectively. The transcript of lupeol synthase was highly expressed in the cultured cells and root nodules. The transcript of beta-amyrin synthase, an OSC responsible for oleanane-type triterpene biosynthesis, was highly expressed in the cultured cells, root nodules and germinating seeds, where soyasaponin accumulates, and in the thickened roots where glycyrrhizin accumulates. In the cultured cells, the addition of methyl jasmonate up-regulated beta-amyrin synthase mRNA and soyasaponin biosynthesis, but down-regulated lupeol synthase mRNA. Furthermore, the addition of gibberellin A(3) down-regulated beta-amyrin synthase mRNA but not lupeol synthase mRNA in the cultured cells. The mRNA levels of cycloartenol synthase, an additional OSC responsible for sterol biosynthesis, in the intact plant and cultured cells were relatively constant in these experiments.