High expression of ethylene response factor BcERF98 delays the flowering time of non-heading Chinese cabbage.

MAIN CONCLUSION: BcERF98 is induced by ethylene signaling and inhibits the expression of BcFT by interacting with BcNF-YA2 and BcEIP9, thereby inhibiting plant flowering. Several stresses trigger the accumulation of ethylene, which then transmits the signal to ethylene response factors (ERFs) to participate in the regulation of plant development to adapt to the environment. This study clarifies the function of BcERF98, a homolog of AtERF98, in the regulation of plant flowering time mediated by high concentrations of ethylene. Results indicate that BcERF98 is a nuclear and the cell membrane-localized transcription factor and highly responsive to ethylene signaling. BcERF98 inhibits the expression of BcFT by interacting with BcEIP9 and BcNF-YA2, which are related to flowering time regulation, thereby participating in ethylene-mediated plant late flowering regulation. The results have enriched the theoretical knowledge of flowering regulation in non-heading Chinese cabbage (NHCC), providing the scientific basis and gene reserves for cultivating new varieties of NHCC with different flowering times.

BcWRKY1 confers Botrytis cinerea susceptibility via inhibiting JA biosynthesis.

WRKYs play important roles in plant stress resistance. However, the role of WRKYs in non-heading Chinese cabbage (Brassica campestris ssp. chinensis) against Botrytis cinerea (B. cinerea) remains poorly understood. Herein, the expression of BcWRKY1 was induced by B. cinerea. Further, the role of BcWRKY1 in B. cinerea infection was identified. Silencing of BcWRKY1 in non-heading Chinese cabbage enhanced plant resistance to B. cinerea. After B. cinerea inoculation, BcWRKY1-silencing plants exhibited lower reactive oxygen species (ROS) content, higher jasmonic acid (JA) content, and the expression level of JA biosynthesis genes, BcOPR3, BcLOX3-1 and BcLOX3-2 were upregulated. Overexpression of BcWRKY1 in Arabidopsis exhibited a complementary phenotype. By directly targeting W-boxes in the promoter of BcLOX3-2, BcWRKY1 inhibited the transcription of this gene. In addition, 13 candidate interacting proteins of BcWRKY1 were identified by yeast two-hybrid (Y2H) screening, and the interaction between BcWRKY1 and BcCaM6 weakened the inhibition of BcLOX3-2. In summary, our findings suggest that BcWRKY1 interacts with BcCaM6 to negatively regulate disease resistance.

BcNAC056 Interacts with BcWRKY1 to Regulate Leaf Senescence in Pak Choi.

Senescence is the final stage of leaf development. For leafy vegetables such as pak choi, leaf senescence is adverse to yield due to the harvest period shortening. However, the regulatory mechanisms of leaf senescence are largely unknown in leafy vegetables. Here, we isolated and characterized a NAC gene, BcNAC056, in pak choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis cv. 49caixin]. BcNAC056-GFP was located in the nucleus at the subcellular level, and BcNAC056 was responsive to leaf senescence and different hormones at the transcriptional level. Heterologous overexpression of BcNAC056 in Arabidopsis promoted leaf senescence, accompanied by the increased expression of senescence-associated genes (SAGs), whereas virus-induced gene silencing-based silencing in pak choi delayed leaf senescence. The following transcriptome analysis showed that heterologous overexpression of BcNAC056 enhanced some AtSAG transcripts in Arabidopsis. Electrophoretic mobility shift assay (EMSA) and dual-luciferase (LUC) reporter assay revealed that BcNAC056 activated SAG12 by directly binding to the promoter. In addition, with the LUC reporter and transient overexpression assays, we proposed that BcNAC056-BcWRKY1 interaction promoted the activation of BcSAG12. Taken together, our findings revealed a new regulatory mechanism of leaf senescence in pak choi.

Transcriptome sequencing and gas chromatography-mass spectrometry analyses provide insights into ß-caryophyllene biosynthesis in Brassica campestris.

Sesquiterpenes are important defensive secondary metabolites and aroma components. However, limited information is available on the mechanism of sesquiterpene formation and composition in the non-heading Chinese cabbage (NHCC) leaf. Therefore, headspace solid-phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS) combined with transcriptome analysis was used to study the mechanism of volatile organic compound formation. A total of 26 volatile organic compounds were identified in two NHCC cultivars 'SZQ' and 'XQC' and their F1 hybrids. Among these, sesquiterpene ß-caryophyllene was identified only in 'XQC' and F1. Five genes encoding caryophyllene synthase were identified. The candidate ß-caryophyllene synthase genes BcTPSa11 and BcTPSa21 had high expression levels only in 'XQC' and F1. In addition, several transcription factors of MYB-related, MYB, bHLH, and AP2/ERF families were identified by co-expression, suggesting that they regulate ß-caryophyllene biosynthesis. Our results provide a molecular basis for sesquiterpene biosynthesis as well as insights into the regulatory network of ß-caryophyllene in NHCC.

BcOPR3 Mediates Defense Responses to Biotrophic and Necrotrophic Pathogens in Arabidopsis and Non-heading Chinese Cabbage.

In plants, the salicylic acid (SA) and jasmonic acid (JA) signaling pathways usually mediate the defense response to biotrophic and necrotrophic pathogens, respectively. Our previous work showed that after non-heading Chinese cabbage (NHCC) was infected with the biotrophic pathogen Hyaloperonospora parasitica, expression of the JA biosynthetic gene BcOPR3 is induced; however, its molecular mechanism remains unclear. Here, we overexpressed BcOPR3 in Arabidopsis and silenced BcOPR3 in NHCC001 plants to study the defensive role of BcOPR3 in plants against pathogen invasion. The results showed that overexpression of BcOPR3 increased the susceptibility of Arabidopsis to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) but enhanced its resistance to Botrytis cinerea. BcOPR3-silenced NHCC001 plants with a 50% reduction in BcOPR3 expression increased their resistance to downy mildew by reducing the hyphal density and spores of H. parasitica. In addition, BcOPR3-partly silenced NHCC001 plants were also resistant to B. cinerea, which could be the result of a synergistic effect of JA and SA. These findings indicate a complicated role of BcOPR3 in the mediating defense responses to biotrophic and necrotrophic pathogens.

BcWRKY1 confers salt sensitivity via inhibiting Reactive oxygen species scavenging.

WRKY transcription factors play important roles in abiotic stress by directly regulating stress-related genes. However, the molecular mechanism of its involvement in salt stress in pak-choi is still poorly understood. In this study, we elucidated the function of BcWRKY1 from pak-choi (Brassica rapa ssp. chinensis) in salt stress. The expression level of BcWRKY1 showed the highest in rosette leaves among different tissues and was induced by salt and ABA treatment in pak-choi. Subcellular localization showed that BcWRKY1 was located in nucleus. The transgenic Arabidopsis overexpressing BcWRKY1 exhibited enhanced salt sensitivity and higher H2O2 contents, which were further confirmed by silencing BcWRKY1 in pak-choi. In addition, the expression of ZAT12 was negatively regulated with BcWRKY1 under salt stress both in pak-choi and Arabidopsis. Yeast one-hybrid and dual luciferase reporter assay showed that BcWRKY1 could bind to the promoter of BcZAT12, and BcsAPX expression was activated by BcZAT12. To sum up, we propose a BcWRKY1-BcZAT12-BcsAPX regulatory model that involves in pak-choi salt stress response.

Identification of IgE and IgG epitopes on native Bos d 4 allergen specific to allergic children.

Alpha-lactalbumin (ALA) is one of the major allergens in cow's milk. However, research on its conformational epitopes has been relatively limited. In our study, specific antibodies against cow's milk ALA were purified from eight children by two-step affinity chromatography. Subsequently, mimotopes against IgG and IgE were biopanned from Ph.D.-12 and Ph.D.-C7C, respectively. Based on the mimotopes, linear epitopes were defined with the UniProt alignment tool. Conformational epitopes were computed using the Pepitope Server. Six IgE and seven IgG linear epitopes were identified. Meanwhile, five IgE and three IgG conformational epitopes were revealed with PyMOL. The results showed that common residues were identified in both IgE and IgG epitopes and some residues of the conformational epitopes were composed of linear epitopes on bovine α-lactalbumin. The results indicated that the data could be used for developing hypoallergenic dairy products on the basis of epitopes and providing a diagnostic tool for the assessment of patients who are allergic to cow's milk.

Identification and characterization of the antigenic site (epitope) on bovine ß-lactoglobulin: common residues in linear and conformational epitopes.

BACKGROUND: ß-Lactoglobulin is recognised as one of major allergens in milk and its epitopes include linear and conformational epitopes contributed to milk allergy. RESULTS: In our work, two types of epitopes have been identified. Linear epitopes identified by using SPOT™ peptide arrays approach and three common peptide sequences AA77-82 (KIPAVF), AA126-131 (PEVDNE) and AA142-147 (ALPMHI) were obtained by reacting with specific sera from two rabbits. At the same time, mimotopes were screened by the panning of a phage display peptide library and the corresponding conformational epitopes were calculated by the web tool of Peptiope server with Mapitope algorithm. Three conformational epitopes against two specific sera were identified, in which there were 15 common residues as well and located in the different position and appeared mainly as an α-helix. CONCLUSION: Common residues on the linear and conformational epitopes were identified in the first time, respectively, which could be regarded as informative epitopes for detection of allergen in dairy products.