ETHYLENE RESPONSE FACTOR 070 inhibits flowering in Pak-choi by indirectly impairing BcLEAFY expression.

APETALA2/ethylene responsive factors respond to ethylene and participate in many biological and physiological processes, such as plant morphogenesis, stress resistance, and hormone signal transduction. Ethylene responsive factor 070 (BcERF070) is important in flowering. However, the underlying molecular mechanisms of BcERF070 in floral transition in response to ethylene signaling have not been fully characterized. Herein, we explored the function of BcERF070 in Pak-choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis]. Ethylene treatment induced BcERF070 expression and delayed flowering in Pak-choi. Silencing of BcERF070 induced flowering in Pak-choi. BcERF070 interacted with major latex protein-like 328 (BcMLP328), which forms a complex with helix-loop-helix protein 30 (BcbHLH30) to enhance the transcriptional activity of BcbHLH30 on LEAFY (BcLFY), ultimately promoting flowering. However, BcERF070 impaired the BcMLP328-BcbHLH30 complex activation of LEAFY (BcLFY), ultimately inhibiting flowering in Pak-choi. BcERF070 directly promoted the expression of the flowering inhibitor gene B-box 29 (BcBBX29) and delayed flowering by reducing FLOWERING LOCUS T (BcFT) expression. These results suggest that BcERF070 mediates ethylene-reduced flowering by impairing the BcMLP328-BcbHLH30 complex activation of BcLFY and by directly promoting the gene expression of the flowering inhibition factor BcBBX29 to repress BcFT expression. The findings contribute to understanding the molecular mechanisms underlying floral transition in response to ethylene in plants.

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.

Comparative Transcriptome Analysis Reveals the Protective Role of Melatonin during Salt Stress by Regulating the Photosynthesis and Ascorbic Acid Metabolism Pathways in Brassica campestris.

Salinity stress is a type of abiotic stress which negatively affects the signaling pathways and cellular compartments of plants. Melatonin (MT) has been found to be a bioactive compound that can mitigate these adverse effects, which makes it necessary to understand the function of MT and its role in salt stress. During this study, plants were treated exogenously with 100 µM of MT for 7 days and subjected to 200 mM of salt stress, and samples were collected after 1 and 7 days for different indicators and transcriptome analysis. The results showed that salt reduced chlorophyll contents and damaged the chloroplast structure, which was confirmed by the downregulation of key genes involved in the photosynthesis pathway after transcriptome analysis and qRT-PCR confirmation. Meanwhile, MT increased the chlorophyll contents, reduced the electrolyte leakage, and protected the chloroplast structure during salt stress by upregulating several photosynthesis pathway genes. MT also decreased the H2O2 level and increased the ascorbic acid contents and APX activity by upregulating genes involved in the ascorbic acid pathway during salt stress, as confirmed by the transcriptome and qRT-PCR analyses. Transcriptome profiling also showed that 321 and 441 DEGs were expressed after 1 and 7 days of treatment, respectively. The KEGG enrichment analysis showed that 76 DEGs were involved in the photosynthesis pathway, while 35 DEGs were involved in the ascorbic acid metabolism pathway, respectively. These results suggest that the exogenous application of MT in plants provides important insight into understanding MT-induced stress-responsive mechanisms and protecting Brassica campestris against salt stress by regulating the photosynthesis and ascorbic acid pathway genes.

BcMYB111 Responds to BcCBF2 and Induces Flavonol Biosynthesis to Enhance Tolerance under Cold Stress in Non-Heading Chinese Cabbage.

Flavonols have been shown to respond to a variety of abiotic stresses in plants, including cold stress. Higher total flavonoid content was found in non-heading Chinese cabbage (NHCC, Brassica campestris (syn. Brassica rapa) ssp. chinensis) after cold stress. A non-targeted metabolome analysis showed a significant increase in flavonol content, including that of quercetin and kaempferol. Here, we found that an R2R3-MYB transcription factor, BcMYB111, may play a role in this process. BcMYB111 was up-regulated in response to cold treatment, with an accompanying accumulation of flavonols. Then, it was found that BcMYB111 could regulate the synthesis of flavonols by directly binding to the promoters of BcF3H and BcFLS1. In the transgenic hairy roots of NHCC or stable transgenic Arabidopsis, overexpression of BcMYB111 increased flavonol synthesis and accumulation, while these were reduced in virus-induced gene silencing lines in NHCC. After cold stress, the higher proline content and lower malondialdehyde (MDA) content showed that there was less damage in transgenic Arabidopsis than in the wild-type (WT). The BcMYB111 transgenic lines performed better in terms of antioxidant capacity because of their lower H2O2 content and higher superoxide dismutase (SOD) and peroxidase (POD) enzyme activities. In addition, a key cold signaling gene, BcCBF2, could specifically bind to the DRE element and activate the expression of BcMYB111 in vitro and in vivo. The results suggested that BcMYB111 played a positive role in enhancing the flavonol synthesis and cold tolerance of NHCC. Taken together, these findings reveal that cold stress induces the accumulation of flavonols to increase tolerance via the pathway of BcCBF2-BcMYB111-BcF3H/BcFLS1 in NHCC.

Genome-wide analysis of the Chinese cabbage IQD gene family and the response of BrIQD5 in drought resistance.

KEY MESSAGE: Thirty-five IQD genes were identified and analysed in Chinese cabbage and BrIQD5 transgenic plants enhanced the drought resistance of plants. The IQD (IQ67-domain) family plays an important role in various abiotic stress responses in plant species. However, the roles of IQD genes in the Chinese cabbage response to abiotic stress remain unclear. Here, 35 IQD genes, from BrIQD1 to BrIQD35, were identified in Chinese cabbage (Brassica rapa ssp. pekinensis). Based on the phylogenetic analysis, these genes were clustered into three subfamilies (I-III), and members within the same subfamilies shared conserved exon-intron distribution and motif composition. The 35 BrIQD genes were unevenly distributed on 9 of the 10 chromosomes with 4 segmental duplication events. Ka/Ks ratios showed that the duplicated BrIQDs had mainly experienced strong purifying selection. Quantitative real-time polymerase chain reaction of 35 BrIQDs under PEG6000 indicated that BrIQD5 was significantly induced by PEG6000. To verify BrIQD5 function, BrIQD5 was heterologously overexpressed in tobacco and was silenced in Chinese cabbage. BrIQD5-overexpressed plants showed more tolerance to drought stress than wild-type plants, while BrIQD5-silenced plants in Chinese cabbage showed decreased drought tolerance. Additionally, six BrIQD5 potential interactive proteins were isolated by the yeast two-hybrid assay, including BrCaMa, BrCaMb and four other stress-related proteins. Motif IQ1 of BrIQD5 is important for the interaction with BrCaMa and BrCaMb, and the isoleucine in motif IQ1 is an essential amino acid for calmodulin binding to BrIQD5. The identification and cloning of the new Chinese cabbage drought tolerance genes will promote the drought-resistant breeding of Chinese cabbage and help to better understand the mechanism of IQD involved in the drought tolerance of plants.

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.

BcGR1.1, a Cytoplasmic Localized Glutathione Reductase, Enhanced Tolerance to Copper Stress in Arabidopsis thaliana.

Copper is a mineral element, which is necessary for the normal growth and development of plants, but high levels of copper will seriously damage plants. Studies have shown that AtGR1 improves the tolerance of Arabidopsis to aluminum and cadmium stress. However, the role of GR in the copper stress response of plants is still unclear. Here, we identified four genes (named BcGR1.1, BcGR1.2, BcGR2.1 and BcGR2.2, respectively) encoding glutathione reductase (GR) in non-heading Chinese cabbage (Brassica campestris (syn. Brassica rapa) ssp. chinensis), which could be divided into two types based on the subcellular localization. Among them, BcGR1.1, which belonged to the cytoplasmic localization type, was significantly upregulated under copper stress. Compared to WT (the wild type), Arabidopsis thaliana heterologously overexpressed BcGR1.1 had longer roots, higher fresh weight, higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and accumulated more superoxide dismutase and peroxidase under copper stress. However, in the AsA-GSH cycle under copper stress, the contents of AsA and AsA/DHA were significantly downregulated, and the contents of DHA and T-AsA (total AsA) were upregulated, in the BcGR1.1-overexpressing Arabidopsis. Therefore, BcGR1.1 could improve the scavenging ability of reactive oxygen species (ROS) by increasing the activity of GR, antioxidant enzymes and the utilization of AsA, and then enhance the copper stress tolerance of plants.