A group VIIIa ethylene-responsive factor, CmERF4, negatively regulates waterlogging tolerance in chrysanthemum.

Ethylene-responsive factors (ERF) play an important role in plant responses to waterlogging stress. However, the function and mechanism of action of ERFVIII in response to waterlogging stress remain poorly understood. In this study, we found that expression of the ERF VIIIa gene CmERF4 in chrysanthemum was induced by waterlogging stress. CmERF4 localized to the nucleus when expressed in tobacco leaves. Yeast two-hybrid and luciferase assays showed that CmERF4 is a transcriptional inhibitor. CmERF4 overexpression in chrysanthemum reduced plant waterlogging tolerance, whereas overexpression of the chimeric activator CmERF4-VP64 reversed its transcriptional activity, promoting higher waterlogging tolerance than that observed in wild-type plants, indicating that CmERF4 negatively regulates waterlogging tolerance. Transcriptome profiling showed that energy metabolism and reactive oxygen species (ROS) pathway-associated genes were differentially expressed between CmERF4-VP64 and wild-type plants. RT-qPCR analysis of selected energy metabolism and reactive oxygen species-related genes showed that the gene expression patterns were consistent with the expression levels obtained from RNA-seq analysis. Overall, we identified new functions of CmERF4 in negatively regulating chrysanthemum waterlogging tolerance by modulating energy metabolism and ROS pathway genes.

CmERF5-CmRAP2.3 transcriptional cascade positively regulates waterlogging tolerance in Chrysanthemum morifolium.

Waterlogging stress affects plant growth by limiting root respiration and reducing yield and economic value. Therefore, identifying genes involved in regulating waterlogging stress is vital. This study reports the ethylene-responsive VII transcription factor (CmRAP2.3) in the chrysanthemum. Subcellular localization and transactivation assay analyses revealed that CmRAP2.3 was localized in the nucleus and possessed transactivation activity. Overexpression of CmRAP2.3 in chrysanthemum was found to enhance waterlogging tolerance by decreasing reactive oxygen species (ROS) levels. Furthermore, we found that the transcription factor CmERF5 binds to GCC-like motifs in the CmRAP2.3 promoter region and activates CmRAP2.3 expression. Additionally, CmERF5 overexpression maintained a low ROS level and improved chrysanthemum waterlogging tolerance. Taken together, this study shows a molecular mechanism by which CmERF5 transcriptionally activates CmRAP2.3 to reduce waterlogging stress via the ROS pathway in the chrysanthemum.

The natural antisense transcript NATTD regulates the transcription of decapping scavenger (DcpS) enzyme.

Natural antisense transcripts (NATs) are transcribed from the opposite strand of other genes. Most of them are noncoding RNAs. They have been reported to play important roles in a variety of biological processes. In this study, we identified a novel NAT, NATTD, which is partially complementary to both the TIRAP/Mal and DcpS genes. Interestingly, NATTD only positively regulates the expression of DcpS, a decapping scavenger enzyme which is a promising therapeutic target for spinal muscular atrophy. But it has no obvious effects on the expression of TIRAP/Mal gene. The NATTD transcript primarily resides in the nucleus and does not alter the mRNA stability of DcpS. Instead, it is required for the recruitment of RNA polymerase II at the mouse DcpS promoter. Chromatin immunoprecipitation assays revealed that knocking-down NATTD transcript with shRNA enhanced the H3K27-Me3 modification at the DcpS promoter. In summary, our studies identified NATTD as a regulator of DcpS transcription through epigenetic mechanisms.

Kelch-like Protein 21 (KLHL21) Targets IκB Kinase-ß to Regulate Nuclear Factor κ-Light Chain Enhancer of Activated B Cells (NF-κB) Signaling Negatively.

Activation of IKKß is the key step in canonical activation of NF-κB signaling. Extensive work has provided insight into the mechanisms underlying IKKß activation through the identification of context-specific regulators. However, the molecular processes responsible for its negative regulation are not completely understood. Here, we identified KLHL21, a member of the Kelch-like gene family, as a novel negative regulator of IKKß. The expression of KLHL21 was rapidly down-regulated in macrophages upon treatment with proinflammatory stimuli. Overexpression of KLHL21 inhibited the activation of IKKß and degradation of IκBα, whereas KLHL21 depletion via siRNA showed the opposite results. Coimmunoprecipitation assays revealed that KLHL21 specifically bound to the kinase domain of IKKß via its Kelch domains and that this interaction was gradually attenuated upon TNFα treatment. Furthermore, KLHL21 did not disrupt the interaction between IKKß and TAK1, TRAF2, or IκBα. Also, KLHL21 did not require its E3 ubiquitin ligase activity for IKKß inhibition. Our findings suggest that KLHL21 may exert its inhibitory function by binding to the kinase domain and sequestering the region from potential IKKß inducers. Taken together, our data clearly demonstrate that KLHL21 negatively regulates TNFα-activated NF-κB signaling via targeting IKKß, providing new insight into the mechanisms underlying NF-κB regulation in cells.

[The expression and purification of TRPM2 protein in E.coli and preparation of its polyclonal antibody].

OBJECTIVE: To purify the prokaryotically expressed GST-TRPM2N fusion protein and prepare specific polyclonal antibody against human transient receptor potential melastatin 2(TRPM2) protein. METHODS: The DNA fragment encoding evolutionarily conserved N-terminus (1-334 amino acids) of TRPM2 was amplified by PCR. The amplicon was then subcloned into prokaryotic expression plasmid pGEX-4T-3 and the recombinant plasmid was transformed into BL21 (DE3) cells. The expression of GST-TRPM2N fusion protein with a molecular weight about 70 000 Da was induced with 1 mmol/L IPTG and purified by GST affinity chromatography. The purified protein was mixed with complete Freund's adjuvant and used to immunize the New Zealand white rabbits with classical 4-injection protocol to generate specific anti-TRPM2 polyclonal antibody. The specificity and titer of the anti-TRPM2 antibody was analyzed by Western blotting. RESULTS: The cDNA fragment encoding N-terminus of human TRPM2 was amplified by PCR and directionally subcloned into pGEX-4T3 plasmid. Under the induction of IPTG, we observed the expression of GST-TRPM2N fusion protein. Polyclonal antibody against human TRPM2 protein was successfully prepared in the rabbits immunized with the purified GST-TRPM2N fusion protein. And the preliminary analysis showed that the anti-TRPM2 antibody could specifically identify transiently expressed TRPM2-EE in HEK293 cells. CONCLUSION: The specific polyclonal antibody against human TRPM2 protein has been successfully prepared, which facilitates our future research on the function of TRPM2 channel.

[Treatment of skeletal Class II adult patients with microscrew implant anchorage and multi-loop edgewise arch wire].

OBJECTIVE: To evaluate the effects and mechanisms of the microscrew implant anchorage (MIA) combined with multi-loop edgewise arch wire (MEAW) technique in the treatment of skeletal Class II adult patients. METHODS: Eleven adult patients with skeletal Class II high-angle malocclusions were treated with fixed appliances. The spaces were closed by the springs from the MIA to the hook on the archwire. The height of the hook and the direction of the force were different according to the intrusion and retraction of upper anterior teeth. In the finishing stage, MEAW technique and modified class II elastics (from the first loop of MEAW to the MIA) were used for final detailing. Cephalometric analysis was used to evaluate the effect after treatment. RESULTS: After treatment, the decrease of SNA, ANB and FMA were (2.86 +/- 1.05) degrees , (2.82 +/- 0.96) degrees and (2.95 +/- 1.35) degrees , respectively. The torque control of upper anterior teeth was good. The protrusion of lower incisors and the molar extrusion were avoided. The upper molars were moved distally by (3.00 +/- 2.19) mm. CONCLUSIONS: The treatment of adult patients with skeletal Class II high angle malocclusions with MIA and MEAW technique could not only improve the facial esthetics but also avoided the common side effects of traditional Class II elastics.