BnaC06.WIP2-BnaA09.STM transcriptional regulatory module promotes leaf lobe formation in Brassica napus.

The lobed leaves of rapeseed (Brassica napus L.) offer significant advantages in dense planting, leading to increased yield. Although AtWIP2, a C2H2 zinc finger transcription factor, acts as a regulator of leaf development in Arabidopsis thaliana, the function and regulatory mechanisms of BnaWIP2 in B. napus remain unclear. Here, constitutive expression of the BnaC06.WIP2 paralog, predominantly expressed in leaf serrations, produced lobed leaves in both A. thaliana and B. napus. We demonstrated that BnaC06.WIP2 directly repressed the expression of BnaA01.TCP4, BnaA03.TCP4, and BnaC03.TCP4 and indirectly inhibited the expression of BnaA05.BOP1 and BnaC02.AS2 to promote leaf lobe formation. On the other hand, we discovered that BnaC06.WIP2 modulated the levels of endogenous gibberellin, cytokinin, and auxin, and controlled the auxin distribution in B. napus leaves, thus accelerating leaf lobe formation. Meanwhile, we revealed that BnaA09.STM physically interacted with BnaC06.WIP2, and ectopic expression of BnaA09.STM generated smaller and lobed leaves in B. napus. Furthermore, we found that BnaC06.WIP2 and BnaA09.STM synergistically promoted leaf lobe formation through forming transcriptional regulatory module. Collectively, our findings not only facilitate in-depth understanding of the regulatory mechanisms underlying lobed leaf formation, but also are helpful for guiding high-density breeding practices through improving leaf morphology in B. napus.

Transcription factor DIVARICATA1 positively modulates seed germination in response to salinity stress.

Seed germination is a critical checkpoint for plant growth under unfavorable environmental conditions. In Arabidopsis (Arabidopsis thaliana), the abscisic acid (ABA) and gibberellic acid (GA) signaling pathways play important roles in modulating seed germination. However, the molecular links between salinity stress and ABA/GA signaling are not well understood. Herein, we showed that the expression of DIVARICATA1 (DIV1), which encodes a MYB-like transcription factor, was induced by GA and repressed by ABA, salinity, and osmotic stress in germinating seeds. DIV1 positively regulated seed germination in response to salinity stress by directly regulating the expression of DELAY OF GERMINATION 1-LIKE 3 (DOGL3) and GA-STIMULATED ARABIDOPSIS 4 (GASA4) and indirectly regulating the expression of several germination-associated genes. Moreover, NUCLEAR FACTOR-YC9 (NF-YC9) directly repressed the expression of DIV1 in germinating seeds in response to salinity stress. These results help reveal the function of the NF-YC9-DIV1 module and provide insights into the regulation of ABA and GA signaling in response to salinity stress during seed germination in Arabidopsis.

Antagonistic MADS-box transcription factors SEEDSTICK and SEPALLATA3 form a transcriptional regulatory network that regulates seed oil accumulation.

Transcriptional regulation is essential for balancing multiple metabolic pathways that influence oil accumulation in seeds. Thus far, the transcriptional regulatory mechanisms that govern seed oil accumulation remain largely unknown. Here, we identified the transcriptional regulatory network composed of MADS-box transcription factors SEEDSTICK (STK) and SEPALLATA3 (SEP3), which bridges several key genes to regulate oil accumulation in seeds. We found that STK, highly expressed in the developing embryo, positively regulates seed oil accumulation in Arabidopsis (Arabidopsis thaliana). Furthermore, we discovered that SEP3 physically interacts with STK in vivo and in vitro. Seed oil content is increased by the SEP3 mutation, while it is decreased by SEP3 overexpression. The chromatin immunoprecipitation, electrophoretic mobility shift assay, and transient dual-luciferase reporter assays showed that STK positively regulates seed oil accumulation by directly repressing the expression of MYB5, SEP3, and SEED FATTY ACID REDUCER 4 (SFAR4). Moreover, genetic and molecular analyses demonstrated that STK and SEP3 antagonistically regulate seed oil production and that SEP3 weakens the binding ability of STK to MYB5, SEP3, and SFAR4. Additionally, we demonstrated that TRANSPARENT TESTA 8 (TT8) and ACYL-ACYL CARRIER PROTEIN DESATURASE 3 (AAD3) are direct targets of MYB5 during seed oil accumulation in Arabidopsis. Together, our findings provide the transcriptional regulatory network antagonistically orchestrated by STK and SEP3, which fine tunes oil accumulation in seeds.

The MYB59 transcription factor negatively regulates salicylic acid- and jasmonic acid-mediated leaf senescence.

Leaf senescence is the final stage of leaf development and is affected by various exogenous and endogenous factors. Transcriptional regulation is essential for leaf senescence, however, the underlying molecular mechanisms remain largely unclear. In this study, we report that the transcription factor MYB59, which was predominantly expressed in early senescent rosette leaves, negatively regulates leaf senescence in Arabidopsis (Arabidopsis thaliana). RNA sequencing revealed a large number of differentially expressed genes involved in several senescence-related biological processes in myb59-1 rosette leaves. Chromatin immunoprecipitation and transient dual-luciferase reporter assays demonstrated that MYB59 directly repressed the expression of SENESCENCE ASSOCIATED GENE 18 and indirectly inhibited the expression of several other senescence-associated genes to delay leaf senescence. Moreover, MYB59 was induced by salicylic acid (SA) and jasmonic acid (JA). MYB59 inhibited SA production by directly repressing the expression of ISOCHORISMATE SYNTHASE 1 and PHENYLALANINE AMMONIA-LYASE 2 and restrained JA biosynthesis by directly suppressing the expression of LIPOXYGENASE 2, thus forming two negative feedback regulatory loops with SA and JA and ultimately delaying leaf senescence. These results help us understand the novel function of MYB59 and provide insights into the regulatory network controlling leaf senescence in Arabidopsis.

Overexpression of BnaAGL11, a MADS-Box Transcription Factor, Regulates Leaf Morphogenesis and Senescence in Brassica napus.

Previous studies have reported that SEEDSTICK/AGAMOUS-LIKE 11 (AtSTK/AtAGL11), a MADS-box transcription factor, plays important roles in many biological processes in Arabidopsis thaliana. However, the function of BnaAGL11, an AtAGL11 homologous gene from Brassica napus, in leaf development remains unknown. Here, we found that the ectopic expression of any copy of Bna.C09.AGL11, Bna.A03.AGL11, and Bna.A09.AGL11 in A. thaliana led to smaller and curly leaves and promoted leaf senescence. Consistently, the overexpression of Bna.C09.AGL11 in B. napus also caused smaller and curly leaves and accelerated leaf senescence. Furthermore, we demonstrated that Bna.C09.AGL11 controlled leaf morphogenesis by indirectly downregulating the genes of Bna.A01.DWF4 and Bna.C07.PGX3 and promoted leaf senescence by indirectly upregulating the genes of Bna.A04.ABI5, Bna.A05.ABI5, Bna.C04.ABI5-1, and Bna.C01.SEN4 and directly activating the transcription of Bna.C04.ABI5-2 and Bna.C03.SEN4 genes. Our results provide new insights into the underlying regulatory mechanism of BnaAGL11 during leaf development in B. napus.

DEC1 deficiency results in accelerated osteopenia through enhanced DKK1 activity and attenuated PI3KCA/Akt/GSK3ß signaling.

BACKGROUND: Human differentiated embryonic chondrocyte expressed gene 1 (DEC1) has been implicated in enhancing osteogenesis, a desirable outcome to counteract against deregulated bone formation such as retarded bone development, osteopenia and osteoporosis. METHODS AND RESULTS: DEC1 knockout (KO) and the age-matched wild-type (WT) mice were tested for the impact of DEC1 deficiency on bone development and osteopenia as a function of age. DEC1 deficiency exhibited retarded bone development at the age of 4 weeks and osteopenic phenotype in both 4- and 24-week old mice. However, the osteopenia was more severe in the 24-week age groups. Mechanistically, DEC1 deficiency downregulated the expression of bone-enhancing genes such as Runx2 and ß-catenin accompanied by upregulating DKK1, an inhibitor of the Wnt/ß-catenin signaling pathway. Consistently, DEC1 deficiency favored the attenuation of the integrated PI3KCA/Akt/GSK3ß signaling, a pathway targeting ß-catenin for degradation. Likewise, the attenuation was greater in the 24-week age group. These changes, however, were reversed by in vivo treatment with lithium chloride, a stabilizer of ß-catenin, and confirmed by gain-of-function study with DEC1 transfection into DEC1 KO bone marrow mesenchymal stem cells and loss-of-function study with siDEC1 lentiviral infection into the corresponding WT cells. CONCLUSION: DEC1 is a positive regulator with a broad activity spectrum in both bone development and maintenance, and the osteopenic phenotype accelerated by DEC1 deficiency is achieved by enhanced DKK1 activity and attenuated PI3KCA/Akt/GSK3ß signaling.

Melatonin Represses Oil and Anthocyanin Accumulation in Seeds.

Previous studies have clearly demonstrated that the putative phytohormone melatonin functions directly in many aspects of plant growth and development. In Arabidopsis (Arabidopsis thaliana), the role of melatonin in seed oil and anthocyanin accumulation, and corresponding underlying mechanisms, remain unclear. Here, we found that serotonin N-acetyltransferase1 (SNAT1) and caffeic acid O-methyltransferase (COMT) genes were ubiquitously and highly expressed and essential for melatonin biosynthesis in Arabidopsis developing seeds. We demonstrated that blocking endogenous melatonin biosynthesis by knocking out SNAT1 and/or COMT significantly increased oil and anthocyanin content of mature seeds. In contrast, enhancement of melatonin signaling by exogenous application of melatonin led to a significant decrease in levels of seed oil and anthocyanins. Further gene expression analysis through RNA sequencing and reverse-transcription quantitative PCR demonstrated that the expression of a series of important genes involved in fatty acid and anthocyanin accumulation was significantly altered in snat1-1 comt-1 developing seeds during seed maturation. We also discovered that SNAT1 and COMT significantly regulated the accumulation of both mucilage and proanthocyanidins in mature seeds. These results not only help us understand the function of melatonin and provide valuable insights into the complicated regulatory network controlling oil and anthocyanin accumulation in seeds, but also divulge promising gene targets for improvement of both oil and flavonoids in seeds of oil-producing crops and plants.

17ß-estradiol suppresses carboxylesterases by activating c-Jun/AP-1 pathway in primary human and mouse hepatocytes.

In order to study the influence of estrogen on carboxylesterases, we investigated the effects of 17ß-estradiol on CES1 (Ces1d) and CES2 (Ces1e) in human and mouse hepatocytes. After being treated with 17ß-estradiol, the mRNA levels of CES1 and CES2 decreased by 29-39% and 28-55%, respectively, in the human hepatocytes from four donors. Consistently, the hydrolysis of para-nitrophenylacetate decreased markedly by 32% induced by 17ß-estradiol. Moreover, 17ß-estradiol decreased CES1 and CES2 by 45% and 47% respectively at protein levels. The response of altered expression of Ces1d (CES1) and Ces1e (CES2) to 17ß-estradiolin in mouse hepatocytes was very similar to that in the human hepatocytes. Further, the decreased Ces1d and Ces1e expression induced by 17ß-estradiol could be abolished by SP600125, an inhibitor of AP-1, both at mRNA and protein levels. Likewise, the increased c-Jun expression induced by 17ß-estradiol could almost be abolished by SP600125. In vivo, the expression of Ces1d, Ces1e and the hydrolytic activity of liver were higher in the ovariectomized female mice(OVX) than those in control mice(SHAM). However, when 17ß-estradiol was administrated, the expression of Ces1d, Ces1e and the hydrolytic activity of liver in the ovariectomized female mice (OVX+E2) became restored to their normal levels. Taken together, 17ß-estradiol suppresses carboxylesterases by activating c-Jun/AP-1 pathway in primary human and mouse hepatocytes. The findings can offer the potential gains in the safety and efficacy of pharmacotherapy for women, especially for pregnant and menopausal women.

Interleukin-6 Induces DEC1, Promotes DEC1 Interaction with RXRα and Suppresses the Expression of PXR, CAR and Their Target Genes.

Inflammatory burden is a primary cellular event in many liver diseases, and the overall capacity of drug elimination is decreased. PXR (pregnane X receptor) and CAR (constitutive androstane receptor) are two master regulators of genes encoding drug-metabolizing enzymes and transporters. DEC1 (differentiated embryonic chondrocyte-expressed gene 1) is a ligand-independent transcription factor and reportedly is induced by many inflammatory cytokines including IL-6. In this study, we used primary hepatocytes (human and mouse) as well as HepG2 cell line and demonstrated that IL-6 increased DEC1 expression and decreased the expressions of PXR, CAR, and their target genes. Overexpression of DEC1 had similar effect as IL-6 on the expression of these genes, and knockdown of DEC1 reversed their downregulation by IL-6. Interestingly, neither IL-6 nor DEC1 altered the expression of RXRα, a common dimerization partner for many nuclear receptors including PXR and CAR. Instead, DEC1 was found to interact with RXRα and IL-6 enhanced the interaction. These results conclude that DEC1 uses diverse mechanisms of action and supports IL-6 downregulation of drug-elimination genes and their regulators.

Icariin protects against glucocorticoid induced osteoporosis, increases the expression of the bone enhancer DEC1 and modulates the PI3K/Akt/GSK3ß/ß-catenin integrated signaling pathway.

Osteoporosis is a serious public health concern worldwide. Herba epimedii has been used for centuries and even thousands of years to treat osteoporotic conditions. Icariin, a flavonol glycoside, is one of the major active ingredients. In this study, we have shown that icariin protected against glucocorticoid-induced osteoporotic changes in SaoS-2 cells and mice. We have also shown that dexamethasone (a glucocorticoid) suppressed and icariin induced DEC1, a structurally distinct helix-loop-helix protein. DEC1 overexpression promoted whereas DEC1 knockdown decreased osteogenic activity. Likewise, DEC1 overexpression and knockdown inversely regulated the expression of ß-catenin and PIK3CA, an essential player in the Wnt/ß-catenin and PI3K/Akt signaling pathways, respectively. Interestingly, DKK1, an inhibitor of Wnt/ß-catenin signaling inhibitor, and LY294002, an inhibitor of PI3K/Akt signaling, abolished the induction of DEC1 by icariin. It is established that these two pathways are interconnected by the phosphorylation status of GSK3ß. Dexamethasone decreased but icariin increased GSK3ß phosphorylation. Finally, DEC1 deficient mice developed osteoporotic phenotypes. Taken together, it is concluded that DEC1 likely supports the action of icariin against glucocorticoid induced osteoporosis with an involvement of the PI3K/Akt/GSK3ß/ß-catenin integrated signaling pathway.

Involvement of pregnane X receptor in the suppression of carboxylesterases by metformin in vivo and in vitro, mediated by the activation of AMPK and JNK signaling pathway.

Type 2 diabetes mellitus (T2D) is a complex metabolic disorder requiring polypharmacy treatment in clinic, with metformin being widely used antihyperglycemic drug. However, the mechanisms of metformin as a perpetrator inducing potential drug-drug interactions and adverse drug reactions are scarcely known to date. Carboxylesterases (CESs) are major hydrolytic enzymes highly expressed in the liver, including mouse carboxylesterase 1d (Ces1d) and Ces1e. In the present study, experiments are designed to investigate the effects and mechanisms of metformin on Ces1d and Ces1e in vivo and in vitro. In results, metformin suppresses the expression and activity of Ces1d and Ces1e in a dose- and time-dependent manner. The decreased expression of nuclear receptor PXR and its target gene P-gp indicates the involvements of PXR in the suppressed expression of carboxylesterases by metformin. Furthermore, metformin significantly suppresses the phosphorylation of AMPK and JNK, and the suppression of carboxylesterases induced by metformin is repeatedly abolished by AMPK inhibitor Compound C and JNK inhibitor SP600125. It implies that the activation of AMPK and JNK pathways mediates the suppression of carboxylesterases by metformin. The findings deserve further elucidation including clinical trials and have a potential to make contribution for the rational medication in the treatment of T2D patients.