Ip3r-Grp75-Vdac and Relevant Ca2+ Signaling Regulate Dietary Palmitic Acid-Induced De Novo Lipogenesis by Mitochondria-Associated ER Membrane (MAM) Recruiting Seipin in Yellow Catfish.

BACKGROUND: The mitochondria-associated endoplasmic reticulum membrane (MAM) is the central hub for endoplasmic reticulum and mitochondria functional communication. It plays a crucial role in hepatic lipid homeostasis. However, even though MAM has been acknowledged to be rich in enzymes that contribute to lipid biosynthesis, no study has yet investigated the exact role of MAM on hepatic neutral lipid synthesis. OBJECTIVES: To address these gaps, this study investigated the systemic control mechanisms of MAM on neutral lipids synthesis by recruiting seipin, focusing on the role of the inositol trisphosphate receptor-1,4,5(Ip3r)-75 kDa glucose-regulated protein (Grp75)-voltage-dependent anion channel (Vdac) complex and their relevant Ca2+ signaling in this process. METHODS: To this end, a model animal for lipid metabolism, yellow catfish (Pelteobagrus fulvidraco), were fed 6 different diets containing a range of palmitic acid (PA) concentrations from 0-150 g/kg in vivo for 10 wk. In vitro, experiments were also conducted to intercept the MAM-mediated Ca2+ signaling in isolated hepatocytes by transfecting them with si-mitochondrial calcium uniporter (mcu). Because mcu was placed in the inner mitochondrial membrane (IMM), si-mcu cannot disrupt MAM's structural integrity. RESULTS: 1. Hepatocellular MAM subproteome analysis indicated excessive dietary PA intake enhanced hepatic MAM structure joined by activating Ip3r-Grp75-Vdac complexes. 2. Dietary PA intake induced hepatic neutral lipid accumulation through MAM recruiting Seipin, which activated lipid droplet biogenesis. Our findings also revealed a previously unidentified mechanism whereby MAM-recruited seipin and controlled hepatic lipid homeostasis, depending on Ip3r-Grp75-Vdac-controlled Ca2+ signaling and not only MAM's structural integrity. CONCLUSIONS: These results offer a novel insight into the MAM-recruited seipin in controlling hepatic lipid synthesis in a MAM structural integrity-dependent and Ca2+ signaling-dependent manner, highlighting the critical contribution of MAM in maintaining hepatic neutral lipid homeostasis.

Choline-mediated hepatic lipid homoeostasis in yellow catfish: unravelling choline's lipotropic and methyl donor functions and significance of ire-1α signalling pathway.

Choline plays a crucial role in hepatic lipid homeostasis by acting as a major methyl-group donor. However, despite this well-accepted fact, no study has yet explored how choline's methyl-donor function contributes to preventing hepatic lipid dysregulation. Moreover, the potential regulatory role of Ire-1α, an ER-transmembrane transducer for the unfolded protein response (UPRer), in choline-mediated hepatic lipid homeostasis remains unexplored. Thus, this study investigated the mechanism by which choline prevents hepatic lipid dysregulation, focusing on its role as a methyl-donor and the involvement of Ire-1α in this process. To this end, a model animal for lipid metabolism, yellow catfish (Pelteobagrus fulvidraco) were fed two different diets (adequate or deficient choline diets) in vivo for 10 weeks. The key findings of studies are as follows: 1. Dietary choline, upregulated selected lipolytic and fatty acid ß-oxidation transcripts promoting hepatic lipid homeostasis. 2. Dietary choline ameliorated UPRer and prevented hepatic lipid dysregulation mainly through ire-1α signalling, not perk or atf-6α signalling. 3. Choline inhibited the transcriptional expression level of ire-1α by activating site-specific DNA methylations in the promoter of ire-1α. 4. Choline-mediated ire-1α methylations reduced Ire-1α/Fas interactions, thereby further inhibiting Fas activity and reducing lipid droplet deposition. These results offer a novel insight into the direct and indirect regulation of choline on lipid metabolism genes and suggests a potential crosstalk between ire-1α signalling and choline-deficiency-induced hepatic lipid dysregulation, highlighting the critical contribution of choline as a methyl-donor in maintaining hepatic lipid homeostasis.

Astragalus polysaccharide promotes autophagy and alleviates diabetic nephropathy by targeting the lncRNA Gm41268/PRLR pathway.

BACKGROUND: Astragalus polysaccharide (APS) is a major bioactive component of the Chinese herb astragalus, with well-established protective effects on the kidney. However, the effect of APS on diabetic nephropathy (DN) is unclear. METHODS: Long non-coding RNA (lncRNA) expression profiles in kidney samples from control, db/db, and APS-treated db/db mice were evaluated using RNA high-throughput sequencing techniques. Additionally, rat renal tubular epithelial (NRK-52E) cells were cultured in high glucose (HG) media. We inhibited the expression of Gm41268 and prolactin receptor (PRLR) by transfecting NRK-52E cells with Gm41268-targeting antisense oligonucleotides and PRLR siRNA. RESULTS: We found that APS treatment reduced 24-h urinary protein levels and fasting blood glucose and improved glucose intolerance and pathological renal damage in db/db mice. Furthermore, APS treatment enhanced autophagy and alleviated fibrosis in the db/db mice. We identified a novel lncRNA, Gm41268, which was differentially expressed in the three groups, and the cis-regulatory target gene PRLR. APS treatment induced autophagy by reducing p62 and p-mammalian target of rapamycin (mTOR) protein levels and increasing the LC3 II/I ratio. Furthermore, APS alleviated fibrosis by downregulating fibronectin (FN), transforming growth factor-ß (TGF-ß), and collagen IV levels. In addition, APS reversed the HG-induced overexpression of Gm41268 and PRLR. Reduction of Gm41268 decreased PRLR expression, restored autophagy, and ameliorated renal fibrosis in vitro. Inhibition of PRLR could enhance the protective effect of APS. CONCLUSIONS: In summary, we demonstrated that the therapeutic effect of APS on DN is mediated via the Gm41268/PRLR pathway. This information contributes to the exploration of bioactive constituents in Chinese herbs as potential treatments for DN.

Inhibition of ferroptosis ameliorates hypertensive nephropathy through p53/Nrf2/p21 pathway by Taohongsiwu decoction: Based on network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypertensive nephropathy (HN) is a complication of hypertension. Taohongsiwu decoction (THSWD) is used clinically but its application in the prevention and treatment of HN remains unelucidated. AIM OF STUDY: This study aims to explore the potential targets and molecular mechanisms of THSWD in the treatment of HN. MATERIALS AND METHODS: A network pharmacology approach was used to predict the components and targets of THSWD for treating HN. Animal experiments were performed to verify the network pharmacology findings. RESULTS: 205 targets were identified and regarded as potential targets of THSWD in HN treatment. Subsequently, we screened 17 hub genes and identified TP53 as the most critical one. KEGG enrichment analysis showed that p53 signaling pathway might play a significant role. In vivo experiments indicated that high-salt diets can lead to high blood pressure, kidney injury, inflammation, and fibrosis. Furthermore, the altered levels of biomarkers (Iron, malondialdehyde, catalase, ferritin, transferrin, Superoxide dismutase and Glutathione Peroxidase 4) provided evidence of ferroptosis. We found that the ferroptosis inhibitor ferrostatin-1 (Fer-1) and THSWD could significantly alleviate HN by suppressing ferroptosis. THSWD and Fer-1 treatment downregulated the protein and mRNA expression of p53, p21, RB, and CTNNB1, which were upregulated by high salt. Meanwhile, THSWD and Fer-1 reversed the downregulation of Nrf2 caused by high-salt diet. CONCLUSIONS: Our results suggested that THSWD attenuate HN induced by a high-salt diet through inhibiting ferroptosis via the p53/Nrf2/p21 pathway.

A dummy molecularly imprinted ratiometric fluorescence nanosensor for the sensitive detection of guanidyl-microcystins in environmental water.

An effective strategy is proposed to construct a highly sensitive ratiometric fluorescence sensing platform for microcystins (MCs) based on a dummy molecularly imprinted polymer using metformin as a template. The imprinted nanohybrids of carbon dots (CDs) combined with fluorescein isothiocyanate (FITC) are synthesized (CDs-FITC-SiO2@MIP), in which the CDs and FITC serve as assisted response signals and reference enhancement signals, respectively. Metformin can be used as a dummy template for MCs due to its partially similar molecular fragments to MCs that can form a specific recognition site cavity. MCs can simultaneously induce an obvious fluorescence quenching effect for the CDs and a reference fluorescence enhancement for FITC-SiO2, enabling ratiometric fluorescence detection of MCs. Thus, CDs-FITC-SiO2@MIP used as a signal probe has favorable sensitivity, stability, and selectivity. More importantly, a good linear relationship between the fluorescence intensity ratio (I620/450) and the concentration of MCs in the range of 0.5-500 µg L-1 is obtained with a LOD of 0.013 µg L-1 and 0.022 µg L-1 for MC-RR and MC-LR, respectively, under the optimum conditions. This method has great application potential in water quality monitoring by using CDs-FITC-SiO2@MIP as a promising candidate for monitoring MCs in complex systems.

Molecular imprinted electrochemical sensor for ovalbumin detection based on boronate affinity and signal amplification approach.

Ovalbumin (OVA), a class of glycoproteins, is the main allergen in hen egg white that often causes allergies in humans, especially in babies. Therefore, it is pivotal to be able to detect and separate OVA. This work presents an ingenious sandwich-structured magnetic molecular imprinted electrochemical sensor for OVA detection by utilizing boronate affinity and signal amplification strategy. With anti-OVA antibody-modified gold nanoparticles (AuNPs) as amplifiers, the imprinted cavities in the probe could capture protein to form a sandwich structure. Due to its specific recognition of antibody and molecular imprinted polymers and the signal amplification of AuNPs, the sensor had good selectivity and sensitivity toward OVA and a low detection limit of 3.0 fg/mL. The sensor also had excellent stability and could satisfactorily detect OVA in real red wine samples.

Dietary Choline Mitigates High-Fat Diet-Impaired Chylomicrons Assembly via UPRer Modulated by perk DNA Methylation.

High-fat diets (HFD) lead to impairment of chylomicrons (CMs) assembly and adversely influence intestinal lipid homeostasis. However, the mechanisms of HFD impairing CMs assembly have yet to be fully understood. Additionally, although choline, as a lipid-lowering agent, has been widely used and its deficiency has been closely linked to non-alcoholic steatohepatitis (NASH), the contribution of choline by functioning as a methyl donor in alleviating HFD-induced intestinal lipid deposition is unknown. Thus, this study was conducted to determine the mechanism of HFD impairing CMs assembly and also tested the effect of choline acting as a methyl donor in this process. To this end, in this study, four diets (control, HFD, choline and HFD + choline diet) were fed to yellow catfish for 10 weeks in vivo and their intestinal epithelial cells were isolated and incubated for 36 h in fatty acids (FA) with or without choline solution combining si-perk transfection in vitro. The key findings from this study as follows: (1) HFD caused impairment of CMs assembly main by unfolded protein response (UPRer). HFD activated perk and then induced UPRer, which led to endoplasmic reticulum dysfunction and further impaired CMs assembly via protein-protein interactions between Perk and Apob48. (2) Choline inhibited the transcriptional expression level of perk via activating the -211 CpG methylation site, which initiated the subsequent ameliorating effect on HFD-impaired CMs assembly and intestinal lipid dysfunction. These results provide a new insight into direct crosstalk between UPRer and CMs assembly, and also emphasize the critical contribution of choline acting as a methyl donor and shed new light on choline-deficient diet-induced NASH.

An integrated RNA sequencing and network pharmacology approach reveals the molecular mechanism of dapagliflozin in the treatment of diabetic nephropathy.

Dapagliflozin, an inhibitor of sodium-glucose cotransporter 2 (SGLT2), is a new type of oral hypoglycemic drugs which can promote glucose excretion in the kidney. Studies have shown that dapagliflozin has renoprotective effect in the treatment of type 2 diabetes. However, the underlying mechanism remains unclear. Here, we combined integrated RNA sequencing and network pharmacology approach to investigate the molecular mechanism of dapagliflozin for diabetic nephropathy (DN). Dapagliflozin significantly relieved glucose intolerance, urinary albumin/creatinine ratio (UACR) and renal pathological injuries of db/db mice. The LncRNA and mRNA expression in kidney tissues from control group (CR), db/db group (DN) and dapagliflozin group (DG) were assessed by RNA sequencing. We identified 7 LncRNAs and 64 mRNAs common differentially expressed in CR vs DN and DN vs DG, which were used to construct co-expression network to reveal significantly correlated expression patterns in DN. In addition, network pharmacology was used to predict the therapeutic targets of dapagliflozin and we constructed component-target-pathway network according to the results of RNA sequencing and network pharmacology. We found that SMAD9, PPARG, CD36, CYP4A12A, CYP4A12B, CASP3, H2-DMB2, MAPK1, MAPK3, C3 and IL-10 might be the pivotal targets of dapagliflozin for treating DN and these genes were mainly enriched in pathways including TGF-ß signaling pathway, PPAR signaling pathway, Chemokine signaling pathway, etc. Our results have important implication and provide novel insights into the protective mechanism of dapagliflozin for treating DN.

Fluorescence and electrochemical integrated dual-signal sensor for the detection of iron ions in water based on an ITO substrate.

A novel type of fluorescent and electrochemical dual-signal sensor was constructed for the sensitive and selective detection of iron ions (Fe3+) based on a fluorescent material (Chi-FITC-4MU), which was synthesized by combining the organic dye 4-methylumbelliferone (4-MU), chitosan, and fluorescein isothiocyanate (FITC) in a simple step process. The 4-MU could bind to Fe3+ to form a complex, and clearly improved the selectivity of Chi-FITC-4MU for Fe3+ detection. FITC showed excellent fluorescence performance and chitosan was beneficial to the curing of the material. By solidifying the fluorescent material on an ITO surface, the dual-signal detection of Fe3+ could be realized with excellent selectivity, stability, and anti-interference ability. Based on the unique fluorescence properties of this sensor, the concentration of Fe3+ could be visualized in the linear range of 0.1-100 µM based on the degree of fluorescence quenching. Moreover, the highly sensitive and rapid analysis of low concentrations of Fe3+ was achieved through the electrochemical properties of the ITO sensor. The limit of detection (LOD) and the corresponding linear range were 0.0184 nM and 0.1-500 nM, respectively. Furthermore, this dual-signal sensor was effectively used for the detection of Fe3+ in actual water.

Deciphering the Pharmacological Mechanisms of Qidan Dihuang Decoction in Ameliorating Renal Fibrosis in Diabetic Nephropathy through Experimental Validation In Vitro and In Vivo.

Objective: QiDan DiHuang decoction (QDD) has been proven to have good efficacy in decreasing albuminuria levels, improving renal function, and inhibiting renal fibrosis in diabetic nephropathy (DN). However, the potential mechanism remains unclear. The purpose of this study was to explore the underlying mechanism of QDD for treating DN in vitro and in vivo. Methods: Db/db mice were treated with QDD or saline intragastrically for 12 weeks. Non-diabetic db/m mice were used as controls. Rat renal tubular epithelial cells (NRK-52E) were cultured in high glucose conditions. ATF4 siRNA was transfected into NRK-52E cells. Different indicators were detected via UPLC, RT-PCR, western blotting, cell viability assays and apoptosis, transmission electron microscopy, histology, and immunofluorescence staining. Results: Db/db mice experienced severe kidney damage and fibrosis, increased levels of PERK, eIF2α, and ATF4, and suppression of renal autophagy compared with db/m mice. The results showed a significant improvement in glucose intolerance, blood urea nitrogen, urine albumin, serum creatinine, and renal fibrosis in db/db mice with QDD treatment. Meanwhile, the application of QDD resulted in the downregulation of PERK, eIF2α, and ATF4 and the upregulation of autophagy in diabetic kidneys. In vitro, the exposure of NRK-52E cells to high glucose resulted in downregulation of the ratio of LC3-II/LC3-I and upregulation of P62, a reduction in the number of autophagosomes and upregulation of fibronectin (FN), collagen IV and TGF-ß1 protein, which was reversed by QDD treatment through inhibiting ATF4 expression. Conclusions: Taken together, our results suggest that QDD effectively alleviates diabetic renal injuries and fibrosis by inhibiting the PERK-eIF2α-ATF4 pathway and promoting autophagy in diabetic nephropathy.

The Physicochemical Properties and Antioxidant Activity of Spirulina (Artrhospira platensis) Chlorophylls Microencapsulated in Different Ratios of Gum Arabic and Whey Protein Isolate.

Spirulina (Artrhospira platensis) is rich in chlorophylls (CH) and is used as a potential natural additive in the food industry. In this study, the CH content was extracted from spirulina powder after ultrasound treatment. Microcapsules were then prepared at different ratios of gum Arabic (GA) and whey protein isolate (WPI) through freeze-drying to improve the chemical stability of CH. As a result, a* and C* values of the microcapsules prepared from GA:WPI ratios (3:7) were -8.94 ± 0.05 and 15.44 ± 0.08, respectively. The GA fraction increased from 1 to 9, and encapsulation efficiency (EE) of microcapsules also increased by 9.62%. Moreover, the absorption peaks of CH at 2927 and 1626 cm-1 in microcapsules emerged as a redshift detected by FT-IR. From SEM images, the morphology of microcapsules changed from broken glassy to irregular porous flake-like structures when the GA ratio increased. In addition, the coated microcapsules (GA:WPI = 3:7) showed the highest DPPH free radical scavenging activity (SADPPH) (56.38 ± 0.19) due to low moisture content and better chemical stability through thermogravimetric analysis (TGA). Conclusively, GA and WPI coacervates as the wall material may improve the stability of CH extracted from spirulina.

Dietary Choline Alleviates High-Fat Diet-Induced Hepatic Lipid Dysregulation via UPRmt Modulated by SIRT3-Mediated mtHSP70 Deacetylation.

The mitochondrial unfolded protein response (UPRmt) is known as a conservative mechanism in response to mitochondrial dysfunction. Thus, based on UPRmt, this study was conducted to determine the mechanism of a high-fat diet (HFD) inducing mitochondrial dysfunction and its role in stimulating hepatic lipid dysregulation. The choline-activated alleviating effect was also evaluated. In vivo, yellow catfish were fed three diets (control, HFD, and HFD + choline diet) for 10 weeks. In vitro, hepatocytes isolated from yellow catfish and the HepG2 cell line were cultured and incubated with fatty acid (FA) for 48 h. (1) HFD-induced mitochondrial dysfunction via SIRT3/mtHSP70-mediated UPRmt. HFD inhibited the subcellular localization of SIRT3 into the mitochondrion, resulting in the up-regulating of mtHSP70 acetylation via lysine residues 493 and 507. The mtHSP70 acetylation promoted the stability of mtHSP70, which then led to the UPRmt and further mitochondrial dysfunction. (2) SIRT3/mtHSP70-mediated UPRmt regulated HFD/FA-induced hepatic lipid dysregulation. SIRT3/mtHSP70-mediated UPRmt reduced FA ß-oxidation via mitochondrial dysfunction and then led to lipid dysregulation. Additionally, the mtHSP70-ACOX1 interaction was confirmed. (3) Choline alleviated HFD-induced UPRmt via up-regulating the localization of SIRT3 into the mitochondrion, which in turn led to the subsequent ameliorating effect on HFD-induced hepatic lipid dysregulation. Through SIRT3-mediated mtHSP70 deacetylation, dietary choline alleviates HFD-induced hepatic lipid dysregulation via UPRmt. This provides the first proof of acetylation regulating UPRmt and the crosstalk between UPRmt and FA ß-oxidation.

Genomic Identification of RNA Editing Through Integrating Omics Datasets and the Clinical Relevance in Hepatocellular Carcinoma.

RNA editing is a widespread post-transcriptional mechanism to introduce single nucleotide changes to RNA in human cancers. Here, we characterized the global RNA editing profiles of 373 hepatocellular carcinoma (HCC) and 50 adjacent normal liver samples from The Cancer Genome Atlas (TCGA) and revealed that most editing events tend to occur in minor percentage of samples with moderate editing degrees (20-30%). Moreover, these RNA editing prefer to be A-to-I RNA editing in protein coding genes, especially in 3'UTR regions. Considering the association between DNA mutation and RNA editing, our analysis found that RNA editing maybe a complementary event for DNA mutation of HCC risk genes in HCC patients. We next identified 454 HCC-related editing sites, and many locate on the same genes with the same editing patterns. The functional consequences of editing revealed 2,086 functional editing sites and demonstrated that most editing in coding regions are non-synonymous variations. Furthermore, our results showed that editing in the 3'UTR regions tend to influence miRNA-target binding, and the editing degree seems to be negatively correlated with gene expression. Finally, we found that 46 HCC-related editing sites with consequence are able to distinguish the prognosis differences of HCC patients, suggesting their clinical relevance. Together, our results highlight RNA editing as a valuable molecular resource for investigating HCC mechanisms and clinical treatments.

Transcriptomic analysis of Verbena bonariensis roots in response to cadmium stress.

BACKGROUND: Cadmium (Cd) is a serious heavy metal (HM) soil pollutant. To alleviate or even eliminate HM pollution in soil, environmental-friendly methods are applied. One is that special plants are cultivated to absorb the HM in the contaminated soil. As an excellent economical plant with ornamental value and sound adaptability, V. bonariensis could be adapted to this very situation. In our study, the Cd tolerance in V. bonariensis was analyzed as well as an overall analysis of transcriptome. RESULTS: In this study, the tolerance of V. bonariensis to Cd stress was investigated in four aspects: germination, development, physiological changes, and molecular alterations. The results showed that as a non-hyperaccumulator, V. bonariensis did possess the Cd tolerance and the capability to concentration Cd. Under Cd stress, all 237, 866 transcripts and 191, 370 unigenes were constructed in the transcriptome data of V. bonariensis roots. The enrichment analysis of gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed that differentially expressed genes (DEGs) under Cd stress were predominately related to cell structure, reactive oxygen species (ROS) scavenging system, chelating reaction and secondary metabolites, transpiration and photosynthesis. DEGs encoding lignin synthesis, chalcone synthase (CHS) and anthocyanidin synthase (ANS) were prominent in V. bonariensis under Cd stress. The expression patterns of 10 DEGs, validated by quantitative real-time polymerase chain reaction (qRT-PCR), were in highly accordance with the RNA-Sequence (RNA-Seq) results. The novel strategies brought by our study was not only benefit for further studies on the tolerance of Cd and functional genomics in V. bonariensis, but also for the improvement molecular breeding and phytoremediation.

Recent Progress in Electrically Rechargeable Zinc-Air Batteries.

Over the past decade, the surging interest for higher-energy-density, cheaper, and safer battery technology has spurred tremendous research efforts in the development of improved rechargeable zinc-air batteries. Current zinc-air batteries suffer from poor energy efficiency and cycle life, owing mainly to the poor rechargeability of zinc and air electrodes. To achieve high utilization and cyclability in the zinc anode, construction of conductive porous framework through elegant optimization strategies and adaptation of alternate active material are employed. Equally, there is a need to design new and improved bifunctional oxygen catalysts with high activity and stability to increase battery energy efficiency and lifetime. Efforts to engineer catalyst materials to increase the reactivity and/or number of bifunctional active sites are effective for improving air electrode performance. Here, recent key advances in material development for rechargeable zinc-air batteries are described. By improving fundamental understanding of materials properties relevant to the rechargeable zinc and air electrodes, zinc-air batteries will be able to make a significant impact on the future energy storage for electric vehicle application. To conclude, a brief discussion on noteworthy concepts of advanced electrode and electrolyte systems that are beyond the current state-of-the-art zinc-air battery chemistry, is presented.

Transcriptome analysis of chrysanthemum (Dendranthema grandiflorum) in response to low temperature stress.

BACKGROUND: Chrysanthemum is one kind of ornamental plant well-known and widely used in the world. However, its quality and production were severely affected by low temperature conditions in winter and early spring periods. Therefore, we used the RNA-Seq platform to perform a de novo transcriptome assembly to analyze chrysanthemum (Dendranthema grandiflorum) transcription response to low temperature. RESULTS: Using Illumina sequencing technology, a total of 86,444,237 high-quality clean reads and 93,837 unigenes were generated from four libraries: T01, controls; T02, 4 °C cold acclimation (CA) for 24 h; T03, - 4 °C freezing treatments for 4 h with prior CA; and T04, - 4 °C freezing treatments for 4 h without prior CA. In total, 7583 differentially expressed genes (DEGs) of 36,462 annotated unigenes were identified. We performed GO and KEGG pathway enrichment analyses, and excavated a group of important cold-responsive genes related to low temperature sensing and signal transduction, membrane lipid stability, reactive oxygen species (ROS) scavenging and osmoregulation. These genes encode many key proteins in plant biological processes, such as protein kinases, transcription factors, fatty acid desaturase, lipid-transfer proteins, antifreeze proteins, antioxidase and soluble sugars synthetases. We also verified expression levels of 10 DEGs using quantitative real-time polymerase chain reaction (qRT-PCR). In addition, we performed the determination of physiological indicators of chrysanthemum treated at low temperature, and the results were basically consistent with molecular sequencing results. CONCLUSION: In summary, our study presents a genome-wide transcript profile of Dendranthema grandiflorum var. jinba and provides insights into the molecular mechanisms of D. grandiflorum in response to low temperature. These data contributes to our deeper relevant researches on cold tolerance and further exploring new candidate genes for chilling-tolerance and freezing-tolerance chrysanthemum molecular breeding.

Overexpression of DgWRKY4 Enhances Salt Tolerance in Chrysanthemum Seedlings.

High salinity seriously affects the production of chrysanthemum, so improving the salt tolerance of chrysanthemum becomes the focus and purpose of our research. The WRKY transcription factor (TF) family is highly associated with a number of processes of abiotic stress responses. We isolated DgWRKY4 from Dendranthema grandiflorum, and a protein encoded by this new gene contains two highly conserved WRKY domains and two C2H2 zinc-finger motifs. Then, we functionally characterized that DgWRKY4 was induced by salt, and DgWRKY4 overexpression in chrysanthemum resulted in increased tolerance to high salt stress compared to wild-type (WT). Under salt stress, the transgenic chrysanthemum accumulated less malondialdehyde, hydrogen peroxide (H2O2), and superoxide anion ([Formula: see text]) than WT, accompanied by more proline, soluble sugar, and activities of antioxidant enzymes than WT; in addition, a stronger photosynthetic capacity and a series of up-regulated stress-related genes were also found in transgenic chrysanthemum. All results demonstrated that DgWRKY4 is a positive regulatory gene responding to salt stress, via advancing photosynthetic capacity, promoting the operation of reactive oxygen species-scavenging system, maintaining membrane stability, enhancing the osmotic adjustment, and up-regulating transcript levels of stress-related genes. So, DgWRKY4 can serve as a new candidate gene for salt-tolerant plant breeding.

Chrysanthemum WRKY gene DgWRKY5 enhances tolerance to salt stress in transgenic chrysanthemum.

WRKY transcription factors play important roles in plant growth development, resistance and substance metabolism regulation. However, the exact function of the response to salt stress in plants with specific WRKY transcription factors remains unclear. In this research, we isolated a new WRKY transcription factor DgWRKY5 from chrysanthemum. DgWRKY5 contains two WRKY domains of WKKYGQK and two C2H2 zinc fingers. The expression of DgWRKY5 in chrysanthemum was up-regulated under various treatments. Meanwhile, we observed higher expression levels in the leaves contrasted with other tissues. Under salt stress, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) enzymes in transgenic chrysanthemum were significantly higher than those in WT, whereas the accumulation of H2O2, O2- and malondialdehyde (MDA) was reduced in transgenic chrysanthemum. Several parameters including root length, root length, fresh weight, chlorophyll content and leaf gas exchange parameters in transgenic chrysanthemum were much better compared with WT under salt stress. Moreover, the expression of stress-related genes DgAPX, DgCAT, DgNCED3A, DgNCED3B, DgCuZnSOD, DgP5CS, DgCSD1 and DgCSD2 was up-regulated in DgWRKY5 transgenic chrysanthemum compared with that in WT. These results suggested that DgWRKY5 could function as a positive regulator of salt stress in chrysanthemum.

Whole-transcriptome sequence analysis of differentially expressed genes in Phormium tenax under drought stress.

Phormium tenax is a kind of drought resistant garden plant with its rich and colorful leaves. To clarify the molecular mechanism of drought resistance in Phormium tenax, transcriptome was sequenced by the Illumina sequencing technology under normal and drought stress, respectively. A large number of contigs, transcripts and unigenes were obtained. Among them, only 30,814 unigenes were annotated by comparing with the protein databases. A total of 4,380 genes were differentially expressed, 2,698 of which were finally annotated under drought stress. Differentially expression analysis was also performed upon drought treatment. In KEGG pathway, the mechanism of drought resistance in Phormium tenax was explained from three aspects of metabolism and signaling of hormones, osmotic adjustment and reactive oxygen species metabolism. These results are helpful to understand the drought tolerance mechanism of Phormium tenax and will provide a precious genetic resource for drought-resistant vegetation breeding and research.

Overexpression of a chrysanthemum transcription factor gene DgNAC1 improves the salinity tolerance in chrysanthemum.

KEY MESSAGE: DgNAC1, a transcription factor of chrysanthemum, was functionally verified to confer salt stress responses by regulating stress-responsive genes. NAC transcription factors play effective roles in resistance to different abiotic stresses, and overexpressions of NAC TFs in Arabidopsis have been proved to be conducive in improving salinity tolerance. However, functions of NAC genes in chrysanthemum continue to be poorly understood. Here, we performed physiology and molecular experiments to evaluate roles of DgNAC1 in chrysanthemum salt stress responses. In this study, DgNAC1-overexpressed chrysanthemum was obviously more resistant to salt over the WT (wild type). Specifically, the transgenic chrysanthemum showed a higher survival rate and lower EC (electrolyte conductivity) than WT under salt stress. The transgenic chrysanthemum also showed fewer accumulations of MDA (malondialdehyde) and reactive oxygen species (H2O2 and O2-), greater activities of SOD (superoxide dismutase), POD (peroxidase) and CAT (catalase), as well as more proline content than WT under salt stress. Furthermore, stress-responsive genes in transgenic chrysanthemum were greater up-regulated than in WT under salinity stress. Thus, all results revealed that DgNAC1 worked as a positive regulator in responses to salt stress and it may be an essential gene for molecular breeding of salt-tolerant plants.