Transcriptome analysis during axillary bud growth in chrysanthemum (chrysanthemum×morifolium).

The chrysanthemum DgLsL gene, homologous with tomato Ls, is one of the earliest expressed genes controlling axillary meristem initiation. In this study, the wild-type chrysanthemum (CW) and DgLsL-overexpressed line 15 (C15) were used to investigate the regulatory mechanism of axillary bud development in chrysanthemum. Transcriptome sequencing was carried out to detect the differentially expressed genes of the axillary buds 0 h, 24 h and 48 h after decapitation. The phenotypic results showed that the number of axillary buds of C15 was significantly higher than CW. A total of 9,224 DEGs were identified in C15-0 vs. CW-0, 10,622 DEGs in C15-24 vs. CW-24, and 8,929 DEGs in C15-48 vs. CW-48.GO and KEGG pathway enrichment analyses showed that the genes of the flavonoid, phenylpropanoids and plant hormone pathways appeared to be differentially expressed, indicating their important roles in axillary bud germination. DgLsL reduces GA content in axillary buds by promoting GA2ox expression.These results confirmed previous studies on axillary bud germination and growth, and revealed the important roles of genes involved in plant hormone biosynthesis and signal transduction, aiding in the study of the gene patterns involved in axillary bud germination and growth.

Fluorinated poly(p-triphenyl piperidine) anion exchange membranes with robust dimensional stability for fuel cells.

Anion exchange membrane fuel cells (AEMFCs), which are more economical than proton exchange membrane fuel cells (PEMFCs), stand out in the context of the rapid development of renewable energy. Superacid-catalyzed ether-free aromatic polymers have recently received a lot of attention due to their exceptional performance, but their development has been hampered by the trade-off between the dimensional stability and ionic conductivity of anion exchange membranes (AEMs). Here, we introduced fluoroketones containing different numbers of fluorinated groups (x = 0, 3 and 6) in the main chain of p-terphenyl piperidine because of the favorable hydrophobic properties of fluorinated groups. The results show that fluorinated AEMs can enhance OH- conductivity by building more aggregated hydrophilic channels while ensuring dimensional stability. The PTF6-QAPTP AEM with more fluorinated groups has the most excellent performance at 80 °C with an OH- conductivity of 142.7 mS cm-1 and a swelling ratio (SR) of only 4.55 %. Additionally, it exhibits good alkali durability, with the OH- conductivity and quaternary ammonium (QA) cation retaining at 93.45% and 92.6%, respectively, after immersion in a 2 M NaOH solution at 80 °C for 1200 h. In addition, the power density of the PTF6-QAPTP based single cell reaches 849 mW cm-2 when the current density is 1600 mA cm-2. The PTF6-QAPTP based cell has a voltage retention of 88% after 80 h of stability testing at a constant current density of 300 mA cm-2 at 80 °C.

Extranodal natural killer/T-cell lymphoma with hepatosplenic involvement: a retrospective study of a consecutive 14-year case series.

Extranodal natural killer/T-cell lymphoma (ENKTL) with hepatosplenic involvement is rare, accounting for approximately 0.2% of ENKTL cases. The clinicopathologic features of ENKTL with hepatosplenic involvement are still poorly understood. Seven cases of ENKTL with hepatosplenic involvement were investigated retrospectively by clinical features, pathology, immunophenotype, genotype, Epstein-Barr virus (EBV) status, and survival analysis. The median age was 36 years; three patients (3/7) had a history of primary nasal ENKTL. Six cases (6/7) presented liver or spleen structures that were replaced by neoplasms, and the neoplastic cells displayed diffuse infiltration; one case (1/7) displayed neoplastic cells scattered in hepatic sinuses and portal areas. The cellular morphology and immunohistochemical features were similar to those of ENKTL involving other sites. Follow-up data were available in five of the seven patients. All five patients received first-line chemotherapy based on L-asparaginase. Three patients died, and two were still alive by the last follow-up. The median overall survival (OS) was 21 months. ENKTL with hepatosplenic involvement is rare, regardless of whether it is initial or secondary. There are two histopathologic patterns of ENKTL with hepatosplenic involvement, and L-asparaginase-based chemotherapy combined with AHSCT might yield good efficacy. Morphological features of ENKTL in the spleen and liver A The architecture of the spleen was affected, and dense infiltration of the neoplastic cells was observed in the left part; B Focal infiltration of the neoplastic cells was located in the red pulp; C Dense infiltration of the neoplastic cells in the liver, accompanied by fatty change of hepatocytes and congestion; D More neoplastic cells accumulated in sinusoidal region.

Effects of the crown ether cavity on the performance of anion exchange membranes.

Anion exchange membrane fuel cells (AEMFCs) have emerged as a promising alternative to proton exchange membrane fuel cells (PEMFCs) due to their adaptability to low-cost stack components and non-noble-metals catalysts. However, the poor alkaline resistance and low OH- conductivity of anion exchange membranes (AEMs) have impeded the large-scale implementation of AEMFCs. Herein, the preparation of a new type of AEMs with crown ether macrocycles in their main chains via a one-pot superacid catalyzed reaction was reported. The study aimed to examine the influence of crown ether cavity size on the phase separation structure, ionic conductivity and alkali resistance of anion exchange membranes. Attributed to the self-assembly of crown ethers, the poly (crown ether) (PCE) AEMs with dibenzo-18-crown-6-ether (QAPCE-18-6) exhibit an obvious phase separated structure and a maximum OH- conductivity of 122.5 mS cm-1 at 80 °C (ionic exchange capacity is 1.51 meq g-1). QAPCE-18-6 shows a good alkali resistance with the OH- conductivity retention of 94.5% albeit being treated in a harsh alkali condition. Moreover, the hydrogen/oxygen single cell equipped with QAPCE-18-6 can achieve a peak power density (PPD) of 574 mW cm-2 at a current density of 1.39 A cm-2.

Highly conductive branched poly(aryl piperidinium) anion exchange membranes with robust chemical stability.

Poly(aryl piperidinium) (PAP) anion exchange membranes (AEMs) furnish an important avenue for the commercialization of anion exchange membrane fuel cells (AEMFCs), but their ionic conductivity and alkali resistance still need to be improved. Here, we report the synthesis of PAP AEMs with a branched structure by the acid-catalyzed reaction and compare them with the main-chain AEMs. The experimental results show that the branched AEMs have higher OH- conductivity and alkaline resistance than the poly(terphenyl piperidine) (PTPQ1) AEM. The alkaline stability and OH- conductivity of the AEMs were further improved by a flexible multi-cation crosslinker. The results show that the branched poly(p-terphenyl triphenylmethane 1-methyl piperidine) membrane crosslinked by multi-cation (PTTPQ4-40) shows an excellent OH- conductivity (155.3 mS cm-1) at 80 °C. The OH- conductivity of the PTTPQ4-40 membrane was maintained at 92.1% after soaking in 2 M NaOH for 1080 h at 80 °C. In addition, the peak power density (PPD) of the crosslinked PTTPQ4-40 membrane can reach 656.7 mW cm-2. Compared to the PTPQ1 AEM, the PPD of the crosslinked PTTPQ4-40 AEM is increased by 38.6% in H2-O2. All of the results confirm that the PTTPQ4-40 AEM has excellent fuel cell application prospects.

The rapid appearance of homostyly in a cultivated distylous population of Primula forbesii.

Evolutionary breakdown from rigorous outbreeding to self-fertilization frequently occurs in angiosperms. Since the pollinators are not necessary, self-compatible populations often reduce investment in floral display characteristics and pollination reward. Primula forbesii is a biennial herb with distribution restricted to southwest China; it was initially a self-incompatible distylous species, but after 20 years of artificial domestication, homostyly appeared. This change in style provides an ideal material to explore the time required for plant mating systems to adapt to new environmental changes and test whether flower attraction has reduced following transitions to selfing. We did a survey in wild populations of P. forbesii where its seeds were originally collected 20 years ago and recorded the floral morph frequencies and morphologies. The floral morphologies, self-incompatibility, floral scent, and pollinator visitation between distyly and homostyly were compared in greenhouse. Floral morph frequencies of wild populations did not change, while the cultivated population was inclined to L-morph and produced homostyly. Evidence from stigma papillae and pollen size supports the hypothesis that the homostyly possibly originated from mutations of large effect genes in distylous linkage region. Transitions to self-compatible homostyly are accompanied by smaller corolla size, lower amounts of terpenoids, especially linalool and higher amounts of fatty acid derivatives. The main pollinators in the greenhouse were short-tongued Apis cerana. However, homostyly had reduced visiting frequency. The mating system of P. forbesii changed rapidly in just about 20 years of domestication, and our findings confirm the hypothesis that the transition to selfing is accompanied by decreased flower attraction.

External application of nitrogen alleviates toxicity of cadmium on poplars via starch and sucrose metabolism.

Phytoremediation technology can help achieve moderate cost and considerable effect with respect to the remediation of heavy metal (HM) pollution in soil and water. Many previous studies have suggested the role of nitrogen (N) in the alleviation of effects of HM on plants. Herein, we sought to determine the molecular mechanisms by which additional N supplementation mitigates cadmium (Cd) toxicity in poplars using a combination of physiological, transcriptomic and phosphoproteomic analyses. The application of N can alleviate the toxicity of Cd to Populus by reducing chlorophyll degradation, maintaining the stability of ions inside and outside the cell membrane and increasing the soluble sugar content. Plant samples from the control, Cd stress and Cd_N treatments were used for an integrated analysis of the transcriptome, as well as for phosphoproteomics analysis. Moreover, 1314 differentially expressed genes and 119 differentially expressed kinase genes were discovered. Application of additional N under Cd stress promoted the phosphorylation process. Furthermore, 51 significantly enriched phosphorylated protein sites and 23 differentially expressed kinases were identified using phosphoproteomic and proteomic analyses. Importantly, transcriptomic and phosphoproteomic analyses jointly determined that the application of N could activate corresponding gene expression [UDP-glucose-dehydrogenase (UGD), GAUT, PME, pectin lyase, UDP-glucose-pyrophosphorylase 2 (UGP2), sucrose phosphate synthase (SPS), SUS and SPP2] and protein phosphorylation (UGP2 and SPS) in the sugar and starch synthesis pathways, which promoted the synthesis of sucrose and soluble sugar and subsequently alleviated the damage caused by Cd.

Two-dimensional metal-organic framework-graphene oxide hybrid nanocomposite proton exchange membranes with enhanced proton conduction.

A novel two-dimensional (2D) zeolitic imidazolate framework-graphene oxide hybrid nanocomposite (ZIF-L@GO) is designed as an inorganic filler in sulfonated poly(ether ether ketone) (SPEEK). ZIF-L with unique leaf-like morphology is grown in-situ on the GO sheet in aqueous media at room temperature. The terminal imidazole linker in ZIF-L@GO and the -SO3H in SPEEK can form acid-base pairs in the membrane interface to produce low energy proton conduction highway. Benefiting from the unique structural advantage, the hybrid SP-ZIF-L@GO membranes displayed promoted physicochemical and electrochemical performances over the pure SPEEK. The SP-ZIF-L@GO-5 achieved a proton conductivity of 0.265 and 0.0364 S cm-1 at 70 °C-100% RH and 90 °C-40% RH, 1.76- and 6.24-fold higher than pure SPEEK, respectively. Meanwhile, a single cell based on SP-ZIF-L@GO-5 had an output power up to 652.82 mW cm-2 at 60 °C, 1.45 times higher than the pure SPEEK. In addition, the durability test was performed by holding open circuit voltage (OCV) for 24 h. The SP-ZIF-L@GO-5 provided better long-term stability than the pure SPEEK. These superior performance suggests a promising application in PEMFC.

Proteome-wide and lysine crotonylation profiling reveals the importance of crotonylation in chrysanthemum (Dendranthema grandiforum) under low-temperature.

BACKGROUND: Low-temperature severely affects the growth and development of chrysanthemum which is one kind of ornamental plant well-known and widely used in the world. Lysine crotonylation is a recently identified post-translational modification (PTM) with multiple cellular functions. However, lysine crotonylation under low-temperature stress has not been studied. RESULTS: Proteome-wide and lysine crotonylation of chrysanthemum at low-temperature was analyzed using TMT (Tandem Mass Tag) labeling, sensitive immuno-precipitation, and high-resolution LC-MS/MS. The results showed that 2017 crotonylation sites were identified in 1199 proteins. Treatment at 4 °C for 24 h and - 4 °C for 4 h resulted in 393 upregulated proteins and 500 downregulated proteins (1.2-fold threshold and P < 0.05). Analysis of biological information showed that lysine crotonylation was involved in photosynthesis, ribosomes, and antioxidant systems. The crotonylated proteins and motifs in chrysanthemum were compared with other plants to obtain orthologous proteins and conserved motifs. To further understand how lysine crotonylation at K136 affected APX (ascorbate peroxidase), we performed a site-directed mutation at K136 in APX. Site-directed crotonylation showed that lysine decrotonylation at K136 reduced APX activity, and lysine complete crotonylation at K136 increased APX activity. CONCLUSION: In summary, our study comparatively analyzed proteome-wide and crotonylation in chrysanthemum under low-temperature stress and provided insights into the mechanisms of crotonylation in positively regulated APX activity to reduce the oxidative damage caused by low-temperature stress. These data provided an important basis for studying crotonylation to regulate antioxidant enzyme activity in response to low-temperature stress and a new research ideas for chilling-tolerance and freezing-tolerance chrysanthemum molecular breeding.

Multiple Enhancement Effects of Crown Ether in Tröger's Base Polymers on the Performance of Anion Exchange Membranes.

The development of anion exchange membranes (AEMs) is hindered by the trade-off of ionic conductivity, alkaline stability, and mechanical properties. Tröger's base polymers (Tb-polymers) are recognized as promising membrane materials to overcome these obstacles. Herein, the AEMs made from Tb-poly(crown ether)s (Tb-PCEs) show good comprehensive performance. The influence of crown ether on the conductivity and alkaline stability of AEMs has been investigated in detail. The formation of hydronium ion-crown ether complexes and an obvious microphase-separated structure formed by the existence of crown ether can enhance the conductivity of the AEMs. The maximum OH- conductivity of 141.5 mS cm-1 is achieved from the Tb-PCEs based AEM (Tb-PCE-1) at 80 °C in ultrapure water. The ion-dipole interaction of the Na+ with crown ether can protect the quaternary ammonium from the attack of OH- to improve the alkaline stability of AEMs. After 675 h of alkaline treatment, the OH- conductivity of Tb-PCE-1 decreases by only 6%. The Tb-PCE-1-based single cell shows a peak power density of 0.202 W cm-2 at 80 °C. The prominent physicochemical properties are attributed to the well-developed microstructure of the Tb-PCEs, as revealed by TEM, AFM, and SAXS observations.

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.

Transcriptomic Analysis of Verbena bonariensis Leaves Under Low-Temperature Stress.

Verbena bonariensis is a valuable plant for both ornament and flower border. As a major constraint, low temperature affects the growing development and survival of V. bonariensis. However, there are few systematic studies in terms of molecular mechanism on the tolerance of low temperature in V. bonariensis. In this study, Illumina sequencing technology was applied to analyze the cold resistance mechanism of plants. Six cDNA libraries were obtained from two samples of two groups, the cold-treated group and the control group. A total of 271,920 unigenes were produced from 406,641 assembled transcripts. Among these, 19,003 differentially expressed genes (DEGs) (corrected p-value <0.01, |log2(fold change) | >3) were obtained, including 9852 upregulated and 9151 downregulated genes. The antioxidant enzyme system, photosynthesis, plant hormone signal transduction, fatty acid metabolism, starch and sucrose metabolism pathway, and transcription factors were analyzed. Based on these results, series of candidate genes related to cold stress were screened out and discussed. The physiological indexes related to response mechanism of low temperature were tested. Eleven upregulated DEGs were validated by Quantitative Real-time PCR. In this study, we provided the transcriptome sequence resource of V. bonariensis and used these data to realize its molecular mechanism under cold stress. The results contributed to valuable clues for genetic studies and helped to screen candidate genes for cold-resistance breeding.

The Overexpression of a Transcription Factor Gene VbWRKY32 Enhances the Cold Tolerance in Verbena bonariensis.

Cold stress poses a serious threat to the survival and bloom of Verbena bonariensis. The enhancement of the cold tolerance of V. bonariensis is the central concern of our research. The WRKY transcription factor (TF) family was paid great attention to in the field of abiotic stress. The VbWRKY32 gene was obtained from V. bonariensis. The VbWRKY32 predicted protein contained two typical WRKY domains and two C2H2 zinc-finger motifs. Under cold stress, VbWRKY32 in leaves was more greatly induced than that in stems and roots. The overexpression (OE) in V. bonariensis increased cold tolerance compared with wild-type (WT). Under cold stress, the OE lines possessed showed greater recovery after cold-treatment restoration ratios, proline content, soluble sugar content, and activities of antioxidant enzymes than WT; the relative electrolyte conductivity (EL), the accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2 -) are lower in OE lines than that in WT. In addition, a series of cold-response genes of OE lines were compared with WT. The results revealed that VbWRKY32 worked as a positive regulator by up-regulating transcription levels of cold-responsive genes. The genes above can contribute to the elevation of antioxidant activities, maintain the membrane stability, and raise osmotic regulation ability, leading to the enhancement of the survival capacity under cold stress. According to this work, VbWRKY32 could serve as an essential gene to confer enhanced cold tolerance in plants.

Transcriptome Comparative Analysis of Salt Stress Responsiveness in Chrysanthemum (Dendranthema grandiflorum) Roots by Illumina- and Single-Molecule Real-Time-Based RNA Sequencing.

Salt response has long been considered a polygenic-controlled character in plants. Under salt stress conditions, plants respond by activating a great amount of proteins and enzymes. To develop a better understanding of the molecular mechanism and screen salt responsive genes in chrysanthemum under salt stress, we performed the RNA sequencing (RNA-seq) on both salt-processed chrysanthemum seedling roots and the control group, and gathered six cDNA databases eventually. Moreover, to overcome the Illumina HiSeq technology's limitation on sufficient length of reads and improve the quality and accuracy of the result, we combined Illumina HiSeq with single-molecule real-time sequencing (SMRT-seq) to decode the full-length transcripts. As a result, we successfully collected 550,823 unigenes, and from which we selected 48,396 differentially expressed genes (DEGs). Many of these DEGs were associated with the signal transduction, biofilm system, antioxidant system, and osmotic regulation system, such as mitogen-activated protein kinase (MAPK), Acyl-CoA thioesterase (ACOT), superoxide (SOD), catalase (CAT), peroxisomal membrane protein (PMP), and pyrroline-5-carboxylate reductase (P5CR). The quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 15 unigenes was performed to test the data validity. The results were highly consistent with the RNA-seq results. In all, these findings could facilitate further detection of the responsive molecular mechanism under salt stress. They also provided more accurate candidate genes for genetic engineering on salt-tolerant chrysanthemums.

Chrysanthemum DgWRKY2 Gene Enhances Tolerance to Salt Stress in Transgenic Chrysanthemum.

WRKY transcription factors (TFs) play a vital part in coping with different stresses. In this study, DgWRKY2 was isolated from Dendranthema grandiflorum. The gene encodes a 325 amino acid protein, belonging to the group II WRKY family, and contains one typical WRKY domain (WRKYGQK) and a zinc finger motif (C-X4-5-C-X22-23-H-X1-H). Overexpression of DgWRKY2 in chrysanthemum enhanced tolerance to high-salt stress compared to the wild type (WT). In addition, the activities of antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT)), proline content, soluble sugar content, soluble protein content, and chlorophyll content of transgenic chrysanthemum, as well as the survival rate of the transgenic lines, were on average higher than that of the WT. On the contrary, hydrogen peroxide (H2O2), superoxide anion (O2-), and malondialdehyde (MDA) accumulation decreased compared to WT. Expression of the stress-related genes DgCAT, DgAPX, DgZnSOD, DgP5CS, DgDREB1A, and DgDREB2A was increased in the DgWRKY2 transgenic chrysanthemum compared with their expression in the WT. In conclusion, our results indicate that DgWRKY2 confers salt tolerance to transgenic chrysanthemum by enhancing antioxidant and osmotic adjustment. Therefore, this study suggests that DgWRKY2 could be used as a reserve gene for salt-tolerant plant breeding.

Whole-Transcriptome Sequence Analysis of Verbena bonariensis in Response to Drought Stress.

Drought is an important abiotic factor that threatens the growth and development of plants. Verbena bonariensis is a widely used landscape plant with a very high ornamental value. We found that Verbena has drought tolerance in production practice, so in order to delve into its mechanism of drought resistance and screen out its drought-resistance genes, we used the RNA-Seq platform to perform a de novo transcriptome assembly to analyze Verbena transcription response to drought stress. By high-throughput sequencing with Illumina Hiseq Xten, a total of 44.59 Gb clean data was obtained from T01 (control group) and T02 (drought experiment group). After assembly, 111,313 unigenes were obtained, and 53,757 of them were annotated by compared databases. In this study, 4829 differentially expressed genes were obtained, of which 4165 were annotated. We performed GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses, and explored a lot of differently expressed genes related to plant energy production, hormone synthesis, cell signal transduction, and metabolism to understand the stress response of Verbena in drought stress. In addition, we also found that a series of TFs related to drought-resistance of Verbena and provide excellent genetic resources for improving the drought tolerance of crops.

Anion Conductive Triblock Copolymer Membranes with Flexible Multication Side Chain.

To achieve highly conductive and stable anion exchange membranes (AEMs) for fuel cells, novel triblock copolymer AEMs bearing flexible side chain were synthesized. The triblock structure and flexible side chain are responsible for the developed hydrophilic/hydrophobic phase separated morphology and well-connected ion conducting channels, as confirmed by transmission electron microscopy. As a result, the triblock copolymer AEMs with flexible side chain (ABA-TQA- x) demonstrated considerably higher conductivities, up to 130.5 mS cm-1 at 80 °C, than the AEMs with monocation side chain (ABA-MQA). Furthermore, the long alkyl spacer between the backbone and quaternary ammonium groups, as well as long intercation spacer limits the water swelling of the membranes to some degree, resulting in good alkaline stability. The ABA-TQA-44 membrane retained 84.7% and 83.1% of its original conductivity and ionic exchange capacity, respectively, after immersed in a 1 M aqueous KOH solution at 80 °C for 480 h. Furthermore, the peak power density of a H2/O2 single cell using ABA-TQA-44 is 204.6 mW cm-2 at a current density of 500 mA cm-2 at 80 °C.

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.