Genome-Wide Identification and Expression Analysis of the bHLH Transcription Factor Family in Wintersweet (Chimonanthus praecox).

Wintersweet (Chimonanthus praecox (L.) Link, Calycanthaceae) is an esteemed ornamental flowering shrub known for its distinct blooming period in winter, vibrant color petals, and captivating floral fragrance. Basic helix-loop-helix (bHLH) transcription factors (TFs) play pivotal roles as key regulators in secondary metabolites biosynthesis, growth, and development in plants. However, the systematic analysis of the bHLH family members and their role in the regulation of floral traits in Wintersweet remains insufficiently understood. To bridge this knowledge gap, we conducted a comprehensive genome-wide analysis of the C. praecox bHLH (CpbHLH) gene family, identifying a total of 131 CpbHLH genes across 11 chromosomes. Phylogenetic analysis classified these CpbHLH genes into 23 subfamilies, wherein most members within the same subfamily exhibited analogous intron/exon patterns and motif composition. Moreover, the expansion of the CpbHLH gene family was primarily driven by segmental duplication, with duplicated gene pairs experiencing purifying selection during evolution. Transcriptomic analysis revealed diverse expression patterns of CpbHLH genes in various tissues and distinct stages of Wintersweet flower development, thereby suggesting their involvement in a diverse array of physiological processes. Furthermore, yeast 2-hybrid assay demonstrated interaction between CpbHLH25 and CpbHLH59 (regulators of floral scent and color) as well as with CpbHLH112 and CpMYB2, suggesting potential coordinately regulation of secondary metabolites biosynthesis in Wintersweet flowers. Collectively, our comprehensive analysis provides valuable insights into the structural attributes, evolutionary dynamics, and expression profiles of the CpbHLH gene family, laying a solid foundation for further explorations of the multifaceted physiological and molecular roles of bHLH TFs in Wintersweet.

CpCAF1 from Chimonanthus praecox Promotes Flowering and Low-Temperature Tolerance When Expressed in Arabidopsis thaliana.

CCR4-associated factor I (CAF1) is a deadenylase that plays a critical role in the initial step of mRNA degradation in most eukaryotic cells, and in plant growth and development. Knowledge of CAF1 proteins in woody plants remains limited. Wintersweet (Chimonanthus praecox) is a highly ornamental woody plant. In this study, CpCAF1 was isolated from wintersweet. CpCAF1 belongs to the DEDDh (Asp-Glu-Asp-Asp-His) subfamily of the DEDD (Asp-Glu-Asp-Asp) nuclease family. The amino acid sequence showed highest similarity to the homologous gene of Arabidopsis thaliana. In transgenic Arabidopsis overexpressing CpCAF1, the timing of bolting, formation of the first rosette, and other growth stages were earlier than those of the wild-type plants. Root, lateral branch, rosette leaf, and silique growth were positively correlated with CpCAF1 expression. FLOWERING LOCUS T (FT) and SUPPRESSOROF OVEREXPRESSION OF CO 1 (SOC1) gene expression was higher while EARLY FLOWERING3 (ELF3) and FLOWERING LOCUS C (FLC) gene expression of transgenic Arabidopsis was lower than the wild type grown for 4 weeks. Plant growth and flowering occurrences were earlier in transgenic Arabidopsis overexpressing CpCAF1 than in the wild-type plants. The abundance of the CpCAF1 transcript grew steadily, and significantly exceeded the initial level under 4 °C in wintersweet after initially decreasing. After low-temperature exposure, transgenic Arabidopsis had higher proline content and stronger superoxide dismutase activity than the wild type, and the malondialdehyde level in transgenic Arabidopsis was decreased significantly by 12 h and then increased in low temperature, whereas it was directly increased in the wild type. A higher potassium ion flux in the root was detected in transgenic plants than in the wild type with potassium deficiency. The CpCAF1 promoter was a constitutive promoter that contained multiple cis-acting regulatory elements. The DRE, LTR, and MYB elements, which play important roles in response to low temperature, were identified in the CpCAF1 promoter. These findings indicate that CpCAF1 is involved in flowering and low-temperature tolerance in wintersweet, and provide a basis for future genetic and breeding research on wintersweet.

Optimizing DUS testing for Chimonanthus praecox using feature selection based on a genetic algorithm.

Chimonanthus praecox is a famous traditional flower in China with high ornamental value. It has numerous varieties, yet its classification is highly disorganized. The distinctness, uniformity, and stability (DUS) test enables the classification and nomenclature of various species; thus, it can be used to classify the Chimonanthus varieties. In this study, flower traits were quantified using an automatic system based on pattern recognition instead of traditional manual measurement to improve the efficiency of DUS testing. A total of 42 features were quantified, including 28 features in the DUS guidelines and 14 new features proposed in this study. Eight algorithms were used to classify wintersweet, and the random forest (RF) algorithm performed the best when all features were used. The classification accuracy of the outer perianth was the highest when the features of the different parts were used for classification. A genetic algorithm was used as the feature selection algorithm to select a set of 22 reduced core features and improve the accuracy and efficiency of the classification. Using the core feature set, the classification accuracy of the RF model improved to 99.13%. Finally, K-means was used to construct a pedigree cluster tree of 23 varieties of wintersweet; evidently, wintersweet was clustered into a single class, which can be the basis for further study of genetic relationships among varieties. This study provides a novel method for DUS detection, variety identification, and pedigree analysis.

Overexpression of a Senescence-Related Gene CpSRG1 from Wintersweet (Chimonanthus praecox) Promoted Growth and Flowering, and Delayed Senescence in Transgenic Arabidopsis.

Plant senescence is a complex process that is controlled by developmental regulation and genetic programs. A senescence-related gene CpSRG1, which belongs to the 2OG-Fe(II) dioxygenase superfamily, was characterized from wintersweet, and the phylogenetic relationship of CpSRG1 with homologs from other species was investigated. The expression analysis by qRT-PCR (quantitative real-time PCR) indicated that CpSRG1 is abundant in flower organs, especially in petals and stamens, and the highest expression of CpSRG1 was detected in stage 6 (withering period). The expression patterns of the CpSRG1 gene were further confirmed in CpSRG1pro::GUS (ß-glucuronidase) plants, and the activity of the CpSRG1 promoter was enhanced by exogenous Eth (ethylene), SA (salicylic acid), and GA3 (gibberellin). Heterologous overexpression of CpSRG1 in Arabidopsis promoted growth and flowering, and delayed senescence. Moreover, the survival rates were significantly higher and the root lengths were significantly longer in the transgenic lines than in the wild-type plants, both under low nitrogen stress and GA3 treatment. This indicated that the CpSRG1 gene may promote the synthesis of assimilates in plants through the GA pathway, thereby improving growth and flowering, and delaying senescence in transgenic Arabidopsis. Our study has laid a satisfactory foundation for further analysis of senescence-related genes in wintersweet and wood plants. It also enriched our knowledge of the 2OG-Fe(II) dioxygenase superfamily, which plays a variety of important roles in plants.

A tandem CCCH type zinc finger protein gene CpC3H3 from Chimonanthus praecox promotes flowering and enhances drought tolerance in Arabidopsis.

BACKGROUND: CCCH-type zinc finger proteins play important roles in plant development and biotic/abiotic stress responses. Wintersweet (Chimonanthus praecox) is a popular ornamental plant with strong resistance to various stresses, which is a good material for exploring gene resource for stress response. In this study, we isolated a CCCH type zinc finger protein gene CpC3H3 (MZ964860) from flower of wintersweet and performed functional analysis with a purpose of identifying gene resource for floral transition and stress tolerance. RESULTS: CpC3H3 was predicted a CCCH type zinc finger protein gene encoding a protein containing 446 amino acids with five conserved C-X8-C-X5-C-X3-H motifs. CpC3H3 was localized in the cell membrane but with a nuclear export signal at the N-terminal. Transcripts of CpC3H3 were significantly accumulated in flower buds at floral meristem formation stage, and were induced by polyethylene glycol. Overexpression of CpC3H3 promoted flowering, and enhanced drought tolerance in transgenic A. thaliana. CpC3H3 overexpression affects the expression level of genes involved in flower inducement and stress responses. Further comparative studies on physiological indices showed the contents of proline and soluble sugar, activity of peroxidase and the rates of electrolyte leakage were significantly increased and the content of malondialdehyde and osmotic potential was significantly reduced in transgenic A. thaliana under PEG stress. CONCLUSION: Overall, CpC3H3 plays a role in flowering inducement and drought tolerance in transgenic A. thaliana. The CpC3H3 gene has the potential to be used to promote flowering and enhance drought tolerance in plants.

Anti-oxidative and anti-inflammatory activities of the ethanol extract of edible flower from Chimonanthus praecox.

Chimonanthi Praecocis Flos, namely wintersweet flower, is the edible flower or flower bud of Chimonanthus praecox (L.) Link which is a deciduous shrub plant originated from China and is widely cultivated as a garden or ornamental plant all over the world. However, few studies focused on its anti-inflammatory property. In the present study, we explored the anti-inflammatory and anti-oxidative activities of ethanol extract of Chimonanthi Praecocis Flos (CPE) which contained 7.980% ± 0.176% total flavonoids and 1.461% ± 0.041% total alkaloids. In LPS-stimulated RAW264.7 macrophages, CPE significantly decreased the production of NO and prostaglandin E2 (PGE2) through reducing the expressions of their synthases-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). It also suppressed the transcription and translation of pro-inflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Further research revealed that CPE impeded the phosphorylation and degradation of IκBα, thus restraining the nuclear translocation of p65, and consequently dampening NF-κB signaling. In endotoxemia mice, several pro-inflammatory cytokines in serum were also decreased after CPE treatment. Besides anti-inflammatory activity, anti-oxidative activity is another important capacity of wintersweet flower. Indeed, CPE reduced LPS-elevated intracellular total reactive oxygen species (ROS) level by weakening NADPH oxidase activity in cell system. Moreover, it directly scavenged DPPH radical and superoxide anion, and exerted ferric reducing ability in cell-free system. Our findings demonstrate that wintersweet flower can be used as a beneficial natural product or an additive by virtue of its anti-oxidative and anti-inflammatory properties.

CpMAX1a, a Cytochrome P450 Monooxygenase Gene of Chimonanthus praecox Regulates Shoot Branching in Arabidopsis.

Strigolactones (SLs) are a class of important hormones in the regulation of plant branching. In the model plant Arabidopsis, AtMAX1 encodes a cytochrome P450 protein and is a crucial gene in the strigolactone synthesis pathway. Yet, the regulatory mechanism of MAX1 in the shoot branching of wintersweet (Chimonanthus praecox) remains unclear. Here we identified and isolated three MAX1 homologous genes, namely CpMAX1a, CpMAX1b, and CpMAX1c. Quantitative real-time PCR (qRT-PCR) revealed the expression of CpMAX1a in all tissues, being highest in leaves, whereas CpMAX1b was only expressed in stems, while CpMAX1c was expressed in both roots and stem tips. However, CpMAX1a's expression decreased significantly after decapitation; hence, we verified its gene function. CpMAX1a was located in Arabidopsis chloroplasts. Overexpressing CpMAX1a restored the phenotype of the branching mutant max1−3, and reduced the rosette branch number, but resulted in no significant phenotypic differences from the wild type. Additionally, expression of AtBRC1 was significantly upregulated in transgenic lines, indicating that the CpMAX1a gene has a function similar to the homologous gene of Arabidopsis. In conclusion, our study shows that CpMAX1a plays a conserved role in regulating the branch development of wintersweet. This work provides a molecular and theoretical basis for better understanding the branch development of wintersweet.

Sustainable and Green Synthesis of Waste-Biomass-Derived Carbon Dots for Parallel and Semi-Quantitative Visual Detection of Cr(VI) and Fe3.

Carbon dot (CD)-based multi-mode sensing has drawn much attention owing to its wider application range and higher availability compared with single-mode sensing. Herein, a simple and green methodology to construct a CD-based dual-mode fluorescent sensor from the waste biomass of flowers of wintersweet (FW-CDs) for parallel and semi-quantitative visual detection of Cr(VI) and Fe3+ was firstly reported. The FW-CD fluorescent probe had a high sensitivity to Cr(VI) and Fe3+ with wide ranges of linearity from 0.1 to 60 µM and 0.05 to 100 µM along with low detection limits (LOD) of 0.07 µM and 0.15 µM, respectively. Accordingly, the FW-CD-based dual-mode sensor had an excellent parallel sensing capacity toward Cr(VI) and Fe3+ with high selectivity and strong anti-interference capability by co-using dual-functional integration and dual-masking strategies. The developed parallel sensing platform was successfully applied to Cr(VI) and Fe3+ quantitative detection in real samples with high precision and good recovery. More importantly, a novel FW-CD-based fluorescent hydrogel sensor was fabricated and first applied in the parallel and semi-quantitative visual detection of Cr(VI) and ferrous ions in industrial effluent and iron supplements, further demonstrating the significant advantage of parallel and visual sensing strategies.

Functional Characterization of the CpNAC1 Promoter and Gene from Chimonanthus praecox in Arabidopsis.

The NAC (NAM, ATAF, and CUC) gene family is one of the largest plant-specific transcription factor families. Its members have various biological functions that play important roles in regulating plant growth and development and in responding to biotic and abiotic stresses. However, their functions in woody plants are not fully understood. In this study, we isolated an NAC family member, the CpNAC1 promoter and gene, from wintersweet. CpNAC1 was localized to the nucleus and showed transcriptional activation activity. qRT-PCR analyses revealed that the gene was expressed in almost all tissues tested, with the highest levels found in mature leaves and flower buds. Moreover, its expression was induced by various abiotic stresses and ABA treatment. Its expression patterns were further confirmed in CpNAC1pro:GUS (ß-glucuronidase) plants. Among all the transgenic lines, CpNAC1pro-D2 showed high GUS histochemical staining and activity in different tissues of Arabidopsis. Furthermore, its GUS activity significantly increased in response to various abiotic stresses and ABA treatment. This may be related to the stress-related cis-elements, such as ABRE and MYB, which clustered in the CpNAC1pro-D2 segment, suggesting that CpNAC1pro-D2 is the core segment that responds to abiotic stresses and ABA. In addition, CpNAC1-overexpressed Arabidopsis plants had weaker osmosis tolerance than the wild-type plants, demonstrating that CpNAC1 may negatively regulate the drought stress response in transgenic Arabidopsis. Our results provide a foundation for further analyses of NAC family genes in wintersweet, and they broaden our knowledge of the roles that NAC family genes may play in woody plants.

Carotenoid Cleavage Dioxygenase Genes of Chimonanthus praecox, CpCCD7 and CpCCD8, Regulate Shoot Branching in Arabidopsis.

Strigolactones (SLs) regulate plant shoot development by inhibiting axillary bud growth and branching. However, the role of SLs in wintersweet (Chimonanthus praecox) shoot branching remains unknown. Here, we identified and isolated two wintersweet genes, CCD7 and CCD8, involved in the SL biosynthetic pathway. Quantitative real-time PCR revealed that CpCCD7 and CpCCD8 were down-regulated in wintersweet during branching. When new shoots were formed, expression levels of CpCCD7 and CpCCD8 were almost the same as the control (un-decapitation). CpCCD7 was expressed in all tissues, with the highest expression in shoot tips and roots, while CpCCD8 showed the highest expression in roots. Both CpCCD7 and CpCCD8 localized to chloroplasts in Arabidopsis. CpCCD7 and CpCCD8 overexpression restored the phenotypes of branching mutant max3-9 and max4-1, respectively. CpCCD7 overexpression reduced the rosette branch number, whereas CpCCD8 overexpression lines showed no phenotypic differences compared with wild-type plants. Additionally, the expression of AtBRC1 was significantly up-regulated in transgenic lines, indicating that two CpCCD genes functioned similarly to the homologous genes of the Arabidopsis. Overall, our study demonstrates that CpCCD7 and CpCCD8 exhibit conserved functions in the CCD pathway, which controls shoot development in wintersweet. This research provides a molecular and theoretical basis for further understanding branch development in wintersweet.

Overexpression of CpWRKY75 from Chimonanthus praecox Promotes Flowering Time in Transgenic Arabidopsis.

WRKY transcription factors play critical roles in the physiological processes of plants. Although the roles of WRKYs have been characterized in some model plants, their roles in woody plants, especially wintersweet (Chimonanthus praecox), are largely unclear. In this study, a wintersweet WRKY gene named CpWRKY75 belonging to group IIc was isolated and its characteristics were identified. CpWRKY75 is a nucleus-localized protein, and exhibited no transcriptional activation activity in yeast. CpWRKY75 was highly expressed in flowers at different bloom stages. Ectopic expression of CpWRKY75 significantly promoted the flowering time of transgenic Arabidopsis (Arabidopsis thaliana), as determined by the rosette leaf number and first flower open time. The expression levels of flowering-related genes were quantified by qRT-PCR, and the results suggested that CpWRKY75 had obvious influence on the expression level of MICRORNA156C (MIR156C), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9), FLOWERING LOCUS T (FT), LEAFY (LFY), SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), APETALA1 (AP1), CAULIFLOWER (CAL), and FRUITFULL (FUL). These results suggest that CpWRKY75 might have a flowering time regulation function, and additionally provide a new gene resource for the genetic engineering of woody flowering plants.

The chromosome-level wintersweet (Chimonanthus praecox) genome provides insights into floral scent biosynthesis and flowering in winter.

BACKGROUND: Wintersweet (Chimonanthus praecox), an important ornamental plant, has evolved unique fragrant aroma and winter-flowering properties, which are critical for its successful sexual reproduction. However, the molecular mechanisms underlying these traits are largely unknown in this species. In addition, wintersweet is also a typical representative species of the magnoliids, where the phylogenetic position of which relative to eudicots and monocots has not been conclusively resolved. RESULTS: Here, we present a chromosome-level wintersweet genome assembly with a total size of 695.36 Mb and a draft genome assembly of Calycanthus chinensis. Phylogenetic analyses of 17 representative angiosperm genomes suggest that Magnoliids and eudicots are sister to monocots. Whole-genome duplication signatures reveal two major duplication events in the evolutionary history of the wintersweet genome, with an ancient one shared by Laurales, and a more recent one shared by the Calycantaceae. Whole-genome duplication and tandem duplication events have significant impacts on copy numbers of genes related to terpene and benzenoid/phenylpropanoid (the main floral scent volatiles) biosynthesis, which may contribute to the characteristic aroma formation. An integrative analysis combining cytology with genomic and transcriptomic data reveals biological characteristics of wintersweet, such as floral transition in spring, floral organ specification, low temperature-mediated floral bud break, early blooming in winter, and strong cold tolerance. CONCLUSIONS: These findings provide insights into the evolutionary history of wintersweet and the relationships among the Magnoliids, monocots, and eudicots; the molecular basis underlying floral scent biosynthesis; and winter flowering, and highlight the utility of multi-omics data in deciphering important ornamental traits in wintersweet.

Molecular Cloning and Functional Characterization of CpMYC2 and CpBHLH13 Transcription Factors from Wintersweet (Chimonanthus praecox L.).

Wintersweet (Chimonanthus praecox L.) is an ornamental and economically significant shrub known for its unique flowering characteristics, especially the emission of abundant floral volatile organic compounds. Thus, an understanding of the molecular mechanism of the production of these compounds is necessary to create new breeds with high volatile production. In this study, two bHLH transcription factors (CpMYC2 and CpbHLH13) of Wintersweet H29 were functionally characterized to illustrate their possible role in the production of volatile compounds. The qRT-PCR results showed that the expression of CpMYC2 and CpbHLH13 increased from the flower budding to full bloom stage, indicating that these two genes may play an essential role in blooming and aroma production in wintersweet. Gas chromatography-mass spectroscopy (GC-MS) analysis revealed that the overexpression of CpMYC2 in arabidopsis (Arabidopsis thaliana) AtMYC2-2 mutant (Salk_083483) and tobacco (Nicotiana tabaccum) genotype Petit Havana SR1 significantly increased floral volatile monoterpene, especially linalool, while the overexpression of CpbHLH13 in Arabidopsis thaliana ecotype Columbia-0 (Col-0) and tobacco genotype SR1 increased floral sesquiterpene ß-caryophyllene production in both types of transgenic plants respectively. High expression of terpene synthase (TPS) genes in transgenic A. thaliana along with high expression of CpMYC2 and CpbHLH13 in transgenic plants was also observed. The application of a combination of methyl jasmonic acid (MeJA) and gibberellic acid (GA3) showed an increment in linalool production in CpMYC2-overexpressing arabidopsis plants, and the high transcript level of TPS genes also suggested the involvement of CpMYC2 in the jasmonic acid (JA) signaling pathway. These results indicate that both the CpMYC2 and CpbHLH13 transcription factors of wintersweet are possibly involved in the positive regulation and biosynthesis of monoterpene (linalool) and sesquiterpene (ß-caryophyllene) in transgenic plants. This study also indicates the potential application of wintersweet as a valuable genomic material for the genetic modification of floral scent in other flowering plants that produce less volatile compounds.

Identification and Molecular Characterization of Geranyl Diphosphate Synthase (GPPS) Genes in Wintersweet Flower.

Geranyl diphosphate synthase (GPPS) is a plastid localized enzyme that catalyzes the biosynthesis of Geranyl diphosphate (GPP), which is a universal precursor of monoterpenes. Wintersweet (Chimonanthus praecox L.), a famous deciduous flowering shrub with a strong floral scent character, could have GPPS-like homologs that are involved in monoterpenes biosynthesis, but it remains unclear. In the present study, five full-length GPPS and geranylgeranyl diphosphate synthases (GGPPS) genes were identified in the wintersweet transcriptome database. The isolated cDNAs showed high protein sequence similarity with the other plants GPPS and GGPPS. The phylogenetic analysis further classified these cDNAs into four distinct clades, representing heterodimeric GPPS small subunits (SSU1 and SSU2), homodimeric GPPS, and GGPPS. Analysis of temporal expression revealed that all genes have the highest transcript level at the full-open flower stage. From tissue-specific expression analysis, CpGPPS.SSU1 and CpGGPPS1 were predominantly expressed in petal and flower, whereas CpGPPS.SSU2, GPPS, and GGPPS2 showed a constitutive expression. Additionally, the subcellular localization assay identified the chloroplast localization of SSUs and GGPPSs proteins, and the yeast two-hybrid assay showed that both CpGPPS.SSU1 and CpGPPS.SSU2 can interact with the GGPPS proteins. Taken together, these preliminary results suggest that the heterodimeric GPPS can regulate floral scent biosynthesis in wintersweet flower.

Expression of the subgroup IIIf bHLH transcription factor CpbHLH1 from Chimonanthus praecox (L.) in transgenic model plants inhibits anthocyanin accumulation.

KEY MESSAGE: Overexpression of CpbHLH1 in Arabidopsis and tobacco resulted in a dramatic decrease in anthocyanin accumulation by repressing the expression of late biosynthesis genes in the flavonoid biosynthesis pathway. Many basic helix-loop-helix (bHLH) transcription factors (TFs) of subgroup IIIf have been characterized as anthocyanin-associated activators in higher plants, but information regarding bHLH TFs that inhibit anthocyanin accumulation remains scarce. In this study, the subgroup IIIf bHLH TF CpbHLH1 from Chimonanthus praecox (L.) was identified as a negative regulator of anthocyanin accumulation. Our results showed that overexpression of CpbHLH1 in model plant species, Arabidopsis and tobacco, resulted in a dramatic decrease in anthocyanin content, whereas the content of proanthocyanidin was little affected. Quantitative RT-PCR (qRT-PCR) assays of the structural genes in the flavonoid biosynthesis pathway revealed that CpbHLH1 inhibits anthocyanin accumulation mainly through repressing the expression of late biosynthesis genes (LBGs). Interactions between CpbHLH1 protein and AtPAP1/NtAN2 protein were detected via yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. This is the first bHLH repressor of anthocyanin biosynthesis identified in dicotyledons. These results can help us better understand the anthocyanin regulatory network in plants and may provide insights into the diverse functions of bHLH proteins.

CpWRKY71, a WRKY Transcription Factor Gene of Wintersweet (Chimonanthus praecox), Promotes Flowering and Leaf Senescence in Arabidopsis.

The WRKY transcription factors are one of the most important plant-specific transcription factors and play vital roles in various biological processes. However, the functions of WRKY genes in wintersweet (Chimonanthus praecox) are still unknown. In this report, a group IIc WRKY gene, CpWRKY71, was isolated from wintersweet. CpWRKY71 was localized to the nucleus and possessed transcriptional activation activity. qRT-PCR (quantitative real-time PCR) analysis showed that CpWRKY71 was expressed in all tissues tested, with higher expression in flowers and senescing leaves. During the flower development, the highest expression was detected in the early-withering stage, an obvious expression of CpWRKY71 was also observed in the flower primordia differentiation and the bloom stage. Meanwhile, the expression of CpWRKY71 was influenced by various abiotic stress and hormone treatments. The expression patterns of the CpWRKY71 gene were further confirmed in CpWRKY71pro:GUS (ß-glucuronidase) plants. Heterologous overexpression of CpWRKY71 in Arabidopsis caused early flowering. Consistent with the early flowering phenotype, the expression of floral pathway integrators and floral meristem identity (FMI) genes were significantly up-regulated in transgenic plants. In addition, we also observed that the transgenic plants of CpWRKY71 exhibited precocious leaf senescence. In conclusion, our results suggested that CpWRKY71 may be involved in the regulation of flowering and leaf senescence in Arabidopsis. Our study provides a foundation for further characterization of CpWRKY genes function in wintersweet, and also enrich our knowledge of molecular mechanism about flowering and senescence in wintersweet.

Squalene synthase cloning and functional identification in wintersweet plant (Chimonanthus zhejiangensis).

BACKGROUND: Three species of wintersweets: Chimonanthus salicifolius S. Y. Hu, Chimonanthus zhejiangensis M. C. Liu and Chimonanthus grammalus M. C. Liu are widely distributed in China. The three wintersweets belonging to the genus of Chimonanthus that can synthesize abundant terpenoids that are beneficial to human health. Their buds and leaves are traditional Chinese herb applied by the 'She' ethnic minority in southeast of China. Squalene is a multi-functional and ubiquitous triterpene in plants, which is biosynthesized by squalene synthase (SQS) using farnesyl diphosphate (FPP) as the substrate. The synthesis of squalene in wintersweet was not clearly. This work would provide us much help to further understand the terpene metabolism in wintersweet and its health function to people at phytochemistry and molecular levels. RESULTS: In this study, we identified squalene component in the extractions of leaves of three wintersweets and isolated SQS genes from leaf transcriptomes. The three SQSs were highly conservative, so CzSQS from C. zhejiangensis was just determined the enzymatic activity. The in vitro expressed CzSQS that deleted two transmembrane domains could catalyze FPP to generate squalene with the presence of NADPH and Mg2+. CONCLUSIONS: The squalene was one of wintersweet leaves phytochemicals. The squalene synthases of three wintersweet plants were highly conserved. The CzSQS was capable to catalyze two FPP molecules to squalene.

Comprehensive analysis of wintersweet flower reveals key structural genes involved in flavonoid biosynthetic pathway.

Wintersweet (Chimonanthus praecox (L.)), with an over-one-thousand-years long history in cultivation, is still a popular ornamental woody plant in China. The tepals of wintersweet flower are waxy in nature and the overall color of the flower is yellow, while the inner tepals range from yellow to red, which makes it an ideal plant to study floral color formation in ornamental shrubs. In our current work, HPLC analysis revealed that the principal pigments in tepals were the metabolite of flavonoids. All the tepals were containing quercetin, kaempferol 3­O­rutinoside and rutin while cyanidin­3­O­glucoside and cyanidin­3­O­rutinoside were only found in the in the red tepals. Moreover, we found the rutin as the principal component of all the pigments revealed. As well as in this study, a reference transcriptome library constructed from two varieties H29 and H64 flower. Further, 30 proteins of flavonoid biosynthesis pathway were identified in H29 flower using proteome analysis. Based on these dataset, the flavonoid biosynthesis pathway was also speculated. After quantitative analysis of gene expression, we found that ANS act as an on-off switch for the accumulation of red pigments and had positive correlations with various steps genes of the flavonoid pathway. This expression profiling demonstrates that no gene products compete for common substrates to redirect the metabolic flux in wintersweet. It is also demonstrated that high expression of F3'H would provide sufficient content of the precursor, dihydroquercetin, for both flavonol and anthocyanin biosynthesis. The results help us to deepen and enrich the gene resource of color formation in wintersweet flower, and provide specific breeding strategies for increasing diversity of flower color.

Wintersweet (Chimonanthus praecox) toxicity in four goats.

CASE HISTORY: A one-year-old female goat presented with acute onset of recumbency, seizures and vocalisation approximately 5 hours after being given access to branch trimmings from a neighbour's garden. The plant from which the pruned branches came was subsequently identified as wintersweet (Chimonanthus praecox). Three other goats kept in the same paddock displayed similar clinical signs over a period of 4 hours following the initial presentation. CLINICAL FINDINGS: All four goats were ataxic, displayed tetanic seizures and were in lateral recumbency; they had dilated pupils and were hyperaesthetic, with elevated heart and respiratory rates. After symptomatic treatment, including sedation with diazepam, one of the three goats continued to deteriorate and was subjected to euthanasia. The remaining three goats recovered over 1-14 days with nursing care and physiotherapy. DIAGNOSIS: Toxicity due to ingestion of wintersweet, which contains the alkaloid calycanthine. CLINICAL RELEVANCE: Calycanthine is a central nervous system toxin, causing convulsions. Wintersweet shrubs are present in many New Zealand gardens. Practitioners should be aware that the seeds and flowers, and possibly the leaves, of this plant are highly toxic with signs of toxicity including ataxia, hyperaesthesia and seizures.