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Numerous studies have reported phenological changes and their driving mechanisms in spring flowering plants. However, there is little research on the shifts of winter flowering phenology and its response to forcing and chilling requirements. Based on the China Phenological Observation Network (CPON) ground observation data from nine sites over the past 20 years, we explored the spatial and temporal variation patterns of flowering plants and their response to chilling and forcing in wintersweet (Chimonanthus praecox L.), a common winter flowering plant species in temperate and subtropical zones of China. We used three chilling models (chilling hour, Utah, and dynamic models) and the growing degree hours (GDHs) model to calculate each site's daily chilling and forcing. Using the partial least squares (PLSs) regression approach, we established the relationship between the first flowering date (FFD) and pre-season chilling and forcing in wintersweet, based on which we identified chilling and forcing periods and calculated chilling and forcing requirements. This study found that the FFD of wintersweet in China showed an overall advancement trend during the last 20 years. Still, there were temporal and spatial differences in the FFD of wintersweet among different sites. The PLS results showed that wintersweet also had periods of chilling and forcing, both of which co-regulated wintersweet flowering. We found the forcing and chilling requirements of wintersweet varied significantly from site to site. The higher the latitude is, the more chilling requirements are needed. The chilling requirements for wintersweet were about 6.9–34.9 Chill Portions (CPs) and 1.4–21.6 CP in the temperate and subtropical zones, respectively, with corresponding forcing requirements of 3.2–1922.9 GDH and 965.3–8482.6 GDH, respectively. In addition, we found that the temperature requirements of wintersweet were correlated by a negative exponential relationship, suggesting that chilling and forcing requirements have an antagonistic effect on initiating flowering phenology. Our results could help us understand how flowering dates of winter flowering plants respond to climate change. [ABSTRACT FROM AUTHOR]

Wintersweet (Chimonanthus praecox (L.) Link) is a commercial significance woody plant. As a rare winter-blooming plant, C. praecox is widely distributed and highly adaptable to various climates, especially low temperatures. In this study, we identified the COR413 plasma membrane gene CpCOR413PM1 in wintersweet. CpCOR413PM1 was expressed in all tissues of the plant, with the highest expression observed in the root and floral parts. Cultivation under 4 °C and with treatment of ABA led to the up-regulated expression of CpCOR413PM1. The expression of recombinant CpCOR413PM1 protein in Escherichia coli resulted in the tissues' increased resilience to cold and drought stress. In vitro enzyme activity assays confirmed the protective impact of the CpCOR413PM1 protein on superoxide dismutase under low-temperature stress. Furthermore, the overexpression of CpCOR413PM1 in Arabidopsis thaliana resulted in increased cold and drought tolerance and ABA inhibited transgenic Arabidopsis seed germination. The CpCOR413PM1 gene promoter can influence expression of the GUS reporter gene under conditions of 4 °C, 42 °C and abscisic acid. Overall, our study demonstrates that CpCOR413PM1 plays a significate role in cold and drought stress. Our findings strengthen the knowledge of the molecular mechanisms underlying wintersweet's tolerance to stress and lay the groundwork for the future investigation of the functions of the COR gene family. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Wintersweet (Chimonanthus praecox (L.) Link) is a commercial significance woody plant. As a rare winter-blooming plant, C. praecox is widely distributed and highly adaptable to various climates, especially low temperatures. In this study, we identified the COR413 plasma membrane gene CpCOR413PM1 in wintersweet. CpCOR413PM1 was expressed in all tissues of the plant, with the highest expression observed in the root and floral parts. Cultivation under 4 °C and with treatment of ABA led to the up-regulated expression of CpCOR413PM1. The expression of recombinant CpCOR413PM1 protein in Escherichia coli resulted in the tissues’ increased resilience to cold and drought stress. In vitro enzyme activity assays confirmed the protective impact of the CpCOR413PM1 protein on superoxide dismutase under low-temperature stress. Furthermore, the overexpression of CpCOR413PM1 in Arabidopsis thaliana resulted in increased cold and drought tolerance and ABA inhibited transgenic Arabidopsis seed germination. The CpCOR413PM1 gene promoter can influence expression of the GUS reporter gene under conditions of 4 °C, 42 °C and abscisic acid. Overall, our study demonstrates that CpCOR413PM1 plays a significate role in cold and drought stress. Our findings strengthen the knowledge of the molecular mechanisms underlying wintersweet’s tolerance to stress and lay the groundwork for the future investigation of the functions of the COR gene family.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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Abstract 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.

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 (β-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. [ABSTRACT FROM AUTHOR]

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 (β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Wintersweet (Chimonanthus praecox L.) is a traditional winter-flowering plant in China and a popular cut flower in winter. Its unique flowering characteristics under cold stress may involve the regulation of a large number of proteins. Protein post-translational modification is an important regulating type for the gene function. However, little is known about the post-translational modification in wintersweet. SUMOylation is an important post-translational modification in eukaryotes. Small ubiquitin-like modifier (SUMO) E3 ligases perform multiple functional regulatory activities in plants via SUMOylation. Here, we cloned and identified a SIZ/PIAS-type SUMO E3 ligase, CpSIZ1, from wintersweet. CpSIZ1 shared high similarity with other SIZ1 proteins. Quantitative real-time PCR (qRT-PCR) indicated that CpSIZ1 was expressed in all tissues tested, with the highest expression in flower wither period of stage 6, and followed by mature leaves except for different flower development stages. The ectopic expression of CpSIZ1 in Arabidopsis, including the CpSIZ1 overexpression in siz1-2 mutant (HB line) and CpSIZ1 overexpression in WT (OE line), not only promoted vegetative growth, delayed flowering and accelerated leaf senescence, but also improve the cold tolerance in Arabidopsis. Therefore, our studies have enrich the understanding of function of SIZ1 gene in woody plant, and provide a good foundation for further research on the post-translational modification regulation mechanism in this winter-flowering plant. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Wintersweet (Chimonanthus praecox), a classical winter-blooming magnoliids species with great aesthetic value highlighting the on the brilliant yellow flower and pleasant floral aroma, possesses diverse varieties mainly classified in three groups (Concolor, Patens, and Rubrum group) with the most striking variation in the tepal color. The recent release of the genome in two wintersweet varieties belonging to Patens and Rubrum group successfully elucidated the molecular mechanisms underlying some ornamental traits highlighting the flowering time, floral scent and tepal color. In this study, the chromosome-scale genome of C. praecox var. concolor was assembled to further verify the placement of magnoliids and exploit the genetic variation underscoring the diverse flowering traits among different wintersweet groups. Phylogenomic analyses placed magnoliids including C. praecox var. concolor as the sister to eudicots. The C. praecox var. concolor 's genome experienced two rounds of whole genome duplication (WGD). Multiple gene families involved in floral development including ANR1 and PI genes were retained and expanded during these WGD events. The expansion and expression pattern of SVP and FUL genes suggest their pivotal role in unique character of winter-flowering. With the pleasant fragrance as the aesthetic characteristics, linalool and benzyl acetate were identified as the top abundant constituents in the floral scent of Concolor wintersweet. Integrating metabolic, genomic, and transcriptomic analyses with enzyme assays led to the identification of CpTPS3 (CpLIS) and CpBAHD2 (CpBEAT), which are responsible for the biosynthesis of linalool and benzyl acetate, respectively, in the Concolor variety. Taken together, the assembled genome of C. praecox var. concolor enables the comparative analysis of the genetic basis underlying both conserved and variable floral traits and provides a useful foundation for variety improvements by molecular breeding in wintersweet. • A high-quality chromosome-level genome of Concolor wintersweet was assembled. • Phylogenomic analyses placed magnoliids as the sister to eudicots. • CpSVP and CpFUL genes are the key regulators in controlling the unique winter-flowering of Concolor wintersweet. • CpTPS3\CpBEAT were functionally identified to produce linalool and benzyl acetate as floral volatiles in Concolor wintersweet. • Comparative analysis highlights the genetic basis in conserved and varied floral traits among three wintersweet varieties. [ABSTRACT FROM AUTHOR]

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.

The selection of traditional Chinese painting materials is often accompanied by other meanings given by people, and the process of giving meaning is the process of information development. Wintersweet, orchid, bamboo, and chrysanthemum, from their own form and people’s observation of them, have been given a special meaning: wintersweet, proud like snow and decent people; orchid, symbolizing the gentleman’s integrity and independence; bamboo, symbolizing the gentleman’s humility; chrysanthemum, symbolizing the gentleman’s hidden inner world, which is not trapped in the trivial things. This development process deals with the development process of in-itself information, for-itself information, and regenerated information. The wintersweet, orchid, bamboo, and chrysanthemum expressed by the artist in Chinese traditional painting is the social information of the three states of in-itself information, for-itself information, and regenerated information of three-state information. The selection of traditional Chinese painting materials has gone through the same process.

Abstract 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 3‑ O ‑rutinoside and rutin while cyanidin‑3‑ O ‑glucoside and cyanidin‑3‑ O ‑rutinoside 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. Highlights • Three flavonol compounds and two anthocyanins are identified in wintersweet flower. • Immature stage of bud is the peak of flavonoids gene expression. • ANS gene active as an enzymatic on-off of anthocyanin biosynthesis. • No gene-products compete for common substrates in order to redirect the metabolic flux. • Facilitate the design of transgenics for engineering and lay foundation for targeted-breeding approaches of wintersweet. [ABSTRACT FROM AUTHOR]

Expansins are extracellular proteins that are involved in cell wall modifications such as cell wall disassembly, cell separation, and cell expansion. Little is known about expansin gene expression during flower development of wintersweet (Chimonanthus praecox). In the present study, an expansin gene, CpEXPl, was isolated from the wintersweet flower cDNA library through random sequencing; this gene encodes a putative protein of 257 amino acids with the essential features conserved, like in other alpha expansins. The CpEXPl gene exhibited different transcription levels in different tissues and had a significantly higher expression in flowers than other tissues. It is strongly correlated with the development of the flower. The expression of CpEXPl increased in the flower buds or whole flowers from Stage 1 to 4 and decreased from Stage 5 to 6 during natural opening. Ethephon (an ethylene releaser) treatment promoted cut flower senescence, whereas 1-methylcyclopropene (1-MCP) (an ethylene perception inhibitor) delayed the process of flower wilting. This result is associated with the concomitant lower transcript levels of CpEXPl in the ethephon-treated samples as well as the steady expression in the 1-MCP- treated samples compared with that in control flowers. The studies show the interesting observation that the expression of an expansin gene CpEXPl is correlated with the development of Chimonanthus praecox flowers, the upregulation during flower opening vs. the downregulation during senescence. [ABSTRACT FROM AUTHOR]

In this study, a full-length complementary deoxyribunucleic acid (cDNA) of a fatty acyl-acyl carrier protein thioesterase ( CpFATB) was isolated from a Chimonanthus praecox (wintersweet) cDNA library. Wintersweet is a hardy shrub native to Chinese montane regions and is known to be tolerant to many biotic and abiotic stresses including cold, drought, and a variety of plant pathogens. The cDNA is 1,110 nucleotides in length, of which 1,107 bp form a single open-reading frame, predicting a 369 amino acid polypeptide. The deduced amino acid sequence of CpFATB displayed high homology to choroplastic palmitoyl-acyl carrier protein thioesterases from other plants. This gene was subsequently overexpressed in Nicotiana tobaccum using Agrobacterium tumefaciens-mediated transformation. Transcripts of CpFATB were shown to be abundant in leaves of the transformants, but were less highly expressed in roots and least highly expressed in stem tissues. Analysis of the fatty acid composition of transformant leaf tissue showed that the levels of myristic acid (C14:0) in leaf material of transgenic plants was increased by 46% compared with levels observed in wild-type plants. In addition, the level of oleate (C18:1) was reduced by 35%. Of most interest, was the observation that the transformants displayed a far higher degree of drought tolerance than wild type. These data suggest that the CpFATB product may be of utility in the production of transgenic, drought-tolerant crop species. [ABSTRACT FROM AUTHOR]

Abstract: Farnesyl pyrophosphate (FPP) synthase catalyzes the biosynthesis of FPP, which is the precursors of sesquiterpenoids such as floral scent volatiles, from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). cDNA encoding wintersweet (Chimonanthus praecox L.) FPP synthase was isolated by the RT-PCR and RACE methods. The deduced amino acid sequence showed a high identity to plant FPP synthases. Expression of the gene in Escherichia coli yielded FPPS activity that catalyzed the synthesis of FPP as a main product. Tissue-specific and developmental analyses of the mRNA levels of CpFPPS and volatile sesquiterpenoids levels in C. praecox flowers revealed that the FPPS may play a regulatory role in floral volatile sesquiterpenoids of wintersweet. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Chimonanthus praecox (wintersweet) is endemic to China. It has been cultivated there for more than 1000 years as a garden, potted, and cut-flower plant. Many cultivars have been developed during its long history of cultivation, and recently many germplasms were collected in Wuhan and Nanjing, China. The identification and genetic relationship of these resources were studied based mainly on morphological traits. In the current study, intersimple sequence repeat markers (ISSR) and random amplified polymorphic DNA markers (RAPD) were used for the first time to investigate 72 wintersweet clones from the two regions. Eleven ISSR primers amplified 115 bands, 90 (78.26%) of which were polymorphic. Nineteen RAPD primers amplified 165 bands, 105 (63.63%) of which were polymorphic. Either ISSR or RAPD markers were sufficient to distinguish all the clones surveyed. A Dendrogram based on Jaccard's similarity coefficients indicated that the distribution pattern of the 72 clones was coherent with their geographical origins. Most of the genetic variation (85.68% with ISSR data; 86.75% with RAPD data) occurred among clones within each region. However, the difference between Wuhan and Nanjing groups is statistically significant (ΦST = 0.143, P < 0.001, with ISSR data; ΦST = 0.132, P < 0.001, with RAPD data). Morphological variation and classification of wintersweet cultivars were also discussed compared with the genetic relationship based on ISSR and RAPD markers. This is the first report of the partitioning of genetic variability within and between different cultivated wintersweet regions, and it provides useful baseline data for optimizing sampling strategies in breeding. These results are important for future genetic improvement, identification, and conservation of Chimonanthus praecox germplasm. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]