Chimonanthus praecox, a prized ornamental unique to China, is a rare and exceptional winter-flowering tree with a long cultivation history (Sui et al., 2015; Shang et al., 2020). As a landscape plant, C. praecox can be admired for up to 5 months in gardens, and its cut flowers can last over 3 weeks in a vase. When in bloom, the tree is covered with golden-yellow flowers with a rich, fragrant aroma (Chen, 2012; Sui et al., 2012). The floral traits of C. praecox, key to its ornamental appeal, have varied significantly over centuries of cultivation (Zhu et al., 2024). These traits, which are reproductive in nature, show notable variability both between populations and within a single population, serving as an adaptation to environmental changes (Carr and Fenster, 1994; Williams and Conner, 2001; E-Vojtkó et al., 2022). C. praecox is a predominantly outcrossing species with both dioecious and heterostylous traits that rely on insect pollination (Zhou et al., 2006). Outcrossing not only maintains high genetic diversity within the plant population but also enhances the adaptability of plants to environmental conditions. It is considered the most advantageous mating system for flowering plants because it helps to avoid inbreeding depression (Bellusci et al., 2009; Travers et al., 2018). It is widely believed that most plant reproductive traits, particularly floral structural traits, are the result of natural selection mediated by pollinators (Carr and Fenster, 1994; Williams and Conner, 2001).

The primary morphological traits of plant flowers have evolved under selective pressures aimed at enhancing male function, specifically the dissemination of pollen to receptive stigmas (Barrett, 2002, 2003). As direct participants in male function, stamens exhibit certain variations in morphology and structure under these selective pressures. Pollen grains from stamens often serve as attractants for pollinators, such as when pollen is offered as a reward (Ashman, 2000). Approximately 20,000 flowering plant species exclusively use pollen as an attractant for insects (Lu et al., 2009). Additionally, anthers may use color as an indicator to attract pollinators (Lau and Galloway, 2004). Consequently, differences in anther morphology and color can affect the ability of flowers to attract pollinators and influence pollinator behavior (Escaravage et al., 2001; Kudo, 2003). Anther color differentiation has been widely observed in various plant families, including Acanthaceae, Balsaminaceae, Commelinaceae, Passifloraceae, Fabaceae, and Lythraceae. This differentiation primarily involves the presence of brightly colored stamens or anthers to attract pollinators, thereby enhancing pollen output (Ren, 2009).

The color variation of C. praecox flowers is mainly expressed in the tepals, and the study of C. praecox flower color is also mainly focused on the tepals (Zhou et al., 2007; Ge et al., 2008; Shen et al., 2021). The outer petals of C. praecox have evolved into scales, with the middle petals typically yellow or light yellow and occasionally yellowish-white or yellow-green. The inner petals, located inside the flower, display a wide range of colors, including purple, red, and yellow. Yellow petals primarily derive their color from flavonols, while red petals contain anthocyanins, with the intensity of red coloration directly linked to anthocyanin content (Shen et al., 2022). Anthocyanins are water-soluble flavonoid compounds widely found in plant roots, stems, leaves, flowers, fruits, and seeds, and contribute to red, blue, and purple hues (Wu et al., 2023). Targeted flavonoid metabolomic analysis of different flower color types in C. praecox has shown that the characteristic red metabolite in red-flowered varieties is chrysanthemin, including chrysanthemin-3-O-glucoside, chrysanthemin-3-O-rutinoside, and chrysanthemin-3-O-galactoside (Shen et al., 2023). Color variation in C. praecox (‘Meiren Zui’) results from the combined effects of anthocyanins, flavonoids, and chlorophyll, with anthocyanin content being the primary determinant (Xie et al., 2023).

The physiological mechanisms affecting the color change of C. praecox petals, including pigment types and concentrations, as well as the molecular mechanisms involving major metabolic pathways and key gene functions, have been well-studied (Zhou et al., 2007; Ge et al., 2008; Shen et al., 2021). However, the reddening of C. praecox stamens and its underlying mechanisms remain unexplored. It is unclear whether the processes responsible for stamen reddening are similar to those for petal reddening. The flower characteristics of C.praecox, as important ornamental and reproductive traits, make the discovery of red-stamened germplasm a key resource for modern plant breeding (Zhu et al., 2022; Zheng et al., 2024). Additionally, the reddening of the stamens may affect pollinator behavior and pollination rates, which has significant biological implications (Ren, 2009). In this study, we report for the first time the phenomenon of reddening in C. praecox stamens. Using UPLC-MS/MS and transcriptomic sequencing technologies, we conducted preliminary analyses to identify the main substances and molecular mechanisms responsible for stamen reddening, thereby laying a foundation for further research on the regulatory mechanisms of stamen color changes in C. praecox.

C. praecox, commonly known as wintersweet, is a rare and valuable winter-flowering tree of significant ornamental, medicinal, and cultural importance. Through cultivation, numerous outstanding cultivars have been developed (Chen, 2012; Zhu et al., 2024). Zhao (2008) surveyed C. praecox germplasm resources across 13 provinces in China and found that these resources were primarily distributed south of the Qinling Mountains and east of the Hengduan Mountains, with a broad cultivation area (Zhao, 2008). Subsequent studies investigated and classified C. praecox varieties from various regions, including Sichuan, Hangzhou in Zhejiang, Yanling in Henan, and Wuhan in Hubei (Wu et al., 2009; Lu et al., 2010; Lu and Ren, 2011; Lu and Rong, 2012; Lu and Xie, 2012; Song et al., 2012). Flower color plays a crucial role in the ornamental and economic value of many plants, and breeding new flowers is one of the main goals of ornamental plant breeding. The formation of flower color has also been the focus of research for centuries (To and Wang, 2006; Sawada et al., 2019; Mekapogu et al., 2020). C. praecox predominantly exhibits yellow flowers, with the inner tepals containing primarily cyanidin glycosides (cy-type pigments), resulting in varying degrees of red coloration (Zhou and Chen, 2010; Shen et al., 2022). The flower organs of C. praecox are crucial to cultivar classification, with varieties often distinguished by the color of the inner tepals. They are categorized into the Concolor, Intermedius, and Patens groups. The recent development of the red-flowered Hongyun (C. praecox ‘Hongyun’) cultivar, classified in the Rubrum Group, has further enriched the diversity of C. praecox (Shen et al., 2021).

Flower color is one of the most important phenotypic traits of ornamental plants and serves as a key indicator of their aesthetic and commercial value. Flowers are crucial reproductive organs in plants, and flower color plays a vital role in attracting pollinators and facilitating sexual reproduction (Li and Yue, 2023). Currently, color variations in the floral organs of C.praecox have only been observed in the petals. This study is the first to report changes in the color of the stamens, which not only enriches the diversity of C.praecox varieties but also provides a research foundation for modern plant breeding of C.praecox, enabling precise improvements through gene editing and other technologies (Tan et al., 2023; Zhang et al., 2024). This may have significant implications for studying the reliance of C.praecox on insect pollination for cross-breeding (Ren, 2009). Through morphological observation, we found that the stamens exhibited a red color similar to that of the tepals. Red coloration in the tepals is primarily due to the differential accumulation of anthocyanins. Anthocyanins possess strong antioxidant properties, which can enhance visual quality, reduce photo-inhibition under intense light, minimize damage from ultraviolet radiation, and improve the plant’s resistance to pests and diseases (Dixon et al., 2002; Zhou, 2021). Over 550 types of anthocyanins have been identified, with most derived from six common anthocyanins: cyanidin, delphinidin, malvidin, peonidin, pelargonidin, and petunidin (You et al., 2011). In this study, the analysis of anthocyanin content in C. praecox stamens revealed eight major categories of 49 substances. The red stamens primarily accumulate cyanidin-related compounds, whereas the yellow stamens mainly accumulate flavonoids. Research by Ge et al. (2008) and Li and Yue (2023) on C. praecox flower color and pigments identified two anthocyanins and three flavonols in the red inner tepals: cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside, and quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, and quercetin aglycone (Ge et al., 2008; Yu, 2013). Zhou et al. (2007) study on C. praecox flower color found that the pigment content in the inner tepals correlated positively with color intensity (Zhou et al., 2007). Shen identified cyanidin derivatives as key metabolic compounds responsible for the red coloration of C. praecox tepals, including cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, and cyanidin 3-O-galactoside (Shen et al., 2021). In the present study, the pigment analysis showed similar results, with higher levels of cyanidin 3-O-rutinoside, cyanidin 3-O-glucoside, and other substances in red stamens than in yellow stamens. In yellow stamens, the highest content was found for quercetin-3-O-glucoside, with no significant difference compared with red stamens. Anthocyanins were also detected in the yellow stamens. These findings suggest that the accumulation of cyanidin 3-O-rutinoside and cyanidin 3-O-glucoside is the main metabolic driver of red coloration in C. praecox stamens, and that flavonoid synthesis is not affected by anthocyanin accumulation.

The anthocyanin biosynthetic pathway is divided into four stages. The first stage is the phenylpropanoid biosynthesis pathway (ko00940), which primarily provides precursor substances for the downstream flavonoid pathway. In this stage, phenylalanine is converted to cinnamoyl-CoA through the actions of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL). The second stage is the flavonoid biosynthesis pathway (ko00941), where colorless flavonoid precursors are converted into colored anthocyanins. This transformation occurs through the actions of chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), flavanone 3’-hydroxylase (F3’H), flavanone 3’,5’-hydroxylase (F3’5’H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), resulting in the production of cyanidin, pelargonidin, and delphinidin. Additionally, leukocyanidin is converted into catechin and epicatechin by LAR, and cyanidin is converted into procyanidin by ANR. The third stage involves the anthocyanin biosynthesis pathway (ko00942). At this stage, cyanidin, pelargonidin, and delphinidin are glycosylated to form cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside, and delphinidin-3-O-glucoside via anthocyanidin 3-O-glucosyltransferase (3GT). These compounds undergo further modifications such as acylation and methylation to produce various anthocyanin glycosides. In the fourth stage, anthocyanin glycosides are transported to the vacuole via GST, where they are stored and contribute to coloration. The type of anthocyanin determines the color variation (Fan et al., 2023; Hu et al., 2023; Kang et al., 2023; Zhang et al., 2023; Ma et al., 2024). Differential gene expression analysis in the red stamens of C. praecox at various developmental stages showed an increasing trend in the expression of three genes (PAL, C4H, 4CL) in the first stage. However, the expression of seven genes in the second stage (CHS, CHI, F3H, F3’H, DFR, ANS, LAR) and the 3GT gene in the third stage decreased. Yu found that the expression of CpF3’H was highest in the flower buds of red-flowered C. praecox (Yu, 2017). Our results are consistent with these findings, showing that this gene has the highest expression in PRT1. Yang found that ANS expression plays a crucial role in anthocyanin biosynthesis. CpANS1 showed the highest expression during flower bud maturation, followed by the full bloom stage, and the lowest expression in the immature stage. Additionally, there was no competitive expression between flavonol synthase (FLS) and anthocyanin synthase (ANS) (Yang et al., 2018). Shen concluded that the red coloration of tepals was primarily due to the high expression of the structural gene CpANS1 in the anthocyanin biosynthesis pathway, confirming that CpANS1 is positively correlated with anthocyanin glycosides and is a key structural gene in the red coloration of C. praecox tepals (Shen et al., 2021).

In the present study, both ANS genes were detected during all three stages of red stamen development, with expression trends similar to those reported previously. Expression was the highest during the flower bud maturation stage (PRT1) and the lowest during the full bloom stage (PRT3). Based on these results, it can be inferred that the mechanism of red coloration in C. praecox stamens is similar to that in tepals, primarily due to differential gene expression in the flavonoid pathway, leading to varying anthocyanin accumulation and resulting in red coloration in the stamens. Additionally, the downregulation of genes involved in flavonoid and anthocyanin biosynthesis pathways during the development of red stamens suggests that anthocyanin accumulation is concentrated around the flower bud maturation period, with a gradual decrease in anthocyanin content from the bud stage to full bloom. The phenylpropanoid pathway is a crucial secondary metabolic pathway in plants that serves as the primary source. This pathway also participates in the synthesis of various secondary metabolites and plant hormones such as phenolic acids, flavonoids, lignin, and salicylic acid through multiple downstream branches. These compounds play a significant role in plant stress responses, growth and development, cellulose synthesis, and the accumulation of medicinal components (Dixon et al., 1996; Anwar et al., 2021). It is estimated that over 20% of secondary metabolites in cells are synthesized via this pathway (Grace and Logan, 2000). In the red stamens of C. praecox, the expression levels of genes in the phenylpropanoid biosynthesis pathway increased from the bud stage to full bloom, which may indicate a preparatory step for the synthesis of precursors of anthocyanins and other substances.

This study is the first to report stamen reddening in C. praecox, enriching the genetic resources of this species. Using UPLC-MS/MS, we identified the primary metabolites in red stamens as cyanidin derivatives, specifically cyanidin 3-O-rutinoside and cyanidin 3-O-glucoside, whereas the main metabolite in yellow stamens was quercetin-3-O-glucoside, a flavonol. This significant difference in the cyanidin content is likely the main reason for the reddening of C. praecox stamens. Transcriptome sequencing analysis of red stamens during development revealed the differential expression of genes related to the anthocyanin biosynthesis pathway. It has been preliminarily suggested that the mechanism of red coloration in stamens is similar to that in tepals, primarily due to the differential expression of flavonoid pathway genes, leading to varied anthocyanin accumulation. These findings provide a basis for further research on the mechanisms underlying red coloration in C. praecox floral organs.