Breeding-mediated metabolic changes unexpectedly enhance petal blue fluorescence and shift the attractiveness of tea-oil Camelliato bees

Breeding-mediated metabolic changes unexpectedly enhance petal blue fluorescence and shift the attractiveness of tea-oil Camelliato bees | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Breeding-mediated metabolic changes unexpectedly enhance petal blue fluorescence and shift the attractiveness of tea-oil Camellia to bees Bin Yuan, Yi-huan Li, Xiao-ling Su, Yuan-yuan Lu, Xiao-ming Tian, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7998239/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plant breeding has long focused on improving economic traits, yet its unintended effects on plant–pollinator interactions remain largely overlooked. Here, we report that selective breeding in Camellia oleifera unexpectedly enhances petal blue fluorescence, altering its attractiveness to bees. Field assays and behavioral experiments demonstrated that visual cues, rather than floral scents, play a decisive role in pollinator visitation, with bees showing a strong preference for petals emitting bright blue fluorescence under ultraviolet (UV) light. All six widely cultivated varieties exhibited stronger blue fluorescence than wild C. oleifera , in which the petals were nearly non-fluorescent. Metabolomic profiling revealed that the enhanced fluorescence correlated with the accumulation of hydroxycinnamic acid derivatives, particularly 3-O-p-coumaroylquinic acid. These compounds, originally targeted for improving fruit and oil quality, were found to mediate the formation of petal fluorescence and spatial patterning. This breeding-mediated metabolic shift effectively transformed C. oleifera from an “anti-bee” species into a “bee-attraction” species, suggesting a reversal of its natural pollination syndrome. Our findings uncover a previously unrecognized ecological consequence of plant breeding, linking secondary metabolism with pollination ecology. Recognizing and harnessing such floral traits in future breeding programs may not only improve pollination efficiency but also offer a novel pathway toward sustainable agroecosystem design. Camellia oleifera fluorescence pollination breeding visual signal Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION To ensure the security of the global food supply, people have continued to improve the efficiency of agricultural production through plant domestication and breeding. However, modern agricultural production is facing multiple pressures, including climate change and environmental pollution, and needs to increase production while ensuring sustainability 1. The Food and Agriculture Organization of the United Nations (FAO) predicts that global food production will need to increase by 70% in 2050 to meet the demands of a growing population 2. This daunting goal requires revisiting the existing plant breeding project. While modern breeding has produced new varieties for drought, salt tolerance, and high yield in many crops, there is still a need to consider integrated multifactorial pressures in future breeding to maintain global food security and achieve low-input sustainable agriculture 3. While current plant breeding strategies are attempting to design plants adapted to future environments, the impact of crop domestication and breeding on plant-pollinator interactions, an interaction that is a key prerequisite for stable plant yields, has been overlooked 4. This knowledge gap could lead to serious ecological and economic risks. Up to 75% of the major crops and 35% of food production rely on animal pollination, and the economic value it creates has climbed to $ 235–577 billion per year and continues to grow 5 , 6. Therefore, incorporating plant-pollinator interaction in agroecology into the theoretical framework of future plant breeding is a key scientific prerequisite and urgent practical need to realize sustainable production at the plot and agroecosystem levels. During the domestication and breeding, humans have continuously improved crops through targeted selection for economic traits, a process that has been significantly accelerated by modern breeding techniques 7. However, such selection often produces unintended effects while optimizing the targeted plant traits 8. Numerous empirical studies have shown that domestication and breeding significantly alter the secondary metabolic profiles of plants and affect their chemical defences 9 , 10. More importantly, changes in secondary metabolites may mediate the modification of floral traits such as flower colour and floral scent, which are an important part of the plant-pollinator syndrome 11 , 12. In nature, the flowers of many plants are adapted to a particular group of pollinators and have evolved suitable pollinator syndromes (including colour, shape, and scent) to attract the most efficient pollinators 13 , 14. Thus, breeding-mediated changes in flower traits may affect plant-pollinator interactions. In terms of attraction to pollinators, this may either disrupt existing plant-pollinator interactions, maintain existing interactions, or induce novel interactions 15. A deep understanding of the mechanisms and consequences of such effects is important and interesting for analysing the coevolutionary of plants and pollinators. More importantly, it is meaningful for agroecology, as such effects may directly modulate the pollination effects of self- or cross-pollination plants with respect to the potential fruit yield and quality (including nutrition and flavour) 16. Camellia , including over 280 species, is thought to be involved in the shift from bee-pollinated to bird-pollinated flowers 17. Some of these species have been domesticated and bred over decades to become an important group of economic plants and horticultural plants due to their ornamental flowers and high-quality oil seeds 18 , 19. As one of the world's four major woody oilseed species, C. oleifera can be used as a classical model to study the effects of domestication and breeding on plant-pollinator interactions. Conventional point suggests that C. oleifera relies on specialized pollinators (e.g., Andrena camellia ) and has a low attraction to bees ( Apis carana ) 20; however, recent field observation reveals frequent visits of bees to cultivated C. oleifera 21, suggesting that domestication and breeding may have significantly affected plant-pollinator interactions. To resolve this paradoxical phenomenon, this study utilized cultivars, mutants, and wild sources of C. oleifera to address three key questions: ( 1 ) What are the key flower traits that attract bees to cultivars? ( 2 ) What are the trends in bee-attractive traits in domesticated and breeding from wild to cultivars? ( 3 ) What are the metabolic pathways that drive changes in bee-attractive traits? This study aims to fill the gaps in future plant breeding strategies that point to plant-pollinator interactions, and to assist in the establishment of a sustainable food supply system for agroecology 6. MATERIALS AND METHODS Study species and sites C. oleifera is a woody oil crop with high economic value that originates in China and blooms from October to the following January. Its fruit setting is strictly dependent on insect pollination, including a variety of bees, hoverflies, and flies. This study was conducted in a C. oleifera plantation in the Yuelu and Wangcheng Districts of Changsha City, central China. The two locations are relatively close, and the main species of pollinators are the same (Table S1). Tree traits We selected eight plots measuring 10 m × 10 m in Wangcheng District. The target trees ( n = 40) were selected from the eastern, western, southern, northern, and middle sections of each plot. The following traits of the sampled trees were measured: (a) tree height, namely height from the uppermost point to the surface of ground; (b) base diameter, namely diameter of the trunk 0.2 m above the ground; (c) flatness of crown, that is (east–west crown width –– north–south crown width) / east–west crown width; (d) crown width, namely, the average of the east-west and north-south crown widths; (e) flower number, that is, total number of flowers on the tree; and (f) flower density, namely, flower number/crown area 22. Insect visits Visiting insect data were collected from eight plots in Wangcheng District ( n = 40). Only pollinator data, that is, insects that came into contact with the stigma and anthers, were recorded as available data. Insect visit surveys were conducted when pollinating insects were the most active from 11:00 to 12:00 pm. 23. We selected five target trees in each plot and observed them for 30 min, after which insect visits in the upper and lower layers of the canopy of each tree were recorded separately. The crown located 1.5 m above the ground was called the upper canopy, and the crown located within 1.5 m was called the lower canopy. The average height of the trees was 3 m. All trees in each sample plot were observed simultaneously to ensure data reliability. Behavioral two-choice assay To determine which of the visual and olfactory signals of C. oleifera were more attractive to pollinators, we observed the behavioral choices of the insects. In many studies, black-and-white bottle experiments have been used to explore the preference of insects for signals, but this was difficult to do with C. oleifera . Therefore, plastic bags were used instead of black-and-white bottles. We performed the following experiments: (a) some flowers were covered with transparent plastic bags (TPB) such that they displayed only visual signals, and (b) some flowers were covered with black plastic bags (BPB) with holes such that they emitted only olfactory signals 24. On a clear day, we selected target trees at Site 1 and bagged their flowers. Three trees were used for each treatment, with a total of 120 flowers. Two trees with different treatments comprised the same group in the same position as the insect nests. We observed the number of insects that remained on the bags and explored them. Observations were recorded on four sunny days from 11:00 am to 12:00 pm. Given that the respiration of plants inside the bag produces moisture, which affects the release of odor and visual signals, the bag should be changed regularly, and different trees and branches need to be selected daily to ensure the freshness of the flowers. Flower traits comparison After selective breeding, we have obtained many genetic materials, and 'ST' is one of them. It is obtained by natural intraspecific hybridization of C. oleifera . To understand the difference between C. oleifera 'Huajin' and 'ST', we quantitatively measured their flower size (transverse length and longitudinal length), petal size (transverse length and longitudinal length), at least 30 flowers were measured. Selection experiments of bees in the field Since the previous study found that Apis cerana is a potential dominant pollinator, A. cerana is preferred for different C. oleifera varieties were compared here 21. Firstly, five fresh and blooming flowers of C. oleifera 'Huajin' and 'ST' were collected and placed at the site, 0.5m from the hive entrance. It was ensured that there was no obstruction between the hive and the tested flowers. Recording of choices by bees that came out of the hive to forage was then initiated, and bees that did not make a choice were excluded, with at least 30 bees recorded for valid choice behavior. After every 10 bees made a choice, the position of the flowers was changed to avoid positional effects. It was also necessary to promptly replace flowers that were not fresh (petal oxidation). Y-Tube Olfactometer Assay Behavioral assays were conducted using a glass Y-tube olfactometer (20 cm stem length, 15 cm arm length, 40 mm internal diameter, and 60° arm angle) under species-specific airflow conditions (300mL/min) maintained by atmospheric sampling pump (QC-1B, Beijing Municipal Institute of Labor Protection, Beijing, China). Charcoal-filtered air, humidified with distilled water, was delivered through each arm. Freshly collected C. oleifera flowers (including C. oleifera 'Huajin' and 'TS') of uniform size, maturity, and bloom stage were used as odor sources. For each trial, five intact flowers of a single variety were placed in one arm, while five flowers of different varieties were placed in the opposite arm. Flowers were replaced after each trial to ensure freshness and minimize volatile degradation. Apis cerana were individually introduced at the stem base and observed for 5 min under controlled environmental conditions (25 ± 2°C, 40–60% RH, and red light). A choice was recorded if the insect entered an arm and remained there for at least 5 s. Non-responsive individuals (no choice within the allotted time) were excluded. Each treatment was tested with at least 30 individuals, and arm positions alternated every 10 bees to eliminate positional bias. Floral odor detection by gas chromatography–time of flight mass spectrometry (GC×GC-TOF MS) The analysis of volatile compounds was conducted using a LECO Pegasus® 4D GC instrument (LECO, St. Joseph, MI, USA) consisting of an Agilent 8890A GC×GC-TOF MS (Agilent Technologies, Palo Alto, CA, USA) system equipped with a split/splitless injector and dual-stage cryogenic modulator (LECO) coupled with TOFMS detector (LECO) at Suzhou Panomix Biomedical Technology Co., Ltd. (Suzhou, China). Sample preparation involved solid-phase microextraction (SPME). First, samples of floral odors were prepared using MonoTrap, and three flowers ( C. oleifera 'Huajin' and 'TS') and MonoTrap were placed in the same container to allow natural release of floral odors for 7.5 h at room temperature (25 ± 2°C). Then, MonoTrap was put into a headspace vial. Prior to extraction, the SPME fiber was conditioned at 270°C for 10 min. The headspace vial with samples was incubated at 80°C for 10 min, followed by adsorption of volatiles at 80°C for 40 min. The SPME fiber was then desorbed in the GC injector at 250°C for 5 min. A DB-Heavy Wax column (30 m × 250 µm × 0.5 µm, Agilent) and an Rxi-55il MS column (2 m × 150 µm × 0.15 µm, Restek) were used for separation, with helium as the carrier gas (1.0 mL/min). The oven temperature program started at 50°C (held for 2 min), ramped to 220°C at 4°C/min, and maintained for 13 min; the secondary oven and modulator were set 5°C and 15°C higher than the primary column, respectively, with a 5.0 s modulation period. Mass spectrometry parameters included an ion source temperature of 250°C, electron ionization at 70 eV, a scan range of m/z 35–550, and a detector voltage of 1960 V. Data acquisition was performed at 200 spectra/s. Fluorescence observation and measurement To determine the visual attraction strategy of petals, we picked fresh C. oleifera petals from the tree. We first observed them and compared the petal fluorescence of C. oleifera varieties qualitatively and quantitatively. (a) To determine the fluorescence color of C. oleifera flowers under UV light, the petals were placed on glass slides, observed, and photographed under a fluorescence microscope (OLYMPUS-BX51, Japan). (b) Thirty flowers were randomly selected from C. oleifera 'Huajin', 'Changlin40', 'Ganzhouyou1', 'Huaxin', 'Yilu', 'ST', and wild C. oleifera , respectively; one petal per flower was randomly selected for quantitative analysis (n = 90). The fluorescence of each petal was measured using ImageJ-Java8 software (National Institutes of Health, Bethesda, MD, USA) to determine its color and brightness. An RGB color system was used to quantify the color, decomposing the fluorescent color of the petals under UV light into red, green, and blue components, and obtaining the luminance value for each color. Fluorescence attraction test To test the availability of blue fluorescence for pollinators, we used chlorogenic acid, a substance that releases blue fluorescence under UV light. It has a certain degree of water solubility and is easily soluble in organic solvents. However, given that this study needed to be conducted in a field environment, we chose pure water, which is harmless to wild animals, as the contrast solvent. The water solubility of chlorogenic acid is limited, so we combined our experiments and previous studies, then selected 0.3 mg/g, which is a low concentration that can be observed by insects (Fig. S1). To explore the effect of blue fluorescence on pollinators, we selected four sunny days at Site 1 and observed insects that visited the target trees for 30 minutes each day from 11:00 am to 12:00 pm. The target trees were divided into three groups: (a) CK, untreated; (b) T1, sprayed with pure water; and (c) T2, sprayed with an aqueous solution of 0.3 mg/g chlorogenic acid to enhance the blue fluorescence. Each treated branch was controlled to five flowers, each flower was evenly sprayed, and each treatment was repeated three times. Insect response induced by fluorescent cues under different backgrounds Fluorescence can be used as a food trap to induce pollinator responses. The response of Apis mellifera to paper sprayed with chlorogenic acid above a beaker with sweet sugar has also been tested by others 25 , 26. However, none of these studies have clarified the specific relationship between blue fluorescence and food. To explain this, we prepared four types of paper: (CK) without any treatment; (T1) aqueous solution sprayed with chlorogenic acid (0.3 mg/g); (T2) sprayed with 50% sucrose solution; and (T3) sprayed with a mixture of chlorogenic acid (0.3 mg/g) and sucrose (1 g g-1). Two experiments were conducted in this study. In the first experiment, we placed four types of white paper at the site, which is 0.5m from the hive entrance. And observed them for six hours from 11:00 am to 5:00 pm on each sunny day for 8 days. The number of insect visits per hour was calculated and compared numerically. To effectively evaluate the role of the clues, we treated four types of white, red, blue, and green paper simultaneously, placed at the site, which is 0.5m from the hive entrance. at the same time, and recorded the number of insect visits to each paper, which was the same as before, for five days. The grassland for the experiment was relatively open, where the sun could shine directly. Metabolome of petals The petals of C. oleifera 'Huajin', 'Huashuo' and wild C. oleifera were first cut from the middle, with the section near the anther being the basal part, and the other end being the apical part. The cut petals are immediately frozen in liquid nitrogen and stored in a -80°C refrigerator. Three biological replicates were prepared for each variety. All samples were analyzed using ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) to mitigate batch effects. UPLC-MS/MS analysis was performed at Wuhan Matware Biotechnology Co., Ltd. (Wuhan, China). The biological samples were lyophilized using a vacuum freeze-dryer (Scientz-100F) for 63 hours and ground into powder (30 Hz, 1.5 min) with a grinder (MM 400, Retsch). Approximately 50 mg of the powder was weighed (electronic balance, MS105DM, Mettler Toledo) and mixed with 1200 µL of -20°C pre-cooled 70% methanol aqueous solution containing internal standards (less than 50 mg added at the rate of 1200 µL extractant per 50mg sample). The mixture was vortexed every 30 minutes (30 sec each, 6 times), centrifuged (12,000 rpm, 3 min), and filtered through a 0.22 µm microporous membrane for UPLC-MS/MS analysis. Chromatographic analysis was performed on an UPLC-ESI-MS/MS system (ExionLC™ AD, SCIEX) equipped with an Agilent SB-C18 column (1.8 µm, 2.1 × 100 mm). The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A gradient program was applied: 5% B (0 min) to 95% B (9 min), held for 1 min, returned to 5% B (10–11.1 min), and equilibrated until 14 min. The flow rate, column temperature, and injection volume were 0.35 mL/min, 40°C, and 2 µL, respectively. Effluent was analyzed using an ESI-QTRAP-MS/MS system. Mass spectrometry was conducted using an ESI-QTRAP-MS/MS system (SCIEX) in both positive (IS 5500 V) and negative (IS -4500 V) ion modes. The ion source parameters included a temperature of 500°C, curtain gas (25 psi), and ion source gases I/II (50/60 psi). MRM transitions were optimized for each metabolite with collision energies (CE) and declustering potentials (DP) calibrated using reference standards. Data acquisition and peak integration were performed using Analyst 1.6.3 and MultiQuant software. Data analysis The correlation between tree traits and insect visits was analyzed by calculating the Pearson product–moment correlation coefficient, and the factors most strongly related to insect visits were identified. Correlations among the tree traits were also analyzed. T-tests were used to compare the visits of insects in the upper and lower layers of the crown of three types of trees. Before determining the number of visiting insects to the branches, they were covered with transparent and black plastic bags. These branches were sprayed with chlorogenic acid or left untreated. Statistical significance was set at P < 0.05 . The insect visits of C. oleifera and C. henryi female flowers sprayed with chlorogenic acid aqueous solution, pure water, and untreated branches, the insect visits of white paper under four treatments, and the insect visits of three different colors of paper sprayed with chlorogenic acid aqueous solution, sugar water, and mixed solution were analyzed using the Tukey test. Statistical significance was set at P < 0.05 . RESULTS 3.1 Visual signals are essential in the flower-visiting decision of pollinators While flower display modulates the flower-visiting decision of pollinators, the key signal of C. oleifera flowers attracting pollinators was unclear. To evaluate the differences in the attraction ability of flower visual and olfactory signals to pollinators, we observed differences in the number of insects visiting the branches with different treatments. Throughout the entire observation period, four main types of pollinators were observed (Fig. 1 A). The results of the T -test showed that except for wasps, the other three pollinator types showed a significant preference for the branches that display visual signals (TPB) of flowers (Fig. 1 B, P < 0.05 ). Bees and hoverflies are staunch proponents of visual signals and can only access flowers when visual signals are present (Fig. 1 B and Table S2). Although flies show a preference for visual signals too, they also visit branches that release scent (BPB). Although the wasp population was too small and only visited the branches a limited number of times, no statistical significance was found between the TPB and BPB treatments. However, only the branches that released visual signals were visited by wasps in all the observation records (0.25 ± 0.40). This result highlights the importance of visual signals of C. oleifera flowers in their pollination strategy and may be a prerequisite for pollinators to make flower visit decisions. We have provided a more detailed record of the behavior in the supplementary document. The results show that other pollinators, in addition to wasps, also hesitated in the access decision. They hovered repeatedly over the branch that displayed the visual signal but did not land. Meanwhile, wasps were relatively decisive, landing directly on the branch in each record (Table S2). This shows that although visual signals are essential, they are insufficient to support all pollinators in making immediate decisions. 3.2 Petal blue fluorescence enhances the pollinator attraction of the flower Different C. oleifera varieties have different attractiveness to pollinators. In the field, C. oleifera 'Huajin' attracted more A. carana than C. oleifera 'TS' (Fig. 2 A, P < 0.001 ), and the A. carana selecting proportion of 'Huajin' was as high as 0.79%. To clarify the causes of pollinator preferences, the effects of flower odor on the decision of A. carana to visit flowers were first compared. The results showed no significant difference (Fig. 2 B, P > 0.05 ) in the selection proportion of A. carana choosing the odor of 'Huajin' and 'TS'. The ellipsoid plots of the principal component analysis (PCA) also showed that the chemical composition of the 'Huajin' and 'TS' odors was similar (Fig. 2 B). This result supports the finding of the Y-tube experiment that the olfactory signal (floral scent) is not the cause of pollinator preference. Subsequently, comparisons were made with the visual signals of C. oleifera flowers. Interestingly, the flower and petal sizes of C. oleifera 'Huajin' and 'TS' were similar and could not be effectively separated in the ellipsoids obtained from the PCA (Fig. 2 C). In addition, the color of C. oleifera 'Huajin' and 'TS' petals is all white. This implies that flower size and flower color may not be the cause of the different preferences of pollinators visiting flowers among the C. oleifera varieties. Therefore, we examined the floral organs of C. oleifera under white and UV light. C. oleifera 'Huajin' petals emit blue fluorescence under UV light (Fig. 2 C). However, the intensity of petal fluorescence differed among the C. oleifera varieties, with 'Huajin' having significantly brighter petal fluorescence than 'TS' (Fig. 2 C, P < 0.001 ). The petal fluorescence of 'Huajin' was bright blue under the ultraviolet (UV) light, while the fluorescence of 'TS' was pale and almost invisible under the UV light. This result seems to imply that the blue fluorescence of petals is an essential visual signal that helps to attract pollinators to C. oleifera varieties. To further clarify that this blue fluorescence is available for pollinators, we first sprayed 0.3 mg/g chlorogenic acid solution (Fig. S1) on the branches of C. oleifera to enhance the fluorescence of flowers and sprayed pure water on the branches to shield them from the interference of the solution. The results showed that the number of visiting insects did not decrease significantly after spraying with pure water. However, the number of pollinators visiting flowers increased significantly after the blue fluorescence was enhanced, indicating that the blue fluorescence was attractive to pollinators (Fig. 2 D, P < 0.05 ). 3.3 Pollinator attraction of blue fluorescence on white background surpasses other backgrounds To clarify the effect of blue fluorescence on pollinators alone, we first compared the number of insect visits, including bees and flies, within one hour in the four treatments. The visiting number of pollinators to the paper sprayed with chlorogenic acid and sugar water was significantly higher than that of the untreated paper and significantly lower than that of the paper sprayed with the mixed solution of chlorogenic acid and sugar water. There was no significant difference between the visiting number of pollinators to the paper sprayed with chlorogenic acid and sugar water, suggesting that the sugar water and blue fluorescence acted as two separate signals to attract pollinators (Fig. 3 A, P < 0.05 ). To further test this result, we simultaneously treated blue, red, and green paper with the same four treatments and recorded the pollinators visits. The results showed that the changes of paper color did not mediate the difference in pollinator visits (Fig. 3 B, P < 0.05 ). After confirming that blue fluorescence was a separate flower-visiting signal, we examined whether spraying chlorogenic acid onto paper with different backgrounds could improve insect visits. Before the comparison, we first compared the pollinator visits to untreated paper. There was no difference in the attraction of different colors of paper to insects because there was no significant difference in the number of visitors (Fig. S2). This result provides a necessary premise for comparing the attraction of blue fluorescence with different colored backgrounds. The pollinator visiting numbers of the white, blue, and red papers sprayed with chlorogenic acid (T) were significantly higher than those of the untreated paper. However, there was no difference in insect visits between the treated and untreated green papers (Fig. 3 C, P < 0.05 ). The results show that when provided with a rich number of choices, pollinators do not prioritize the blue fluorescence under the green background but prefer the blue fluorescence under the white background ( P < 0.05 , Fig. S3). 3.4 Cultivated varieties exhibit enhanced blue fluorescence than wild C. oleifera To understand the petal fluorescence phenotypes of different C. oleifera varieties and wild C. oleifera under ultraviolet (UV) light, we observed the petals of 6 cultivated C. oleifera varieties and wild C. oleifera under UV light. We found that the petals of wild C. oleifera hardly emitted blue fluorescence, while the 6 cultivated C. oleifera varieties all showed bright blue fluorescence, but the blue fluorescence brightness of petals of different varieties was different (Fig. 4 A). Interestingly, the fluorescence intensity of petals far away from anthers and petals close to anthers of some varieties is different, for example, 'Huajin' petals close to anthers have dark fluorescence, even invisible; petals far away from anthers show strong blue fluorescence (Fig. 4 A). To accurately compare the fluorescence intensity and pattern of petals of C. oleifera varieties and wild C. oleifera , we quantified the fluorescence intensity of petals of 6 cultivated C. oleifera varieties and wild C. oleifera . And petals far away from anthers and petals close to anthers were quantified separately. Our results indicate that the fluorescence of petals away from anthers and petals close to anthers of the six cultivated C. oleifera varieties was brighter than that of wild C. oleifera (Fig. 4 B, P < 0.05). Although the petals away from the anthers of wild C. oleifera were brighter than those near the anthers ( P < 0.05), the brightness was still low (92.03). Among these six varieties, three C. oleifera varieties (including 'Huajin', 'Changlin40' and 'Changlin53') had fluorescent patterns in their petals, and all showed stronger fluorescence brightness in petals away from anthers than in petals close to anthers (Fig. S4, P < 0.05). Among them, 'Huajin' showed the greatest difference in fluorescence brightness between the two parts of the petals. There was no difference in fluorescence brightness between the petals near the anthers and the petals away from the anthers in the remaining three varieties. Moreover, 'Huashuo' had the brightest fluorescence ( P < 0.05), and the fluorescence brightness of both petals near the anthers and petals far away from the anthers was significantly stronger than that of 'Ganzhouyou1' and 'Yilu'. 3.5 Hydroxycinnamic acid derivatives accumulation correlates with fluorescence intensity enhancement To understand the biochemical basis of petal blue fluorescence variation, we conducted metabolomic analysis of different types of petals: ( 1 ) The C. oleifera variety 'Huajin', its petals far from the anther (HJ_T) emitted blue fluorescence, the petals near the anther (HJ_B) emitted weaker blue fluorescence than HJ_T. Therefore, the petals were divided into two sample types, i.e., HJ_T and HJ_B. ( 2 ) The C. oleifera variety 'Huashuo' (HS), whose whole petals have strong blue fluorescence. ( 3 ) Wild C. oleifera (WCO), whose petals are weakly fluorescent. Untargeted metabolomics demonstrated that these types of samples have significantly different metabolic profiles (Fig. 5 A). A total of 1,988 metabolites were detected, predominantly comprising flavonoids (27.54%), phenolic acids (20.34%), and terpenoids (12.94%, Fig. S5). This result indicated that the blue fluorescence in C. oleifera petals was associated with differential accumulation patterns of metabolites. Comparative analysis ( P 1) suggests that the accumulation of flavonoids and phenolic acids was positively correlated with fluorescence intensity (Fig. 5 B and Table S3,4). In HJ_B vs HJ_T, 329 down-regulated metabolites included 100 flavonoids (23%) and 99 phenolic acids (23%); HS vs WCO showed 241 up-regulated metabolites containing 61 flavonoids (25%) and 53 phenolic acids (22%); HS vs HJ_T exhibited 704 down-regulated metabolites with 181 flavonoids (26%) and 164 phenolic acids (23%). These patterns collectively suggested a positive relationship between flavonoid and phenolic acid content and fluorescence intensity. This hypothesis was further supported by the observation that 42% of 540 up-regulated metabolites in HJ_T vs WCO were flavonoids and phenolic acids, and 42% of 392 up-regulated metabolites in HJ_B vs WCO were flavonoids and phenolic acids. Notably, 52% of 310 down-regulated metabolites in HJ_B vs HS were flavonoids (95, 31%) and phenolic acids (66, 21%). The detected metabolites were classified into 10 clusters of content accumulation patterns by K-means clustering, in which the trend of metabolite accumulation in cluster 8 (64 metabolites, Fig. 5 C) was in high agreement with the quantitative fluorescence intensity data. This cluster contained 14 flavonoids (e.g., 3,7-Di-O-methylquercetin) and 14 phenolic acids (8 hydroxycinnamic acid derivatives, e.g., Isochlorogenic acid b), which accounted for 44% of the metabolites in the cluster (Fig. S6). A total of 27 differential metabolites were obtained after analysis ( P < 0.05 , Fig. 5 D). To further screen the key metabolites from these 27 metabolites, the following screening conditions were set: ( 1 ) Significant down-regulation in HJ_B vs HJ_T and HJ_B vs HS ( P 1); ( 2 ) Concurrent significant up-regulation in HS vs WCO, HJ_B vs WCO, and HJ_T vs WCO ( P 1). These stringent conditions identified three key metabolites (Fig. 6D and Table S5): 3-O-p-Coumaroylquinic acid, 4-p-Coumaroylquinic acid, and Cirsilineol. Among them, 3-O-p-Coumaroylquinic acid, 4-p-Coumaroylquinic acid are hydroxycinnamic acid derivatives. Especially, 3-O-p-Coumaroylquinic acid showed significant up-regulation ( P < 0.05 ) in HJ_B vs HJ_T (|Log 2 FC|=5.75), HS vs WCO (|Log 2 FC|=3.10), HJ_B vs HS (|Log 2 FC|=2.37), HJ_B vs WCO (|Log 2 FC|=0.73), HJ_T vs WCO (|Log 2 FC|=3.25), and its content was at least 1.6-fold higher in strongly fluorescent samples than in weakly fluorescent groups (Table S5). These results suggest that hydroxycinnamic acid derivatives, especially 3-O-p-Coumaroylquinic acid, may be critical for basal fluorescence generation and spatial patterning in C. oleifera petals. DISCUSSION Cultivated C. oleifera was able to effectively attract bees through visual signals from the flowers, but wild C. oleifera not attract bees effectively. This shift in pollination strategy is due to selective breeding that allows the white petals of cultivated C. oleifera to release blue fluorescence under UV light, which is absent in wild C. oleifera . Crucially, bright blue fluorescence was detected in the petals of 6 widely cultivated varieties. We found that the formation and the patterning of this blue fluorescence were positively correlated with the accumulation of hydroxycinnamic acid derivatives (e.g., 3-O-p-Coumaroylquinic acid) within the cultivated C. oleifera petals. Such fluorescent substances are recognized for improving the quality of plant seed oils and have received significant attention from food quality researchers 27. Collectively, selection breeding targeting fruit traits inadvertently influences the plant metabolic network, indirectly enhancing the petal fluorescence of cultivated C. oleifera petals and making ‘bee avoidance’ C. oleifera transform to ‘bee attraction’. This shift highlights that the metabolic network alterations via domestication and breeding can affect and even remodel plant-pollinator interactions. Plants utilize diverse flower signals to attract pollinators to contact the stigma, and pollinators rely on these signals to locate flowers 28. These flower signals have been changed due to the selection of pollinators, especially in the shift from bee-pollinated to bird-pollinated flowers, and plants may undergo changes in multiple traits, such as color, floral scent, and nectar. The study of Mori et al. 29 documented that bird-pollinated and bumblebee-pollinated red camellias use different petal color strategies to attract and avoid different pollinators. Petal blue fluorescence is included in this strategy, and is regarded as an attraction for bees and bumblebees 26 , 29. Interestingly, white wild C. oleifera petals do not emit blue fluorescence under UV light; the non-social Andrena camellia (native pollinator of C. oleifera ) has a similar visual system to bees and bumblebees 30, but prefers filter paper without fluorescence (Fig. S8). Thus, we suggest that the non-fluorescent petals of ‘bee avoidance’ C. oleifera may represent an adaptation shaped by multi-trait coevolution with the native pollinator Andrena camellia. However, selective breeding may inadvertently lead to the partial or total disruption of this adaptation because the plant metabolic networks can be changed by breeding. Meanwhile, the changes of the plant metabolic networks caused by breeding can interfere with floral signals, such as nectar and floral scent components, and may have appreciable levels of genetic variation and heritability like floral reward chemical traits4. This may be attributed to the relationship between genes regulating secondary metabolite biosynthesis and agronomic traits during breeding 31. Generally, some of the genes for secondary metabolite biosynthesis are in close proximity to genes that regulate other traits (such as yield) 32. Here, we report the unexpected finding that 6 C. oleifera varieties that dominate the industry exhibit stronger petal fluorescence than wild C. oleifera , although the petal fluorescence phenotype also differs among varieties. Although current selective breeding of Camellia oleifera and other plants always focuses on economic traits (such as fruit set, fruit oil quality, etc.), none of the research identifies direct selection for the petal fluorescence phenotype 33 , 34. Considering the developmental homology of flowers and fruits, unintentional selection on the content of secondary metabolites in fruits may also influence flower traits 35. Our studies have shown that selective breeding affects the content and spatial distribution of hydroxycinnamic acid derivatives within the petals; these various hydroxycinnamic acid derivatives could emit blue fluorescence under UV light, explaining the fluorescence phenotype change 36 , 37. Hydroxycinnamic acid derivatives are an important class of polyphenolic compounds 38. It has been found that most plant edible oils are rich in hydroxycinnamic acid derivatives, which are associated with oil productivity and oil health benefits during oil plant domestication and breeding 27. Notably, a number of polyphenolic compounds that are beneficial to human health have been favored by researchers of seed oil quality, and may contribute to flower color 39. In addition, hydroxycinnamic acid derivatives are thought to play an important role in protecting plants from UV radiation, herbivores, and pathogens when plants are subjected to biotic or abiotic stresses, ranging from protecting plants 40. Thus, the content of secondary metabolites (e.g., high content of hydroxycinnamic acid derivatives) may be inadvertently selected during oil plant domestication and breeding, because the benefits associated with the accumulation of secondary metabolites are consistent with the goals of selective breeding. As a result, all 6 widely cultivated C. oleifera varieties possessed a bright blue petal fluorescence, which may be a by-product of positive selection for desirable fruit traits. Based on this viewpoint, other secondary metabolites (such as flavonoids) with fluorescence and contributing to fruit quality, substrates for these secondary metabolites, or associated synthases may also be unintentionally selected in this process 41–43. Although breeding and domestication strategies based on secondary metabolites have not been developed for many crops, horticultural plants, and woody perennials, it is well known that breeding and domestication have regulated the network of plant secondary metabolites, and this role has had a profound and neglected impact on plant environmental adaptation and biotic interactions 44. Alterations in floral traits due to agronomic selection frequently yield unintended consequences for pollinator attraction and pollination efficacy, even enabling attraction of novel pollinators 15. In this study, the innovative blue petal fluorescence mediated by selective breeding has made the ‘bee avoidance’ C. oleifera attractive to bees 17 , 45. In the past, flower fluorescence was not considered important for pollinator attraction 46. However, we clarified through a series of experiments that the blue fluorescence of C. oleifera petals can act as an independent visual signal to attract bees to visit flowers. Our previous experiments on Castanea henryi have also demonstrated that blue fluorescence is attractive to flies 47. As one of the visual signals of petals, fluorescence may often be affected by factors such as the petal color and gloss. In past studies, background has been shown to potentially affect the transmission of flower visual signals to pollinators, e.g., background may affect color contrast 48 , 49. Our results reveal that blue fluorescence on a white background has the highest availability for bees, followed by red and blue. These results firstly indicate that the ecological function of blue fluorescence depends on the background color, and the blue fluorescence of white C. oleifera petals is available to bees 50. While the relative importance of fluorescence compared to other visual signals (e.g., flower color and gloss) requires further elucidation, it is evident that inadvertent breeding-induced changes in flower signals may interfere with mutual adaptations of plants and pollinators. This interference even partially or completely reverses the process of pollinator shifts, although such shifts are usually considered irreversible under natural conditions 51. This study highlights a previously unappreciated result in plant domestication and breeding: domestication and breeding that focuses primarily on economic traits (fruit yield and quality) may potentially influence plant metabolic networks, thereby reshaping or altering flower signals and having a profound effect on plant-pollinator interactions 16. Our findings on C. oleifera provide a compelling case study. And unexpectedly, we found that this effect transformed wild ‘bee avoidance’ C. oleifera into ‘bee attraction’, implying a reversal or disruption of pollinator shifts 52. More importantly, the fluorescent traits induced by the accumulation of beneficial secondary metabolites (hydroxycinnamic acid derivatives) in the petals of C. oleifera varieties provide a direct solution to the pollination crisis facing this economically critical oilseed crop, as new flower visitors (bees) are perceived to be effective 21. With the surge in global demand for edible oils, ensuring adequate pollination of insect-dependent crops like C. oleifera is critical, but a challenge in the face of declining pollinators. Thus, petal fluorescence could be consciously selected for in future breeding programs for C. oleifera and related Camellia species to reliably enhance pollination services 53. Our work further supports that future attempts could be made to intentionally design flower traits to attract pollinators or to shape desirable pollination networks by manipulating plant secondary metabolite networks. The current booming biotechnology can fully help us to understand and utilize plant metabolite-related genes, opening avenues for targeted metabolic engineering or breeding strategies 54 , 55. This approach holds significant promise not only for optimizing pollination in existing crops but also for strategically designing plant-pollinator networks within agroecosystems, contributing to more sustainable and resilient agricultural production in the face of biodiversity loss and changing environmental conditions 56. Declarations Conflicts of Interest: The authors declare no conflict of interest. Funding This work was supported by the Modern Agroindustry Technology Research System of the Ministry of Agriculture and Rural Affairs of China (grant number CARS-44) and the National Key R&D Program of China (2023YFD2200301). Author Contributions All authors contributed to the conceptualization and design of this research. B.Y., Y.B.L., F.L.H., Y.H.L., X.L.S., X.M.T., X.M.F., and D.Y.Y. coordinated and collected samples. B.Y., Y.H.L., X.L.S., and Y.Y.L. conducted experiments. B.Y., X.M.T., and Y.Y.L. analyzed the data. 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07:22:01","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":176743,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7998239/v1/4d7c8d6ece013373cc9a261f.html"},{"id":95000691,"identity":"9aa36537-53d6-4733-9272-15a1174b40b1","added_by":"auto","created_at":"2025-11-03 09:00:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":603470,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelection of visual and olfactory signals for four classes of pollinators. \u003c/strong\u003e(A) The major pollinators of\u003cem\u003e Camellia oleifera\u003c/em\u003einclude flies (a), hoverflies (b), bees (c), and wasps (d). (B)Visiting insect numbers of flies (a), hoverflies (b), bees (c), and wasps (d) on the branches of the two treatments. TPB: branches covered with transparent plastic bags; BPB: branches covered with black plastic bags with holes. *T-test for all variables, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05. Data are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7998239/v1/e15cba08b512f3d7681267f5.png"},{"id":94992751,"identity":"86c4e30a-6211-4e8f-ae24-d532112289e9","added_by":"auto","created_at":"2025-11-03 07:22:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":243034,"visible":true,"origin":"","legend":"\u003cp\u003eBlue fluorescence of \u003cem\u003eCamellia oleifera\u003c/em\u003e petals can attract pollinators effectively. (A) Preferences of \u003cem\u003eApis carana\u003c/em\u003e for visiting the flowers of \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' and 'TS'. (B) Attractiveness (a) and chemical composition (b) of the floral odor of \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' and 'TS' flowers to \u003cem\u003eA. carana\u003c/em\u003e. (C) Phenotypic traits (a) of \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' and 'TS', fluorescence color (b), and intensity (c) of flowers under ultraviolet light. (D) Effect of blue fluorescence on pollinator visits by \u003cem\u003eCamellia oleifera\u003c/em\u003e. CK: not treated; T1 group: sprayed with pure water; T2 group: sprayed with 0.3 mg/g chlorogenic acid solution. *T-test for all variables, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. Different letters indicate significant differences at \u003cem\u003eP \u0026lt;0.05\u003c/em\u003e. Data are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7998239/v1/d1362fb7dacc892014d57b4a.png"},{"id":94992754,"identity":"603bb039-4ed6-4d0f-b1ee-26db00a2d34e","added_by":"auto","created_at":"2025-11-03 07:22:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":237106,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlue fluorescence is a separate signal that relies on color contrast to attract pollinators.\u003c/strong\u003e (A) Pollinator visiting numbers for the four white paper treatments. CK, without any treatment; T1, aqueous solution sprayed with chlorogenic acid (0.3 mg/g); T2, solution sprayed with 50% sucrose; T3, solution sprayed with a mixture of chlorogenic acid (0.3 mg/g) and sucrose (1 g/g). Different letters indicate significant differences at \u003cem\u003eP \u0026lt;0.05\u003c/em\u003e. (B) Pollinator visiting numbers for the three treatments: blue (a), red (b), and green (c). T1: aqueous solution sprayed with chlorogenic acid (0.3 mg/g); T2: solution sprayed with 50% sucrose; and T3: solution sprayed with a mixture of chlorogenic acid (0.3 mg/g) and sucrose (1 g/g). Different letters indicate significant differences at \u003cem\u003eP \u0026lt;0.05\u003c/em\u003e. (C)Pollinator visiting numbers on white (a), blue (b), red (c), and green (d) paper sheets with (T) and without (CK) chlorogenic acid aqueous solution. *T-test for all variables, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05. Data are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7998239/v1/8c62d60de3ba3aa97db7bcc0.png"},{"id":94992748,"identity":"0039c3b4-8382-48cc-85c4-9a61e55e8ee4","added_by":"auto","created_at":"2025-11-03 07:22:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1193986,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePetal fluorescence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCamellia oleifera\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e varieties and wild \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. oleifera\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) Petals and petal fluorescence of 6 cultivated \u003cem\u003eC. oleifera\u003c/em\u003e varieties and wild \u003cem\u003eC. oleifera\u003c/em\u003e under ultraviolet (UV) light. (B) Fluorescence intensity of petals away from anthers (a) and petals close to anthers (b) of 6 cultivated \u003cem\u003eC. oleifera\u003c/em\u003evarieties and wild \u003cem\u003eC. oleifera\u003c/em\u003e. WCO refers to wild \u003cem\u003eC. oleifera\u003c/em\u003e, HJ refers to 'Huajin', HS refers to 'Huashuo', CL40 refers to 'Changlin40', CL53refers to 'Changlin53', GZY1 refers to 'Ganzhouyou1', and YL refers to 'Yilu'.Different letters indicate significant differences at \u003cem\u003eP \u0026lt;0.05\u003c/em\u003e. Data are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7998239/v1/a0536e50fa756836251f559f.png"},{"id":95000564,"identity":"73eb859f-40dc-4990-98dc-47cfb6cbe281","added_by":"auto","created_at":"2025-11-03 08:59:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":682446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of key metabolites affecting petal fluorescence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCamellia oleifera\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) Principal component analysis of untargeted metabolomics results for four sample types. (B) Substance composition of differential metabolites obtained from intergroup comparisons. (C) K-means clustering of the untargeted metabolomics results of the four sample types. (D) The contents comparison of 28 flavonoid and phenolic acid metabolites was screened. The red arrows indicate the key metabolites screened. HJ_T represents the petals of the \u003cem\u003eC. oleifera\u003c/em\u003e variety 'Huajin' away from the anthers; HJ_B represents the petals of the \u003cem\u003eC. oleifera\u003c/em\u003e variety 'Huajin' near to the anthers; HS represents the petals of the \u003cem\u003eC. oleifera\u003c/em\u003e variety 'Huashuo'; WCO represents the petals of wild \u003cem\u003eC. oleifera\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7998239/v1/a2a3352533ef9dd6652d376a.png"},{"id":95001663,"identity":"7fb03b82-c597-46c9-b5d3-bf80fbd0d3ab","added_by":"auto","created_at":"2025-11-03 09:02:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4311547,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7998239/v1/5fdc23e7-2c42-401a-9524-592a933f438b.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eBreeding-mediated metabolic changes unexpectedly enhance petal blue fluorescence and shift the attractiveness of tea-oil \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCamellia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eto bees\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTo ensure the security of the global food supply, people have continued to improve the efficiency of agricultural production through plant domestication and breeding. However, modern agricultural production is facing multiple pressures, including climate change and environmental pollution, and needs to increase production while ensuring sustainability 1. The Food and Agriculture Organization of the United Nations (FAO) predicts that global food production will need to increase by 70% in 2050 to meet the demands of a growing population 2. This daunting goal requires revisiting the existing plant breeding project. While modern breeding has produced new varieties for drought, salt tolerance, and high yield in many crops, there is still a need to consider integrated multifactorial pressures in future breeding to maintain global food security and achieve low-input sustainable agriculture 3. While current plant breeding strategies are attempting to design plants adapted to future environments, the impact of crop domestication and breeding on plant-pollinator interactions, an interaction that is a key prerequisite for stable plant yields, has been overlooked 4. This knowledge gap could lead to serious ecological and economic risks. Up to 75% of the major crops and 35% of food production rely on animal pollination, and the economic value it creates has climbed to \u003cspan\u003e$\u003c/span\u003e235\u0026ndash;577\u0026nbsp;billion per year and continues to grow 5\u003csup\u003e,\u003c/sup\u003e6. Therefore, incorporating plant-pollinator interaction in agroecology into the theoretical framework of future plant breeding is a key scientific prerequisite and urgent practical need to realize sustainable production at the plot and agroecosystem levels.\u003c/p\u003e\u003cp\u003eDuring the domestication and breeding, humans have continuously improved crops through targeted selection for economic traits, a process that has been significantly accelerated by modern breeding techniques 7. However, such selection often produces unintended effects while optimizing the targeted plant traits 8. Numerous empirical studies have shown that domestication and breeding significantly alter the secondary metabolic profiles of plants and affect their chemical defences 9\u003csup\u003e,\u003c/sup\u003e10. More importantly, changes in secondary metabolites may mediate the modification of floral traits such as flower colour and floral scent, which are an important part of the plant-pollinator syndrome 11\u003csup\u003e,\u003c/sup\u003e12. In nature, the flowers of many plants are adapted to a particular group of pollinators and have evolved suitable pollinator syndromes (including colour, shape, and scent) to attract the most efficient pollinators 13\u003csup\u003e,\u003c/sup\u003e14. Thus, breeding-mediated changes in flower traits may affect plant-pollinator interactions. In terms of attraction to pollinators, this may either disrupt existing plant-pollinator interactions, maintain existing interactions, or induce novel interactions 15. A deep understanding of the mechanisms and consequences of such effects is important and interesting for analysing the coevolutionary of plants and pollinators. More importantly, it is meaningful for agroecology, as such effects may directly modulate the pollination effects of self- or cross-pollination plants with respect to the potential fruit yield and quality (including nutrition and flavour) 16.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCamellia\u003c/em\u003e, including over 280 species, is thought to be involved in the shift from bee-pollinated to bird-pollinated flowers 17. Some of these species have been domesticated and bred over decades to become an important group of economic plants and horticultural plants due to their ornamental flowers and high-quality oil seeds 18\u003csup\u003e,\u003c/sup\u003e19. As one of the world's four major woody oilseed species, \u003cem\u003eC. oleifera\u003c/em\u003e can be used as a classical model to study the effects of domestication and breeding on plant-pollinator interactions. Conventional point suggests that \u003cem\u003eC. oleifera\u003c/em\u003e relies on specialized pollinators (e.g., \u003cem\u003eAndrena camellia\u003c/em\u003e) and has a low attraction to bees (\u003cem\u003eApis carana\u003c/em\u003e) 20; however, recent field observation reveals frequent visits of bees to cultivated \u003cem\u003eC. oleifera\u003c/em\u003e 21, suggesting that domestication and breeding may have significantly affected plant-pollinator interactions. To resolve this paradoxical phenomenon, this study utilized cultivars, mutants, and wild sources of \u003cem\u003eC. oleifera\u003c/em\u003e to address three key questions: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) What are the key flower traits that attract bees to cultivars? (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) What are the trends in bee-attractive traits in domesticated and breeding from wild to cultivars? (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) What are the metabolic pathways that drive changes in bee-attractive traits? This study aims to fill the gaps in future plant breeding strategies that point to plant-pollinator interactions, and to assist in the establishment of a sustainable food supply system for agroecology 6.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy species and sites\u003c/h2\u003e\u003cp\u003e\u003cem\u003eC. oleifera\u003c/em\u003e is a woody oil crop with high economic value that originates in China and blooms from October to the following January. Its fruit setting is strictly dependent on insect pollination, including a variety of bees, hoverflies, and flies. This study was conducted in a \u003cem\u003eC. oleifera\u003c/em\u003e plantation in the Yuelu and Wangcheng Districts of Changsha City, central China. The two locations are relatively close, and the main species of pollinators are the same (Table S1).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTree traits\u003c/h3\u003e\n\u003cp\u003eWe selected eight plots measuring 10 m \u0026times; 10 m in Wangcheng District. The target trees (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;40) were selected from the eastern, western, southern, northern, and middle sections of each plot. The following traits of the sampled trees were measured: (a) tree height, namely height from the uppermost point to the surface of ground; (b) base diameter, namely diameter of the trunk 0.2 m above the ground; (c) flatness of crown, that is (east\u0026ndash;west crown width \u0026ndash;\u0026ndash; north\u0026ndash;south crown width) / east\u0026ndash;west crown width; (d) crown width, namely, the average of the east-west and north-south crown widths; (e) flower number, that is, total number of flowers on the tree; and (f) flower density, namely, flower number/crown area 22.\u003c/p\u003e\n\u003ch3\u003eInsect visits\u003c/h3\u003e\n\u003cp\u003eVisiting insect data were collected from eight plots in Wangcheng District (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;40). Only pollinator data, that is, insects that came into contact with the stigma and anthers, were recorded as available data. Insect visit surveys were conducted when pollinating insects were the most active from 11:00 to 12:00 pm. 23. We selected five target trees in each plot and observed them for 30 min, after which insect visits in the upper and lower layers of the canopy of each tree were recorded separately. The crown located 1.5 m above the ground was called the upper canopy, and the crown located within 1.5 m was called the lower canopy. The average height of the trees was 3 m. All trees in each sample plot were observed simultaneously to ensure data reliability.\u003c/p\u003e\n\u003ch3\u003eBehavioral two-choice assay\u003c/h3\u003e\n\u003cp\u003eTo determine which of the visual and olfactory signals of \u003cem\u003eC. oleifera\u003c/em\u003e were more attractive to pollinators, we observed the behavioral choices of the insects. In many studies, black-and-white bottle experiments have been used to explore the preference of insects for signals, but this was difficult to do with \u003cem\u003eC. oleifera\u003c/em\u003e. Therefore, plastic bags were used instead of black-and-white bottles. We performed the following experiments: (a) some flowers were covered with transparent plastic bags (TPB) such that they displayed only visual signals, and (b) some flowers were covered with black plastic bags (BPB) with holes such that they emitted only olfactory signals 24. On a clear day, we selected target trees at Site 1 and bagged their flowers. Three trees were used for each treatment, with a total of 120 flowers. Two trees with different treatments comprised the same group in the same position as the insect nests. We observed the number of insects that remained on the bags and explored them. Observations were recorded on four sunny days from 11:00 am to 12:00 pm. Given that the respiration of plants inside the bag produces moisture, which affects the release of odor and visual signals, the bag should be changed regularly, and different trees and branches need to be selected daily to ensure the freshness of the flowers.\u003c/p\u003e\n\u003ch3\u003eFlower traits comparison\u003c/h3\u003e\n\u003cp\u003eAfter selective breeding, we have obtained many genetic materials, and 'ST' is one of them. It is obtained by natural intraspecific hybridization of \u003cem\u003eC. oleifera\u003c/em\u003e. To understand the difference between \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' and 'ST', we quantitatively measured their flower size (transverse length and longitudinal length), petal size (transverse length and longitudinal length), at least 30 flowers were measured.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSelection experiments of bees in the field\u003c/h2\u003e\u003cp\u003eSince the previous study found that \u003cem\u003eApis cerana\u003c/em\u003e is a potential dominant pollinator, \u003cem\u003eA. cerana\u003c/em\u003e is preferred for different \u003cem\u003eC. oleifera\u003c/em\u003e varieties were compared here 21. Firstly, five fresh and blooming flowers of \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' and 'ST' were collected and placed at the site, 0.5m from the hive entrance. It was ensured that there was no obstruction between the hive and the tested flowers. Recording of choices by bees that came out of the hive to forage was then initiated, and bees that did not make a choice were excluded, with at least 30 bees recorded for valid choice behavior. After every 10 bees made a choice, the position of the flowers was changed to avoid positional effects. It was also necessary to promptly replace flowers that were not fresh (petal oxidation).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eY-Tube Olfactometer Assay\u003c/h3\u003e\n\u003cp\u003eBehavioral assays were conducted using a glass Y-tube olfactometer (20 cm stem length, 15 cm arm length, 40 mm internal diameter, and 60\u0026deg; arm angle) under species-specific airflow conditions (300mL/min) maintained by atmospheric sampling pump (QC-1B, Beijing Municipal Institute of Labor Protection, Beijing, China). Charcoal-filtered air, humidified with distilled water, was delivered through each arm. Freshly collected \u003cem\u003eC. oleifera\u003c/em\u003e flowers (including \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' and 'TS') of uniform size, maturity, and bloom stage were used as odor sources. For each trial, five intact flowers of a single variety were placed in one arm, while five flowers of different varieties were placed in the opposite arm. Flowers were replaced after each trial to ensure freshness and minimize volatile degradation. \u003cem\u003eApis cerana\u003c/em\u003e were individually introduced at the stem base and observed for 5 min under controlled environmental conditions (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 40\u0026ndash;60% RH, and red light). A choice was recorded if the insect entered an arm and remained there for at least 5 s. Non-responsive individuals (no choice within the allotted time) were excluded. Each treatment was tested with at least 30 individuals, and arm positions alternated every 10 bees to eliminate positional bias.\u003c/p\u003e\n\u003ch3\u003eFloral odor detection by gas chromatography–time of flight mass spectrometry (GC×GC-TOF MS)\u003c/h3\u003e\n\u003cp\u003eThe analysis of volatile compounds was conducted using a LECO Pegasus\u0026reg; 4D GC instrument (LECO, St. Joseph, MI, USA) consisting of an Agilent 8890A GC\u0026times;GC-TOF MS (Agilent Technologies, Palo Alto, CA, USA) system equipped with a split/splitless injector and dual-stage cryogenic modulator (LECO) coupled with TOFMS detector (LECO) at Suzhou Panomix Biomedical Technology Co., Ltd. (Suzhou, China). Sample preparation involved solid-phase microextraction (SPME). First, samples of floral odors were prepared using MonoTrap, and three flowers (\u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' and 'TS') and MonoTrap were placed in the same container to allow natural release of floral odors for 7.5 h at room temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C). Then, MonoTrap was put into a headspace vial. Prior to extraction, the SPME fiber was conditioned at 270\u0026deg;C for 10 min. The headspace vial with samples was incubated at 80\u0026deg;C for 10 min, followed by adsorption of volatiles at 80\u0026deg;C for 40 min. The SPME fiber was then desorbed in the GC injector at 250\u0026deg;C for 5 min. A DB-Heavy Wax column (30 m \u0026times; 250 \u0026micro;m \u0026times; 0.5 \u0026micro;m, Agilent) and an Rxi-55il MS column (2 m \u0026times; 150 \u0026micro;m \u0026times; 0.15 \u0026micro;m, Restek) were used for separation, with helium as the carrier gas (1.0 mL/min). The oven temperature program started at 50\u0026deg;C (held for 2 min), ramped to 220\u0026deg;C at 4\u0026deg;C/min, and maintained for 13 min; the secondary oven and modulator were set 5\u0026deg;C and 15\u0026deg;C higher than the primary column, respectively, with a 5.0 s modulation period. Mass spectrometry parameters included an ion source temperature of 250\u0026deg;C, electron ionization at 70 eV, a scan range of \u003cem\u003em/z\u003c/em\u003e 35\u0026ndash;550, and a detector voltage of 1960 V. Data acquisition was performed at 200 spectra/s.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eFluorescence observation and measurement\u003c/h2\u003e\u003cp\u003eTo determine the visual attraction strategy of petals, we picked fresh \u003cem\u003eC. oleifera\u003c/em\u003e petals from the tree. We first observed them and compared the petal fluorescence of \u003cem\u003eC. oleifera\u003c/em\u003e varieties qualitatively and quantitatively.\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e(a) To determine the fluorescence color of \u003cem\u003eC. oleifera\u003c/em\u003e flowers under UV light, the petals were placed on glass slides, observed, and photographed under a fluorescence microscope (OLYMPUS-BX51, Japan).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e(b) Thirty flowers were randomly selected from \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin', 'Changlin40', 'Ganzhouyou1', 'Huaxin', 'Yilu', 'ST', and wild \u003cem\u003eC. oleifera\u003c/em\u003e, respectively; one petal per flower was randomly selected for quantitative analysis (n\u0026thinsp;=\u0026thinsp;90). The fluorescence of each petal was measured using ImageJ-Java8 software (National Institutes of Health, Bethesda, MD, USA) to determine its color and brightness. An RGB color system was used to quantify the color, decomposing the fluorescent color of the petals under UV light into red, green, and blue components, and obtaining the luminance value for each color.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eFluorescence attraction test\u003c/h2\u003e\u003cp\u003eTo test the availability of blue fluorescence for pollinators, we used chlorogenic acid, a substance that releases blue fluorescence under UV light. It has a certain degree of water solubility and is easily soluble in organic solvents. However, given that this study needed to be conducted in a field environment, we chose pure water, which is harmless to wild animals, as the contrast solvent. The water solubility of chlorogenic acid is limited, so we combined our experiments and previous studies, then selected 0.3 mg/g, which is a low concentration that can be observed by insects (Fig. S1). To explore the effect of blue fluorescence on pollinators, we selected four sunny days at Site 1 and observed insects that visited the target trees for 30 minutes each day from 11:00 am to 12:00 pm. The target trees were divided into three groups: (a) CK, untreated; (b) T1, sprayed with pure water; and (c) T2, sprayed with an aqueous solution of 0.3 mg/g chlorogenic acid to enhance the blue fluorescence. Each treated branch was controlled to five flowers, each flower was evenly sprayed, and each treatment was repeated three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eInsect response induced by fluorescent cues under different backgrounds\u003c/h2\u003e\u003cp\u003eFluorescence can be used as a food trap to induce pollinator responses. The response of \u003cem\u003eApis mellifera\u003c/em\u003e to paper sprayed with chlorogenic acid above a beaker with sweet sugar has also been tested by others 25\u003csup\u003e,\u003c/sup\u003e26. However, none of these studies have clarified the specific relationship between blue fluorescence and food. To explain this, we prepared four types of paper: (CK) without any treatment; (T1) aqueous solution sprayed with chlorogenic acid (0.3 mg/g); (T2) sprayed with 50% sucrose solution; and (T3) sprayed with a mixture of chlorogenic acid (0.3 mg/g) and sucrose (1 g g-1). Two experiments were conducted in this study. In the first experiment, we placed four types of white paper at the site, which is 0.5m from the hive entrance. And observed them for six hours from 11:00 am to 5:00 pm on each sunny day for 8 days. The number of insect visits per hour was calculated and compared numerically. To effectively evaluate the role of the clues, we treated four types of white, red, blue, and green paper simultaneously, placed at the site, which is 0.5m from the hive entrance. at the same time, and recorded the number of insect visits to each paper, which was the same as before, for five days. The grassland for the experiment was relatively open, where the sun could shine directly.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMetabolome of petals\u003c/h2\u003e\u003cp\u003eThe petals of \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin', 'Huashuo' and wild \u003cem\u003eC. oleifera\u003c/em\u003e were first cut from the middle, with the section near the anther being the basal part, and the other end being the apical part. The cut petals are immediately frozen in liquid nitrogen and stored in a -80\u0026deg;C refrigerator. Three biological replicates were prepared for each variety. All samples were analyzed using ultra-high-performance liquid chromatography\u0026ndash;tandem mass spectrometry (UPLC-MS/MS) to mitigate batch effects. UPLC-MS/MS analysis was performed at Wuhan Matware Biotechnology Co., Ltd. (Wuhan, China).\u003c/p\u003e\u003cp\u003eThe biological samples were lyophilized using a vacuum freeze-dryer (Scientz-100F) for 63 hours and ground into powder (30 Hz, 1.5 min) with a grinder (MM 400, Retsch). Approximately 50 mg of the powder was weighed (electronic balance, MS105DM, Mettler Toledo) and mixed with 1200 \u0026micro;L of -20\u0026deg;C pre-cooled 70% methanol aqueous solution containing internal standards (less than 50 mg added at the rate of 1200 \u0026micro;L extractant per 50mg sample). The mixture was vortexed every 30 minutes (30 sec each, 6 times), centrifuged (12,000 rpm, 3 min), and filtered through a 0.22 \u0026micro;m microporous membrane for UPLC-MS/MS analysis.\u003c/p\u003e\u003cp\u003eChromatographic analysis was performed on an UPLC-ESI-MS/MS system (ExionLC\u0026trade; AD, SCIEX) equipped with an Agilent SB-C18 column (1.8 \u0026micro;m, 2.1 \u0026times; 100 mm). The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A gradient program was applied: 5% B (0 min) to 95% B (9 min), held for 1 min, returned to 5% B (10\u0026ndash;11.1 min), and equilibrated until 14 min. The flow rate, column temperature, and injection volume were 0.35 mL/min, 40\u0026deg;C, and 2 \u0026micro;L, respectively. Effluent was analyzed using an ESI-QTRAP-MS/MS system.\u003c/p\u003e\u003cp\u003eMass spectrometry was conducted using an ESI-QTRAP-MS/MS system (SCIEX) in both positive (IS 5500 V) and negative (IS -4500 V) ion modes. The ion source parameters included a temperature of 500\u0026deg;C, curtain gas (25 psi), and ion source gases I/II (50/60 psi). MRM transitions were optimized for each metabolite with collision energies (CE) and declustering potentials (DP) calibrated using reference standards. Data acquisition and peak integration were performed using Analyst 1.6.3 and MultiQuant software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eThe correlation between tree traits and insect visits was analyzed by calculating the Pearson product\u0026ndash;moment correlation coefficient, and the factors most strongly related to insect visits were identified. Correlations among the tree traits were also analyzed. T-tests were used to compare the visits of insects in the upper and lower layers of the crown of three types of trees. Before determining the number of visiting insects to the branches, they were covered with transparent and black plastic bags. These branches were sprayed with chlorogenic acid or left untreated. Statistical significance was set at \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e. The insect visits of \u003cem\u003eC. oleifera\u003c/em\u003e and \u003cem\u003eC. henryi\u003c/em\u003e female flowers sprayed with chlorogenic acid aqueous solution, pure water, and untreated branches, the insect visits of white paper under four treatments, and the insect visits of three different colors of paper sprayed with chlorogenic acid aqueous solution, sugar water, and mixed solution were analyzed using the Tukey test. Statistical significance was set at \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003e3.1 Visual signals are essential in the flower-visiting decision of pollinators\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhile flower display modulates the flower-visiting decision of pollinators, the key signal of \u003cem\u003eC. oleifera\u003c/em\u003e flowers attracting pollinators was unclear. To evaluate the differences in the attraction ability of flower visual and olfactory signals to pollinators, we observed differences in the number of insects visiting the branches with different treatments. Throughout the entire observation period, four main types of pollinators were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results of the \u003cem\u003eT\u003c/em\u003e-test showed that except for wasps, the other three pollinator types showed a significant preference for the branches that display visual signals (TPB) of flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, P\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Bees and hoverflies are staunch proponents of visual signals and can only access flowers when visual signals are present (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Table S2). Although flies show a preference for visual signals too, they also visit branches that release scent (BPB). Although the wasp population was too small and only visited the branches a limited number of times, no statistical significance was found between the TPB and BPB treatments. However, only the branches that released visual signals were visited by wasps in all the observation records (0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40). This result highlights the importance of visual signals of \u003cem\u003eC. oleifera\u003c/em\u003e flowers in their pollination strategy and may be a prerequisite for pollinators to make flower visit decisions. We have provided a more detailed record of the behavior in the supplementary document. The results show that other pollinators, in addition to wasps, also hesitated in the access decision. They hovered repeatedly over the branch that displayed the visual signal but did not land. Meanwhile, wasps were relatively decisive, landing directly on the branch in each record (Table S2). This shows that although visual signals are essential, they are insufficient to support all pollinators in making immediate decisions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2 Petal blue fluorescence enhances the pollinator attraction of the flower\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDifferent \u003cem\u003eC. oleifera\u003c/em\u003e varieties have different attractiveness to pollinators. In the field, \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' attracted more \u003cem\u003eA. carana\u003c/em\u003e than \u003cem\u003eC. oleifera\u003c/em\u003e 'TS' (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, P\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), and the \u003cem\u003eA. carana\u003c/em\u003e selecting proportion of 'Huajin' was as high as 0.79%. To clarify the causes of pollinator preferences, the effects of flower odor on the decision of \u003cem\u003eA. carana\u003c/em\u003e to visit flowers were first compared. The results showed no significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, P\u0026thinsp;\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) in the selection proportion of \u003cem\u003eA. carana\u003c/em\u003e choosing the odor of 'Huajin' and 'TS'. The ellipsoid plots of the principal component analysis (PCA) also showed that the chemical composition of the 'Huajin' and 'TS' odors was similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This result supports the finding of the Y-tube experiment that the olfactory signal (floral scent) is not the cause of pollinator preference.\u003c/p\u003e\u003cp\u003eSubsequently, comparisons were made with the visual signals of \u003cem\u003eC. oleifera\u003c/em\u003e flowers. Interestingly, the flower and petal sizes of \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' and 'TS' were similar and could not be effectively separated in the ellipsoids obtained from the PCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In addition, the color of \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' and 'TS' petals is all white. This implies that flower size and flower color may not be the cause of the different preferences of pollinators visiting flowers among the \u003cem\u003eC. oleifera\u003c/em\u003e varieties. Therefore, we examined the floral organs of \u003cem\u003eC. oleifera\u003c/em\u003e under white and UV light. \u003cem\u003eC. oleifera\u003c/em\u003e 'Huajin' petals emit blue fluorescence under UV light (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, the intensity of petal fluorescence differed among the \u003cem\u003eC. oleifera\u003c/em\u003e varieties, with 'Huajin' having significantly brighter petal fluorescence than 'TS' (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, P\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). The petal fluorescence of 'Huajin' was bright blue under the ultraviolet (UV) light, while the fluorescence of 'TS' was pale and almost invisible under the UV light. This result seems to imply that the blue fluorescence of petals is an essential visual signal that helps to attract pollinators to \u003cem\u003eC. oleifera\u003c/em\u003e varieties.\u003c/p\u003e\u003cp\u003eTo further clarify that this blue fluorescence is available for pollinators, we first sprayed 0.3 mg/g chlorogenic acid solution (Fig. S1) on the branches of \u003cem\u003eC. oleifera\u003c/em\u003e to enhance the fluorescence of flowers and sprayed pure water on the branches to shield them from the interference of the solution. The results showed that the number of visiting insects did not decrease significantly after spraying with pure water. However, the number of pollinators visiting flowers increased significantly after the blue fluorescence was enhanced, indicating that the blue fluorescence was attractive to pollinators (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, P\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.3 Pollinator attraction of blue fluorescence on white background surpasses other backgrounds\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo clarify the effect of blue fluorescence on pollinators alone, we first compared the number of insect visits, including bees and flies, within one hour in the four treatments. The visiting number of pollinators to the paper sprayed with chlorogenic acid and sugar water was significantly higher than that of the untreated paper and significantly lower than that of the paper sprayed with the mixed solution of chlorogenic acid and sugar water. There was no significant difference between the visiting number of pollinators to the paper sprayed with chlorogenic acid and sugar water, suggesting that the sugar water and blue fluorescence acted as two separate signals to attract pollinators (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, P\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). To further test this result, we simultaneously treated blue, red, and green paper with the same four treatments and recorded the pollinators visits. The results showed that the changes of paper color did not mediate the difference in pollinator visits (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, P\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eAfter confirming that blue fluorescence was a separate flower-visiting signal, we examined whether spraying chlorogenic acid onto paper with different backgrounds could improve insect visits. Before the comparison, we first compared the pollinator visits to untreated paper. There was no difference in the attraction of different colors of paper to insects because there was no significant difference in the number of visitors (Fig. S2). This result provides a necessary premise for comparing the attraction of blue fluorescence with different colored backgrounds. The pollinator visiting numbers of the white, blue, and red papers sprayed with chlorogenic acid (T) were significantly higher than those of the untreated paper. However, there was no difference in insect visits between the treated and untreated green papers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, P\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The results show that when provided with a rich number of choices, pollinators do not prioritize the blue fluorescence under the green background but prefer the blue fluorescence under the white background (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, Fig. S3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.4 Cultivated varieties exhibit enhanced blue fluorescence than wild\u003c/b\u003e \u003cb\u003eC. oleifera\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo understand the petal fluorescence phenotypes of different \u003cem\u003eC. oleifera\u003c/em\u003e varieties and wild \u003cem\u003eC. oleifera\u003c/em\u003e under ultraviolet (UV) light, we observed the petals of 6 cultivated \u003cem\u003eC. oleifera\u003c/em\u003e varieties and wild \u003cem\u003eC. oleifera\u003c/em\u003e under UV light. We found that the petals of wild \u003cem\u003eC. oleifera\u003c/em\u003e hardly emitted blue fluorescence, while the 6 cultivated \u003cem\u003eC. oleifera\u003c/em\u003e varieties all showed bright blue fluorescence, but the blue fluorescence brightness of petals of different varieties was different (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Interestingly, the fluorescence intensity of petals far away from anthers and petals close to anthers of some varieties is different, for example, 'Huajin' petals close to anthers have dark fluorescence, even invisible; petals far away from anthers show strong blue fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eTo accurately compare the fluorescence intensity and pattern of petals of \u003cem\u003eC. oleifera\u003c/em\u003e varieties and wild \u003cem\u003eC. oleifera\u003c/em\u003e, we quantified the fluorescence intensity of petals of 6 cultivated \u003cem\u003eC. oleifera\u003c/em\u003e varieties and wild \u003cem\u003eC. oleifera\u003c/em\u003e. And petals far away from anthers and petals close to anthers were quantified separately. Our results indicate that the fluorescence of petals away from anthers and petals close to anthers of the six cultivated \u003cem\u003eC. oleifera\u003c/em\u003e varieties was brighter than that of wild \u003cem\u003eC. oleifera\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although the petals away from the anthers of wild \u003cem\u003eC. oleifera\u003c/em\u003e were brighter than those near the anthers (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the brightness was still low (92.03). Among these six varieties, three \u003cem\u003eC. oleifera\u003c/em\u003e varieties (including 'Huajin', 'Changlin40' and 'Changlin53') had fluorescent patterns in their petals, and all showed stronger fluorescence brightness in petals away from anthers than in petals close to anthers (Fig. S4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Among them, 'Huajin' showed the greatest difference in fluorescence brightness between the two parts of the petals. There was no difference in fluorescence brightness between the petals near the anthers and the petals away from the anthers in the remaining three varieties. Moreover, 'Huashuo' had the brightest fluorescence (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the fluorescence brightness of both petals near the anthers and petals far away from the anthers was significantly stronger than that of 'Ganzhouyou1' and 'Yilu'.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5 Hydroxycinnamic acid derivatives accumulation correlates with fluorescence intensity enhancement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo understand the biochemical basis of petal blue fluorescence variation, we conducted metabolomic analysis of different types of petals: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) The \u003cem\u003eC. oleifera\u003c/em\u003e variety 'Huajin', its petals far from the anther (HJ_T) emitted blue fluorescence, the petals near the anther (HJ_B) emitted weaker blue fluorescence than HJ_T. Therefore, the petals were divided into two sample types, i.e., HJ_T and HJ_B. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) The \u003cem\u003eC. oleifera\u003c/em\u003e variety 'Huashuo' (HS), whose whole petals have strong blue fluorescence. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Wild \u003cem\u003eC. oleifera\u003c/em\u003e (WCO), whose petals are weakly fluorescent. Untargeted metabolomics demonstrated that these types of samples have significantly different metabolic profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). A total of 1,988 metabolites were detected, predominantly comprising flavonoids (27.54%), phenolic acids (20.34%), and terpenoids (12.94%, Fig. S5). This result indicated that the blue fluorescence in \u003cem\u003eC. oleifera\u003c/em\u003e petals was associated with differential accumulation patterns of metabolites.\u003c/p\u003e\u003cp\u003eComparative analysis (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 or fold change\u0026thinsp;\u0026le;\u0026thinsp;0.5, VIP\u0026thinsp;\u0026gt;\u0026thinsp;1) suggests that the accumulation of flavonoids and phenolic acids was positively correlated with fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and Table S3,4). In HJ_B vs HJ_T, 329 down-regulated metabolites included 100 flavonoids (23%) and 99 phenolic acids (23%); HS vs WCO showed 241 up-regulated metabolites containing 61 flavonoids (25%) and 53 phenolic acids (22%); HS vs HJ_T exhibited 704 down-regulated metabolites with 181 flavonoids (26%) and 164 phenolic acids (23%). These patterns collectively suggested a positive relationship between flavonoid and phenolic acid content and fluorescence intensity. This hypothesis was further supported by the observation that 42% of 540 up-regulated metabolites in HJ_T vs WCO were flavonoids and phenolic acids, and 42% of 392 up-regulated metabolites in HJ_B vs WCO were flavonoids and phenolic acids. Notably, 52% of 310 down-regulated metabolites in HJ_B vs HS were flavonoids (95, 31%) and phenolic acids (66, 21%).\u003c/p\u003e\u003cp\u003eThe detected metabolites were classified into 10 clusters of content accumulation patterns by K-means clustering, in which the trend of metabolite accumulation in cluster 8 (64 metabolites, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) was in high agreement with the quantitative fluorescence intensity data. This cluster contained 14 flavonoids (e.g., 3,7-Di-O-methylquercetin) and 14 phenolic acids (8 hydroxycinnamic acid derivatives, e.g., Isochlorogenic acid b), which accounted for 44% of the metabolites in the cluster (Fig. S6). A total of 27 differential metabolites were obtained after analysis (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). To further screen the key metabolites from these 27 metabolites, the following screening conditions were set: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Significant down-regulation in HJ_B vs HJ_T and HJ_B vs HS (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, fold change\u0026thinsp;\u0026le;\u0026thinsp;0.7, VIP\u0026thinsp;\u0026gt;\u0026thinsp;1); (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Concurrent significant up-regulation in HS vs WCO, HJ_B vs WCO, and HJ_T vs WCO (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, fold change\u0026thinsp;\u0026ge;\u0026thinsp;1.5, VIP\u0026thinsp;\u0026gt;\u0026thinsp;1). These stringent conditions identified three key metabolites (Fig.\u0026nbsp;6D and Table S5): 3-O-p-Coumaroylquinic acid, 4-p-Coumaroylquinic acid, and Cirsilineol. Among them, 3-O-p-Coumaroylquinic acid, 4-p-Coumaroylquinic acid are hydroxycinnamic acid derivatives. Especially, 3-O-p-Coumaroylquinic acid showed significant up-regulation (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) in HJ_B vs HJ_T (|Log\u003csub\u003e2\u003c/sub\u003eFC|=5.75), HS vs WCO (|Log\u003csub\u003e2\u003c/sub\u003eFC|=3.10), HJ_B vs HS (|Log\u003csub\u003e2\u003c/sub\u003eFC|=2.37), HJ_B vs WCO (|Log\u003csub\u003e2\u003c/sub\u003eFC|=0.73), HJ_T vs WCO (|Log\u003csub\u003e2\u003c/sub\u003eFC|=3.25), and its content was at least 1.6-fold higher in strongly fluorescent samples than in weakly fluorescent groups (Table S5). These results suggest that hydroxycinnamic acid derivatives, especially 3-O-p-Coumaroylquinic acid, may be critical for basal fluorescence generation and spatial patterning in \u003cem\u003eC. oleifera\u003c/em\u003e petals.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCultivated \u003cem\u003eC. oleifera\u003c/em\u003e was able to effectively attract bees through visual signals from the flowers, but wild \u003cem\u003eC. oleifera\u003c/em\u003e not attract bees effectively. This shift in pollination strategy is due to selective breeding that allows the white petals of cultivated \u003cem\u003eC. oleifera\u003c/em\u003e to release blue fluorescence under UV light, which is absent in wild \u003cem\u003eC. oleifera\u003c/em\u003e. Crucially, bright blue fluorescence was detected in the petals of 6 widely cultivated varieties. We found that the formation and the patterning of this blue fluorescence were positively correlated with the accumulation of hydroxycinnamic acid derivatives (e.g., 3-O-p-Coumaroylquinic acid) within the cultivated \u003cem\u003eC. oleifera\u003c/em\u003e petals. Such fluorescent substances are recognized for improving the quality of plant seed oils and have received significant attention from food quality researchers 27. Collectively, selection breeding targeting fruit traits inadvertently influences the plant metabolic network, indirectly enhancing the petal fluorescence of cultivated \u003cem\u003eC. oleifera\u003c/em\u003e petals and making \u0026lsquo;bee avoidance\u0026rsquo; \u003cem\u003eC. oleifera\u003c/em\u003e transform to \u0026lsquo;bee attraction\u0026rsquo;. This shift highlights that the metabolic network alterations via domestication and breeding can affect and even remodel plant-pollinator interactions.\u003c/p\u003e\u003cp\u003ePlants utilize diverse flower signals to attract pollinators to contact the stigma, and pollinators rely on these signals to locate flowers 28. These flower signals have been changed due to the selection of pollinators, especially in the shift from bee-pollinated to bird-pollinated flowers, and plants may undergo changes in multiple traits, such as color, floral scent, and nectar. The study of Mori et al. 29 documented that bird-pollinated and bumblebee-pollinated red camellias use different petal color strategies to attract and avoid different pollinators. Petal blue fluorescence is included in this strategy, and is regarded as an attraction for bees and bumblebees 26\u003csup\u003e,\u003c/sup\u003e29. Interestingly, white wild \u003cem\u003eC. oleifera\u003c/em\u003e petals do not emit blue fluorescence under UV light; the non-social \u003cem\u003eAndrena camellia\u003c/em\u003e (native pollinator of \u003cem\u003eC. oleifera\u003c/em\u003e) has a similar visual system to bees and bumblebees 30, but prefers filter paper without fluorescence (Fig. S8). Thus, we suggest that the non-fluorescent petals of \u0026lsquo;bee avoidance\u0026rsquo; \u003cem\u003eC. oleifera\u003c/em\u003e may represent an adaptation shaped by multi-trait coevolution with the native pollinator \u003cem\u003eAndrena camellia.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eHowever, selective breeding may inadvertently lead to the partial or total disruption of this adaptation because the plant metabolic networks can be changed by breeding. Meanwhile, the changes of the plant metabolic networks caused by breeding can interfere with floral signals, such as nectar and floral scent components, and may have appreciable levels of genetic variation and heritability like floral reward chemical traits4. This may be attributed to the relationship between genes regulating secondary metabolite biosynthesis and agronomic traits during breeding 31. Generally, some of the genes for secondary metabolite biosynthesis are in close proximity to genes that regulate other traits (such as yield) 32.\u003c/p\u003e\u003cp\u003eHere, we report the unexpected finding that 6 \u003cem\u003eC. oleifera\u003c/em\u003e varieties that dominate the industry exhibit stronger petal fluorescence than wild \u003cem\u003eC. oleifera\u003c/em\u003e, although the petal fluorescence phenotype also differs among varieties. Although current selective breeding of \u003cem\u003eCamellia oleifera\u003c/em\u003e and other plants always focuses on economic traits (such as fruit set, fruit oil quality, etc.), none of the research identifies direct selection for the petal fluorescence phenotype 33\u003csup\u003e,\u003c/sup\u003e34. Considering the developmental homology of flowers and fruits, unintentional selection on the content of secondary metabolites in fruits may also influence flower traits 35. Our studies have shown that selective breeding affects the content and spatial distribution of hydroxycinnamic acid derivatives within the petals; these various hydroxycinnamic acid derivatives could emit blue fluorescence under UV light, explaining the fluorescence phenotype change 36\u003csup\u003e,\u003c/sup\u003e37. Hydroxycinnamic acid derivatives are an important class of polyphenolic compounds 38. It has been found that most plant edible oils are rich in hydroxycinnamic acid derivatives, which are associated with oil productivity and oil health benefits during oil plant domestication and breeding 27. Notably, a number of polyphenolic compounds that are beneficial to human health have been favored by researchers of seed oil quality, and may contribute to flower color 39. In addition, hydroxycinnamic acid derivatives are thought to play an important role in protecting plants from UV radiation, herbivores, and pathogens when plants are subjected to biotic or abiotic stresses, ranging from protecting plants 40. Thus, the content of secondary metabolites (e.g., high content of hydroxycinnamic acid derivatives) may be inadvertently selected during oil plant domestication and breeding, because the benefits associated with the accumulation of secondary metabolites are consistent with the goals of selective breeding. As a result, all 6 widely cultivated \u003cem\u003eC. oleifera\u003c/em\u003e varieties possessed a bright blue petal fluorescence, which may be a by-product of positive selection for desirable fruit traits. Based on this viewpoint, other secondary metabolites (such as flavonoids) with fluorescence and contributing to fruit quality, substrates for these secondary metabolites, or associated synthases may also be unintentionally selected in this process 41\u0026ndash;43. Although breeding and domestication strategies based on secondary metabolites have not been developed for many crops, horticultural plants, and woody perennials, it is well known that breeding and domestication have regulated the network of plant secondary metabolites, and this role has had a profound and neglected impact on plant environmental adaptation and biotic interactions 44.\u003c/p\u003e\u003cp\u003eAlterations in floral traits due to agronomic selection frequently yield unintended consequences for pollinator attraction and pollination efficacy, even enabling attraction of novel pollinators 15. In this study, the innovative blue petal fluorescence mediated by selective breeding has made the \u0026lsquo;bee avoidance\u0026rsquo; \u003cem\u003eC. oleifera\u003c/em\u003e attractive to bees 17\u003csup\u003e,\u003c/sup\u003e45. In the past, flower fluorescence was not considered important for pollinator attraction 46. However, we clarified through a series of experiments that the blue fluorescence of \u003cem\u003eC. oleifera\u003c/em\u003e petals can act as an independent visual signal to attract bees to visit flowers. Our previous experiments on \u003cem\u003eCastanea henryi\u003c/em\u003e have also demonstrated that blue fluorescence is attractive to flies 47. As one of the visual signals of petals, fluorescence may often be affected by factors such as the petal color and gloss. In past studies, background has been shown to potentially affect the transmission of flower visual signals to pollinators, e.g., background may affect color contrast 48\u003csup\u003e,\u003c/sup\u003e49. Our results reveal that blue fluorescence on a white background has the highest availability for bees, followed by red and blue. These results firstly indicate that the ecological function of blue fluorescence depends on the background color, and the blue fluorescence of white \u003cem\u003eC. oleifera\u003c/em\u003e petals is available to bees 50. While the relative importance of fluorescence compared to other visual signals (e.g., flower color and gloss) requires further elucidation, it is evident that inadvertent breeding-induced changes in flower signals may interfere with mutual adaptations of plants and pollinators. This interference even partially or completely reverses the process of pollinator shifts, although such shifts are usually considered irreversible under natural conditions 51.\u003c/p\u003e\u003cp\u003eThis study highlights a previously unappreciated result in plant domestication and breeding: domestication and breeding that focuses primarily on economic traits (fruit yield and quality) may potentially influence plant metabolic networks, thereby reshaping or altering flower signals and having a profound effect on plant-pollinator interactions 16. Our findings on \u003cem\u003eC. oleifera\u003c/em\u003e provide a compelling case study. And unexpectedly, we found that this effect transformed wild \u0026lsquo;bee avoidance\u0026rsquo; \u003cem\u003eC. oleifera\u003c/em\u003e into \u0026lsquo;bee attraction\u0026rsquo;, implying a reversal or disruption of pollinator shifts 52. More importantly, the fluorescent traits induced by the accumulation of beneficial secondary metabolites (hydroxycinnamic acid derivatives) in the petals of \u003cem\u003eC. oleifera\u003c/em\u003e varieties provide a direct solution to the pollination crisis facing this economically critical oilseed crop, as new flower visitors (bees) are perceived to be effective 21. With the surge in global demand for edible oils, ensuring adequate pollination of insect-dependent crops like \u003cem\u003eC. oleifera\u003c/em\u003e is critical, but a challenge in the face of declining pollinators. Thus, petal fluorescence could be consciously selected for in future breeding programs for \u003cem\u003eC. oleifera\u003c/em\u003e and related \u003cem\u003eCamellia\u003c/em\u003e species to reliably enhance pollination services 53. Our work further supports that future attempts could be made to intentionally design flower traits to attract pollinators or to shape desirable pollination networks by manipulating plant secondary metabolite networks. The current booming biotechnology can fully help us to understand and utilize plant metabolite-related genes, opening avenues for targeted metabolic engineering or breeding strategies 54\u003csup\u003e,\u003c/sup\u003e55. This approach holds significant promise not only for optimizing pollination in existing crops but also for strategically designing plant-pollinator networks within agroecosystems, contributing to more sustainable and resilient agricultural production in the face of biodiversity loss and changing environmental conditions 56.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Modern Agroindustry Technology Research System of the Ministry of Agriculture and Rural Affairs of China (grant number CARS-44) and the National Key R\u0026amp;D Program of China (2023YFD2200301).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\u003cp\u003eAll authors contributed to the conceptualization and design of this research. B.Y., Y.B.L., F.L.H., Y.H.L., X.L.S., X.M.T., X.M.F., and D.Y.Y. coordinated and collected samples. B.Y., Y.H.L., X.L.S., and Y.Y.L. conducted experiments. B.Y., X.M.T., and Y.Y.L. analyzed the data. B.Y., Y.H.L., Y.B.L., and F.L.H. contributed to the writing of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data and analysis routines in this study are available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSalse J, Barnard RL, Veneault-Fourrey C, Rouached H (2024) Strategies for breeding crops for future environments. 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Trends Ecol Evol 38:435\u0026ndash;445. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tree.2022.12.010\u003c/span\u003e\u003cspan address=\"10.1016/j.tree.2022.12.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Camellia oleifera, fluorescence, pollination, breeding, visual signal","lastPublishedDoi":"10.21203/rs.3.rs-7998239/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7998239/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant breeding has long focused on improving economic traits, yet its unintended effects on plant\u0026ndash;pollinator interactions remain largely overlooked. Here, we report that selective breeding in \u003cem\u003eCamellia oleifera\u003c/em\u003e unexpectedly enhances petal blue fluorescence, altering its attractiveness to bees. Field assays and behavioral experiments demonstrated that visual cues, rather than floral scents, play a decisive role in pollinator visitation, with bees showing a strong preference for petals emitting bright blue fluorescence under ultraviolet (UV) light. All six widely cultivated varieties exhibited stronger blue fluorescence than wild \u003cem\u003eC. oleifera\u003c/em\u003e, in which the petals were nearly non-fluorescent. Metabolomic profiling revealed that the enhanced fluorescence correlated with the accumulation of hydroxycinnamic acid derivatives, particularly 3-O-p-coumaroylquinic acid. These compounds, originally targeted for improving fruit and oil quality, were found to mediate the formation of petal fluorescence and spatial patterning. This breeding-mediated metabolic shift effectively transformed \u003cem\u003eC. oleifera\u003c/em\u003e from an \u0026ldquo;anti-bee\u0026rdquo; species into a \u0026ldquo;bee-attraction\u0026rdquo; species, suggesting a reversal of its natural pollination syndrome. Our findings uncover a previously unrecognized ecological consequence of plant breeding, linking secondary metabolism with pollination ecology. Recognizing and harnessing such floral traits in future breeding programs may not only improve pollination efficiency but also offer a novel pathway toward sustainable agroecosystem design.\u003c/p\u003e","manuscriptTitle":"Breeding-mediated metabolic changes unexpectedly enhance petal blue fluorescence and shift the attractiveness of tea-oil Camelliato bees","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-03 07:14:02","doi":"10.21203/rs.3.rs-7998239/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"20741ff1-3f79-4a60-a50e-9e8a26596091","owner":[],"postedDate":"November 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T07:14:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-03 07:14:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7998239","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7998239","identity":"rs-7998239","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}