Materials.

This study examined ‘Zizhenzhu’ (Crataegus pinnatifida) hawthorns that showed consistent growth potential, were free of disease, and were managed in a uniform manner in the Hawthorn Germplasm Resource Nursery of the Institute of Forestry and Fruit Tree Research at Beijing Academy of Agricultural and Forestry Sciences. The trees were more than 10 years old. The experimental design examined five fruit developmental phases: S1, 70 d after bloom (DAB); S2, 90 DAB; S3, 110 DAB; S4, 130 DAB; and S5, 150 DAB (Fig. 1). Fruit samples were collected randomly from each tree from different directions and comprised 40 healthy looking fruit of similar size per tree, and three replications per set. The hawthorn peel was promptly frozen using liquid nitrogen and stored in a refrigerator at −80 °C.

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Colorimeter measurement.

The peel color was determined using a colorimeter (CM-26dG, Konica Minolta, Tokyo, Japan), with measurements taken at the equatorial region of each fruit. Five fruits were randomly selected at each developmental stage, with each fruit measured five times to ensure data reliability. The colorimeter was used to measure the L*, a*, and b* values. L* values indicate lightness, with higher values corresponding to greater lightness; a* values indicate the gradation of the color from green to red, with positive values indicating red and negative values indicating green; b* values indicate the gradation of the color from blue to yellow, with positive values indicating yellowish and negative values indicating bluish.

Reagents and instruments.

High performance liquid chromatography (HPLC)-grade methanol (MeOH), ethanol (EtOH), and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany), and acetone was purchased from Sinopharm (Beijing, China). Formic acid was purchased from Sigma-Aldrich (St Louis, MO, USA). Sodium chloride was from Rhawn (Shanghai, China). Potassium hydroxide was from Hushi (Shanghai, China). Methyl tert-butyl ether and n-hexane were purchased from CNW (Shanghai, China). Hydrochloric acid was obtained from Xinyang Chemical Reagent (Xinyang, China) and butylated hydroxytoluene (BHT) was purchased from Aladdin (Shanghai, China). Anthocyanin standard (50% methanol, 1 mg·mL⁻¹) was from IsoReag (Shanghai, China); the flavonoid standard (70% methanol, 10 mmol·L⁻¹) was from MCE (MedChemExpress, Shanghai, China); and the carotenoid standard (methyl tertiary butyl ether/methanol mixed solution, 1 mg·mL⁻¹) was from BOC (New York, NY, USA) and Sigma-Aldrich (St Louis, MO, USA). The QTRAP 6500 + LC-MS/MS instrument was from SCIEX (MA, Marlborough, USA); the 5424R bench-top high-speed refrigerated centrifuge was from Eppendorf (Hamburg, Germany); and the AS 60/220.R2 electronic balance was from Radwag (Radom, Poland). The MIX-200 multitube vortex oscillator was supplied by Shanghai Jingxin (Shanghai, China). The MM400 ball milling instrument was produced by Retsch (Haan, Germany). The KQ5200E ultrasonic cleaner was from Kunshan Shumei (Kunshan, China). The CentriVap centrifugal concentrator was supplied by Labconco (Kansas City, MO, USA).

Anthocyanin metabolites assay.

After vacuum freeze-drying, the biological samples were ground into a powder using a ball mill at a frequency of 30 Hz. Fifty milligrams of the powdered sample was weighed and dissolved in 500 μL of 50% methanol extract (0.1% hydrochloric acid). Following dissolution of the sample, the sample extract was vortexed and ultrasonicated for 5 min. Then, the mixture was centrifuged at 4 °C and 16,904 g_n for 3 min. The resultant supernatant was aspirated and the remainder was centrifuged again. The two supernatants were combined and filtered by a membrane filter (0.22 μm, Anpel, Shanghai, China). The filtrate was collected in an injection bottle before LC-MS/MS analysis (Acevedo De la Cruz et al. 2012). The liquid-phase and mass-spectrometry conditions followed Li et al. (2022).

Flavonoid metabolite assay.

The frozen samples were ground to powder using a ball mill at 30 Hz. Then, 20-mg samples were weighed and added to 500 μL of 70% methanol with 10 μL of 4000 nmol·L⁻¹ internal standard solution. Following dissolution, the sample was ultrasonicated for 30 min and centrifuged at 4 °C and at 16,904 g_n for 5 min. The upper layer was collected and filtered through a 0.22-μm microporous filter membrane. The filtrate was subsequently collected in the injection vials and used for LC-MS/MS analysis. The extraction of the flavonoids and the liquid-phase and mass-spectrometry conditions followed Shi et al. (2023).

Carotenoid metabolite assay.

The frozen samples were ground to powder using a ball mill at 30 Hz. Then, 50-mg samples were dissolved in 500 μL of a mixture of hexane, acetone, and ethanol (1:1:1 v/v/v), containing 0.01% BHT (g·mL⁻¹), and vortexed for 20 min. The extract sample was then centrifuged for 5 min at 4 °C and 16,904 g_n. The upper layer was aspirated and the above process was repeated for the remainder. The two supernatants were combined and concentrated. Then, they were reconstituted in 100 μL of a 1:1 methanol/methyl tertiary butyl ether mixture solution (v/v), filtered through a 0.22-μm microporous filter membrane, and the filtrate was collected in brown injection vials for LC-MS/MS analysis (Amorim-Carrilho et al. 2014). The liquid-phase and mass-spectrometry conditions followed the methods of Krinsky et al. (2004) and Geyer et al. (2004).

Chlorophyll metabolite assay.

Approximately 0.1 g of hawthorn peel was weighed and ground into powder in liquid nitrogen. Then, the powder was immersed in 10 mL of 95% ethanol solution for 12 h in the dark at room temperature. Next, the mixture was centrifuged at 16,904 g_n for 10 min. The resultant pellet was then removed, and the remaining liquid was collected. The chlorophyll concentration was then determined by measuring the light-absorption values of the extract at 665 nm (D₆₆₅) and 649 nm (D₆₄₉) (Dai et al. 2024). The following formulas were used for calculating the chlorophyll content:$$\begin{array}{l}\text{Chl a\hspace{0.17em}}=(\text{13}.\text{95}\times {\text{D}}_{\text{665}}-\text{\hspace{0.17em}6}.\text{88\hspace{0.17em}}\times {\text{\hspace{0.17em}D}}_{\text{649}})\times \text{\hspace{0.17em}V}/\text{1}000\text{\hspace{0.17em}M}\\ \text{Chl\hspace{0.17em}b\hspace{0.17em}}=\left(\text{24}.\text{96\hspace{0.17em}}\times {\text{\hspace{0.17em}D}}_{\text{649}}-\text{\hspace{0.17em}7}.\text{32\hspace{0.17em}}\times {\text{\hspace{0.17em}D}}_{\text{665}}\right)\times \text{\hspace{0.17em}V}/\text{1}000\text{\hspace{0.17em}M},\\ \text{Total\hspace{0.17em}Chl}\hspace{0.17em}=\hspace{0.17em}\text{Chl\hspace{0.17em}a\hspace{0.17em}}+\text{\hspace{0.17em}Chl\hspace{0.17em}b}\end{array}$$where Chl a is the chlorophyll a concentration (mg·g⁻¹), Chl b is the chlorophyll b concentration (mg·g⁻¹), V is the total volume of extract (mL), and M is the sample mass (g).

Data analysis.

The data were characterized and quantified using a custom Wuhan Mavis Metabolism Biotechnology (MWDB, Wuhan, China) database combined with the multiple reaction monitoring mode of triple quadrupole mass spectrometry. The mass spectrometry data were collected and analyzed by R software and Origin2022 software. Data represent the mean ± standard deviation from at least three experiments. Statistical significance was defined as P < 0.05.

Comparison of color changes during hawthorn fruit development.

The color of the ‘Zizhenzhu’ peel gradually changed from green to purple as the fruit developed (Fig. 1A). During development, the L* value decreased continuously, to a value of 33.79 at S5 (Fig. 1B); the a* value increased continuously, becoming positive at S3 with a value of 16.92, and reached a maximum value of 25.09 at S5 (Fig. 1C). Conversely, the b* value decreased continuously, from 40.65 at S1 to 12.04 at S5, corresponding to the phenotype change of the peel from green to purple (Fig. 1D). S3 was the critical period for color change in ‘Zizhenzhu’, when the peel transitioned from green to red. S4 and S5 corresponded to the mid- and late-coloring stages of fruit color development, respectively.

Comparison of peel metabolites during fruit development.

A total of 135 metabolites were identified in ‘Zizhenzhu’ peel, including 35 anthocyanins, 24 carotenoids, 21 flavones, 21 flavanols, six proanthocyanidins, six flavanols, five flavanones, four flavanonols, four chalcones, four flavone glycosides, three isoflavanones, chlorophyll a, and chlorophyll b (Table 1). Detailed metabolite information is provided in Supplemental Table 1.

Table 1.Qualitative and quantitative results of metabolites in fruit peels of Crataegus pinnatifida ‘Zizhenzhu’.

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During the five developmental periods, 92 metabolites were detected in ‘Zizhenzhu’ peel, with three, two, and nine metabolites specific to S1, S4, and S5, respectively (Fig. 2A). Principal component analysis (PCA) was used to compare the metabolite composition of ‘Zizhenzhu’ peel in the five different developmental periods (Fig. 2B). The quality control samples (mix) were projected to the same region in the PCA plot. Moreover, the three biological replicates from each different period were clustered in the same region, indicating that the replicates shared very similar metabolic profiles, ensuring the reliability and reproducibility of the results. The metabolites from the five periods tended to differentiate along the first (PC1) and second principal component (PC2). Metabolites from adjacent developmental periods segregated about equally on PC1, whereas two periods (S1 and S5) were significantly segregated from S2, S3, and S4 on PC2. These results indicate that the metabolites in ‘Zizhenzhu’ hawthorn peels vary significantly across different developmental periods. Periods S1 and S5 differed the most markedly on PC1, implying that the metabolites present in S1 and S5 differ considerably. These divergent metabolites may play a crucial role in the change in peel color from green to purple.

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Analysis of anthocyanin and proanthocyanidin metabolites in peel during fruit development.

A total of 35 anthocyanins were detected and their abundance varied significantly among samples (Fig. 3A). The total anthocyanin content of the peel increased continuously with fruit development. There were fewer anthocyanins at S1 and S2, which were 20.09 µg·g⁻¹ and 75.70 µg·g⁻¹, respectively. Subsequently, significant anthocyanin accumulation occurred, reaching 384.85 µg·g⁻¹ at S3 and 1479.26 µg·g⁻¹ at S4. Finally, anthocyanin accumulated to a maximum value of 1724.69 µg·g⁻¹ at S5 (Fig. 3B). To identify anthocyanin differential metabolites in the ‘Zizhenzhu’ peel during development, their contents at different stages were compared. Comparing S1 vs. S2, S2 vs. S3, S3 vs. S4, and S4 vs. S5, there were 16, 11, 14, and 7 upregulated anthocyanin metabolites, respectively, and 2, 2, 4, and 7 downregulated anthocyanin metabolites (Fig. 3C). Bar graphs of the differential metabolites were plotted by comparing the multiplicity of differences resulting from quantitative information on anthocyanin metabolites at different times. In the comparison between S1 and S2, this showed that derivatives of cyanidin, petunidin, and delphinidin were significantly upregulated (Fig. 3D). Comparing S2 and S3, the derivatives of cyanidin, petunidin and peonidin were significantly upregulated, whereas some derivatives of petunidin were significantly downregulated (Fig. 3E). In the comparison between S3 and S4, the derivatives of cyanidin, peonidin, delphinidin, pelargonidin, and petunidin were significantly upregulated (Fig. 3F). Comparing S4 and S5, some pelargonidin and cyanidin derivatives were significantly upregulated, and some derivatives of delphinidin, pelargonidin, and cyanidin were significantly downregulated (Fig. 3G). In these four comparisons, the significantly upregulated anthocyanin metabolites were primarily cyanidin derivatives, while the significantly downregulated anthocyanin metabolites were primarily pelargonidin and delphinidin derivatives. Specifically, cyanidin-3-O-galactoside accumulated significantly during ‘Zizhenzhu’ development, accounting for more than 92% of the total anthocyanin content throughout the development process, its content reaching a maximum value (1606.60 µg·g⁻¹) at S5 (Fig. 3H). Furthermore, cyanidin-3-O-arabinoside and peonidin-3-O-arabinoside accumulated continuously, reaching maximum values of 53.60 µg·g⁻¹ (Fig. 3I) and 46.71 µg·g⁻¹ (Fig. 3J), respectively, at S5.

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In summary, the change in the cyanidin-3-O-galactoside content was the main factor leading to the change in the total anthocyanin content, especially at S3, when its accumulation (356.23 µg·g⁻¹) was the main reason for the change in color of ‘Zizhenzhu’ peel from green to red.

The proanthocyanidin content showed a “single peak” tendency, reaching a maximum value of 7659.69 µg·g⁻¹ at S4, and declining to 7328.69 µg·g⁻¹ at S5. The proanthocyanidin content changed smoothly from one period to the next (Fig. 4A). Procyanidin B2 was the predominant proanthocyanidin in the ‘Zizhenzhu’ peel, accounting for 87.86% of the total proanthocyanidin content, followed by procyanidin C1 (11.78%) at S5 (Fig. 4B).

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Analysis of other flavonoid metabolites in peel during fruit development.

The abundance of 68 flavonoid compounds other than anthocyanins and proanthocyanidins in peel varied significantly among samples (Fig. 5A). The total content of these 68 flavonoids showed a “bimodal” trend, with rapid increase to 4250.97 µg·g⁻¹ at S2 and 4621.36 µg·g⁻¹ at S4, and a decrease to 4256.63 µg·g⁻¹ at S5 (Fig. 5B). Comparing these flavonoids in ‘Zizhenzhu’ peel during S1 vs. S2, S2 vs. S3, S3 vs. S4, and S4 vs. S5, 19, 6, 7, and 11 flavonoid metabolites were upregulated, respectively, and 2, 5, 3, and 5 were downregulated (Fig. 5C). Bar graphs of the differential metabolites were plotted by comparing the multiplicity of differences resulting from quantitative information on these flavonoid metabolites at different times. Between S1 and S2, flavonols, flavones, flavone glycosides, and chalcones were significantly upregrated (Fig. 5D). Comparing S2 and S3, flavones, flavonols, and flavanols were significant upregulated, while flavone glycosides and other flavones were significant downregulated (Fig. 5E). Comparing S3 and S4, flavanols and flavones were significantly upregulated (Fig. 5F). Comparing S4 and S5, flavonols, flavones, and some flavanones were significantly upregulated, while flavone glycosides and other flavanols were significantly downregulated (Fig. 5G). Overall, in these four sets of comparisons, the significantly upregulated flavonoid metabolites were mainly flavonols, flavones, and flavanols, while the significantly downregulated flavonoid metabolites were mainly flavone glycosides.

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To analyze the changes in these flavonoid contents, five flavonoids that accounted for ≈92% of the total content of 68 flavonoids were selected. As shown in Fig. 5H, (–)-epicatechin, which accounted for more than 56% of the total content of these flavonoids throughout the development, showed a “bimodal” trend, with the content reaching 2877.15 µg·g⁻¹ at S1, decreasing to a minimum of 2389.97 µg·g⁻¹ at S3, increasing to 2753.61 µg·g⁻¹ at S4, and falling to 2660.93 µg·g⁻¹ at S5 (Fig. 5H). Hyperoside content increased from a minimum value of 298.10 µg·g⁻¹ at S1 to a maximum of 651.46 µg·g⁻¹ at S3, and then decreasing slightly to 604.09 µg·g⁻¹ at S5 (Fig. 5I). Quercetin-3-O-glucoside also showed a “bimodal” trend, increasing to 334.49 µg·g⁻¹ at S2 and 441.23 µg·g⁻¹ at S4, and decreasing to 207.43 µg·g⁻¹ at S5 (Fig. 5J). Baimaside showed a “single peak” trend, reaching minimum and maximum values of 37.82 µg·g⁻¹ and 297.58 µg·g⁻¹ at S1 and S3, respectively, and 224.66 µg·g⁻¹ at S5 (Fig. 5K). Rutin showed a “single peak” trend, with the minimal value of 187.24 µg·g⁻¹ at S1, and rapidly increasing to a maximum value of 261.89 µg·g⁻¹ at S2 and then decreasing to 186.55 µg·g⁻¹ at S5 (Fig. 5L).

Of the five major flavonoids—(–)-epicatechin, hyperoside, quercetin-3-O-glucoside, baimaside, and rutin—(–)-epicatechin does not affect color, whereas the levels of the four metabolites (hyperoside, quercetin-3-O-glucoside, baimaside, and rutin) did not vary markedly between adjacent developmental periods, implying that these metabolites are not the primary drivers of the changes observed in fruit coloration.

Analysis of carotenoid metabolites in peel during fruit development.

A total of 24 carotenoids were detected and their abundance varied significantly among the different samples (Fig. 6A). The total carotenoid content of the peel decreased gradually from a maximum value of 202.26 µg·g⁻¹ at S1 to a minimum value of 73.16 µg·g⁻¹ at S5 (Fig. 6B). Comparing carotenoids in ‘Zizhenzhu’ peel during S1 vs. S2, S2 vs. S3, S3 vs. S4, and S4 vs. S5, the results showed that two, zero, three, and eight carotenoid metabolites were upregulated and one, two, three, and three carotenoid metabolites were downregulated, respectively (Fig. 6C). Bar graphs of the differential metabolites were plotted by comparing the multiplicity of differences resulting from quantitative information on carotenoid metabolites at different times. The comparison between S1 and S2, zeaxanthin and (E/Z)-phytoene were significantly upregulated, whereas α-carotene was significantly downregulated (Fig. 6D). Between S2 and S3, α-carotene was significantly downregulated (Fig. 6E). Between S3 and S4, lutein dilaurate was significantly upregulated and violaxanthin was significantly downregulated (Fig. 6F). Between S4 and S5, violaxanthin dioleate, neochrome palmitate, lutein dimyristate, lutein dilaurate, and lutein palmitate were significantly upregulated and α-carotene was significantly downregulated (Fig. 6G). The contents of these divergent metabolites fluctuated within 0% to 6.34% (Fig. 7A). Despite the substantial variation in the contents over the course of development, they were not deemed significant contributors to the changes in total carotenoid content, due to their minimal levels.

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Therefore, we selected 12 carotenoids that accounted for ∼99% of the total carotenoid content for detailed analysis (Fig. 7B). Among these, lutein accounted for more than 70% of the total carotenoid content. The lutein content decreased gradually from a maximum value of 165.03 µg·g⁻¹ at S1 to a minimum value of 51.83 µg·g⁻¹ at S5. β-Carotene accounted for between 6.31% and 8.57% of total carotenoid content, decreasing from the maximum value (17.33 µg·g⁻¹) at S1 to the minimum value (4.62 µg·g⁻¹) at S5. The contents of other carotenoid metabolites were relatively low, of which α-carotene decreased-increased-decreased; zeaxanthin and β-cryptoxanthin increased-decreased-increased; (E/Z)-phytoene, capsorubin, and violaxanthin dibutyrate increased-decreased; neoxanthin and lutein dilaurate decreased-increased; and violaxanthin and neochrome palmitate decreased and increased, respectively. With the exception of violaxanthin, the trends exhibited by the other carotenoids and total carotenoids were inconsistent, and their contents were minimal, not constituting the primary carotenoid components.

In summary, lutein is the main carotenoid involved in peel coloration, and changes in its content are the main cause of changes in the total carotenoid content. The decline in lutein content resulted in a gradual weakening of the yellowing effect of carotenoids in the process of fruit color change in ‘Zizhenzhu’.

Analysis of chlorophyll metabolites in peel during fruit development.

As shown in Fig. 8, with the development of ‘Zizhenzhu’ hawthorn fruits, the total chlorophyll content of the peel decreased from a maximum value of 297.99 µg·g⁻¹ at S1 to a minimum value of 73.68 µg·g⁻¹ at S5. The chlorophyll a content was slightly higher than that of chlorophyll b, but the contents of both changed in the same manner. Therefore, chlorophyll is the main factor in the green color of hawthorn peel at the early stage of fruit development; as the chlorophyll content decreases, the green color of the peel gradually weakens.

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Analysis of peel-coloration metabolites during fruit development.

Correlation analysis of ‘Zizhenzhu’ peel-coloration metabolites and peel color difference values indicated that anthocyanins were significantly positively correlated with the a* value of peel (r = 0.939, P < 0.01), whereas chlorophyll was significant negatively correlated with the a* value of peel (r = −0.925, P < 0.01); carotenoids were significantly positively correlated with the b* value of peel (r = 0.932, P < 0.01), whereas the correlation of the other flavonoids with the b* value was not significant (r = −0.486, P > 0.05), indicating that there was little correlation between the other flavonoids and fruit color.

To further elucidate the metabolite mechanism underlying the color change in ‘Zizhenzhu’ hawthorn fruit, a correlation analysis of anthocyanins, carotenoids, and chlorophyll was conducted. As shown in Fig. 9, the anthocyanin content increased, while the carotenoid and chlorophyll contents decreased in conjunction with fruit development. The anthocyanin content gradually increased from a value of 20.09 µg·g⁻¹ at S1 to a value of 1724.69 µg·g⁻¹ at S5. The carotenoid content was the maximum value (202.26 µg·g⁻¹) at S1 and was 10.07 times higher than the anthocyanin content at S1; the carotenoid content decreased to the minimum value (73.16 µg·g⁻¹) at S5, when the anthocyanin content was 23.57 times higher than the carotenoid content. The chlorophyll content was the maximum value (297.99 µg·g⁻¹) at S1, which was 14.83 times higher than the anthocyanin content. The chlorophyll content decreased to 73.68 µg·g⁻¹ at S5, at which time the content of anthocyanin was 23.41 times higher than that of chlorophyll. In summary, the accumulation of anthocyanin and decrease in carotenoid and chlorophyll were the main reasons for the change of ‘Zizhenzhu’ fruit color from green to purple.

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