Comprehensive metabolome and transcriptome analyses demonstrate divergent anthocyanin and carotenoid accumulation in fruits of wild and cultivated loquats

Eriobotrya is an evergreen fruit tree native to South-West China and adjacent countries. There are more than 26 loquat species known in this genus, while E. japonica is the only species yet domesticated to produce fresh fruits from late spring to early summer. Fruits of cultivated loquat are usually orange colored, in contrast to the red color of fruits of wild E. henryi (EH). However, the mechanisms of fruit pigment formation during loquat evolution are yet to be elucidated. To understand these, targeted carotenoid and anthocyanin metabolomics as well as transcriptomics analyses were carried out in this study. The results showed that β-carotene, violaxanthin palmitate and rubixanthin laurate, totally accounted for over 60% of the colored carotenoids, were the major carotenoids in peel of the orange colored ‘Jiefangzhong’ (JFZ) fruits. Total carotenoids content in JFZ is about 10 times to that of EH, and the expression levels of PSY, ZDS and ZEP in JFZ were 10.69 to 23.26 folds to that in EH at ripen stage. Cyanidin-3-O-galactoside and pelargonidin-3-O-galactoside were the predominant anthocyanins enriched in EH peel. On the contrary, both of them were almost undetectable in JFZ, and the transcript levels of F3H, F3’H, ANS, CHS and CHI in EH were 4.39 to 73.12 folds higher than that in JFZ during fruit pigmentation. In summary, abundant carotenoid deposition in JFZ peel is well correlated with the strong expression of PSY, ZDS and ZEP, while the accumulation of anthocyanin metabolites in EH peel is tightly associated with the notably upregulated expressions of F3H, F3’H, ANS, CHS and CHI. This study was the first to demonstrate the metabolic background of how fruit pigmentations evolved from wild to cultivated loquat species, and provided gene targets for further breeding of more colorful loquat fruits via manipulation of carotenoids and anthocyanin biosynthesis.


Introduction
Accumulation of pigments including anthocyanins, chlorophylls, and carotenoids is an essential process regulating plant growth and development, as well as for adjusting to environmental stresses.For example, anthocyanin is a popular pigment enriched in organs like fruit, leaf, and petal to attract pollinating insects and foragers to guarantee successful pollination and seed spread, or oppose herbivores (Tanaka et al., 2008).In addition, high enrichment of these pigments also improves fruit appearance and flavor, and supports human beings with health nutrients.Traditional breeding (Bang et al., 2007), biofortification (Römer et al., 2000), and agronomic protocols (Luan et al., 2020) have been long used to enhance pigment accumulation in plant products.The identification of specific chemical basis and gene regulation of specific pigments accumulated in plant tissues is vital for health pigments improvement.
Loquat (Eriobotrya japonica) is a distinctive subtropical fruit tree (apple subfamily, Rosaceae) native to South-West China, which supports human with delicious and nutritious fruits from late spring to early summer (Su et al., 2021b).Carotenoids are one of the most important nutrients in loquat fruits, and cultivated loquats are commonly classified into white-, yellow-, orange-, and orangered fleshed groups, due to differences in carotenoid amount (Zhou et al., 2007).Previously, carotenoid quality and quantity assays identified b-carotene as the predominant pigment of cultivated loquat and segment mutations in EjPSY2A coding region principally confers to the variations of total carotenoid content and flesh color (Fu et al., 2012;Fu et al., 2014).While cultivated loquat usually harbors yellow or orange fruit, fruits from the wild E. henryi are generally in red or purple color (Figure 1) (Su et al., 2021b).However, whether the red and purple colors are derived from higher accumulation of the well-known carotenoids like the cultivated loquat or from the accumulation of other pigments, and how the colorful compounds were enriched in fruit tissues, are still unknown.Here, we performed integrative analyses of targeted metabolomics and transcriptomics to identify key pigment compounds underlying loquat fruit color variation and evolution, and screened candidate important structure genes responsible for in vivo biosynthesis of these pigments in loquat fruits.All these data collectively revealed the metabolic basis of fruit color evolution and shed light on the breeding for more colorful and nutritious loquat fruits.

Plant materials and growth conditions
Ripen fruits of cultivated loquat were collected from 'Jiefangzhong' (JFZ) trees planted in the National Germplasm Bank of Loquat (Fuzhou, Fujian, China) on 15th April, 2021.Trees of the wild loquat species, Eriobotrya henryi (EH), were native grown in Kunming Botanical Garden (Kunming, Yunnan, China), and red-colored ripen fruits were collected on 24th June, 2021.Fruit cortex samples, including pulp and peel tissues, were collected and immediately frozen in liquid nitrogen, thereafter stored at -80°C, three biological repeats were performed for pigment analysis and RNA library construction, five fruits were used in each repeat.Fruits from green, color breaker and ripen stages were collected from JFZ and EH trees during the fruiting season of 2023 for gene expression analyses.

Carotenoids extraction and targeted metabolome analysis
Frozen loquat fruit tissues were freeze-dried for 24 h with a freeze dryer (Labconco, America) and ground at 30 Hz into fine powder using a mixer mill.The fruit carotenoids were extracted as previously described (Song et al., 2023) with some modifications.50 mg fruit powder was added into 500 mL mixed solution of n-hexane: acetone: ethanol (1:1:1, v/v/v), and 10 mL carotenoids internal standard (IS) mixed solution (20 mg/mL) was added into the extracting tube for the quantification.Then they were mixed for 20 min at room temperature with the vortex mixer, centrifuged at 4°C at 12000 r/min for 5 min and the supernatants were collected.The above steps were repeated twice until the samples is colorless.Then the supernatant was evaporated to dryness, and reconstituted in mixed solution of Methanol: Methyl tert-butyl ether (1:1, v/v).The obtained liquid was filtered through 0.22 mm membrane filter (Biosharp) before LC-MS/MS analysis.
A QTRAP ® 6500+ LC-MS/MS System, equipped with an APCI Heated Nebulizer, was operated in positive ion mode and controlled by Analyst 1.6.3software (Sciex).The following source operation parameters were used: ion source, APCI+; source temperature, 350°C; curtain gas (CUR), 25.0 psi; and collision gas (CAD).Carotenoids data acquisitions were performed using Analyst 1.6.3software (Sciex).The integrated peak area of each carotenoid detected in the samples was substituted into the linear equations of standard curves for content calculation (see Table S1 for the standard curves).Three biological repeats were used for each sample.

Anthocyanins extraction and quantification
Total anthocyanin was extracted as formerly performed (Yi et al., 2021).50 mg vacuum freeze-dried fine fruit tissue powder for each sample was weighted and added into 0.5 mL methanol/water/ hydrochloric acid (500:500:1, V/V/V).The samples were vortexed for 30 s then immersed in methanol for 30 min.After six rounds of vortexing-immersing, these samples were placed in a 4°C refrigerator for overnight extraction.Samples were finally centrifuged at 12,000 g for 10 min under 4°C.The supernatants were collected, and filtrated with a microporous membrane (0.22 mm pore size) into injection bottles before subsequent UPLC-MS/MS analysis.
Anthocyanin UPLC-MS/MS analysis was performed according to Huang et al. (2019).The anthocyanins in two kinds of loquat fruits were analyzed by UPLC-MS/MS system comprising the SCIEX ultraperformance liquid chromatography, the Applied Biosystems 6500 Triple Quadrupole mass spectrometry, and the Waters ACQUITY BEH C18 column (1.7 µm, 2.1 mm×100 mm).The mobile phase was made up of phase A (0.1% formic acid in ultrapure water) and phase B (0.1% formic acid in acetonitrile).The column temperature was 40°C , with a 0.35 ml/min flow rate.The sample injection volume was 2 mL.The elution gradient began with 5% phase B at 0 min, then increased to 50% at 6 min, and the proportion of phase B increased to 95% at 12 min and was maintained for 2 min.Anthocyanins contents were measured using Analyst 1.6.3software based on the AB Sciex QTRAP 6500 LC-MS/MS platform.
The ESI source operation parameters were following: ion source, ESI+; source temperature 550°C; ion spray voltage (IS) −4500 V (negative ions) and 5500 V (positive ions); curtain gas (CUR) was set at 35 psi.Anthocyanins were analyzed using multiple reaction monitoring (MRM).Multiquant 3.0.3software (Sciex) was used to quantify all metabolites.The m/z range used in the LC-MS/ MS analysis was 50-1250 Da.The integrated peak area of each detection was substituted into the linear equations of anthocyanins standard curves for sample level calculation (see Table S2 for the standard curves).Three spears were used for each repeat.

RNA extraction and cDNA preparation
Total RNA of these samples was extracted from fine powder fruit tissues as formerly descripted (Su et al., 2017) with EASYspin Plus plant RNA kit (Aidlab, China).The 1.5% agarose gel electrophoresis was performed to evaluate the integrity of the RNA, and the RNA concentration and purity were then assayed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA).A PrimeScript ™ RT reagent Kit with genome DNA wiper (TaKaRa, Japan) was then used to synthesize the first-strand cDNA of the plant samples according to the manufacturer's instructions.

Quantitative real-time PCR assays
Total RNA of fruits from three developmental stages were prepared as above performed for RNA-seq library construction.Quantitative real-time RT-PCR analysis was performed as previously done (Su et al., 2019).Integrated DNA Technologies software (https://sg.idtdna.com/pages) was applied to design the primers for quantitative real-time polymerase chain reaction (qRT-PCR).EjACT2 (AB710173.1)was used as the reference gene.Primer information for all the interested biosynthesis genes were listed in Table S8.Each value confers to the mean of three biological replicates captured by the LightCycler480 Q-PCR system (Roche, Sweden) using iTaq ™ universal SYBR Green Supermix purchased from Bio-Rad.

DNA extraction and PSY2A genotyping
Genomic DNA samples were extracted from young leaves of JFZ and EH with a M5 CTAB plant gDNA extract Kit (Mei5 Biotechnology, Beijing, China) according to the user's protocol.Genotyping of PSY2A, a candidate gene formerly confirm by (Fu et al., 2014) to underlie flesh color and carotenoid variation of cultivated loquat fruits, with forward primer: 5'-ATTTGCCA AC T AC C A C T G C T T T C A -3 ' a n d r e v e r s e p r i m er : 5' -TACACCACATAAGAAACAAGCA-3'.The PCR amplicons were monitored on 1.5% agarose gels.

Fruit appearance of wild and cultivated loquats
In general, fruits of the cultivated Eriobotrya japonica are mostly in yellow to orange-red colors, globose to obovate shape, and 2.0-5.0 cm fruit size.For example, one of the traditional main cultivars, JFZ, is about 5 cm in transverse diameter and both its peel and flesh are orange (Figures 1A, C).While the E. japonica is domesticated for fruit production, most wild loquat species set small fruits with very thin flesh and cannot meet consumption demands.Among these species, E. henryi possess attractive red pigmented fruits in oval shape, and the flesh from their fruit is light yellow or white (Figures 1B, C).

Carotenoid components accumulated in peel of wild and cultivated loquat fruits
To understand what pigments contribute to the color changes of the two loquat species, targeted carotenoid metabolomics was firstly performed on peel of ripen fruits.Obviously, more and higher carotenoid compound peaks were detected in JFZ, among which bcarotene is the highest at 6.21 min by UPLC-MS/MS (Figure 2A).In  S1, 2).And the majority of these compounds existed in both of the two species (little in EH), while the total carotenoid content in JFZ (410.70mg•g - ) was about twelve folds higher to that in EH (Figure 2C).For both of them, b-carotene accounted for more than 42% of the total carotenoids, and b-carotene content in JFZ is thirteen folds to that in EH (Figure 2D).Metabolites derived from g-carotene (including b-carotene, b-cryptoxanthin, violaxanthin, rubixanthin and zeaxanthin) accounted for more than 90% of the total carotenoids in JFZ.The top 15 highly enriched carotenoids compounds, except for lutein, were significantly accumulated at higher levels in JFZ (Figure 2E).

Identification of anthocyanins components enriched in peel of wild loquat fruits
Lower carotenoid accumulation in above detection confirmed that carotenoid was not the major pigment explaining the deeper color of EH fruits.Since anthocyanins are the major pigments for the red color of apple and pear fruits (both of them are relatives of loquat from the apple subfamily) (Espley et al., 2007;Ni et al., 2023), we then carried out flavonoids metabolomics to understand the metabolite basics for red coloring of wild loquat fruits.The UPLC-MS/MS detection showed abundant and high anthocyanin compound peaks in peel of EH fruits, among which cyanidin-3-O-galactoside was the highest at 5.11 min, however the second highest peak at 9.48 min is an unknown compound very similar to naringenin-7-O-glucoside (9.16 min) (Figure 3A).In total, 34 flavonoids components were identified from the two loquat species, including 8 cyanidins, 6 flavonoids, 5 procyanidins, 4 delphinidins, 4 peonidins, 4 petunidins, and 3 pelargonidins (Figure 3B, Tables S3, 4).In sum, the total flavonoids in EH fruits were 4.31 folds to that in JFZ fruits while it is unexpected that the cultivated JFZ contained similar levels of flavonoid in mature fruit compared to the wild loquat (Figures 3C, D).Most of the anthocyanin compounds (cyanidin, delphinidin, peonidin and pelargonidin) were trace or undetectable in peel of JFZ, and cyanidin compounds accounted for 69% of total flavonoids in EH (Figure 3C).Cyanidin (thousands of times higher) and pelargonidin levels were significantly higher accumulated in EH compared with that in JFZ (Figure 3D).Cyanidin-3-O-galactoside (659.07 mg•g-1), cyanidin-3-O-arabinoside (4.47 mg•g-1) and cyanidin-3-Oglucoside (2.53 mg•g-1) accounted for more than 97% of the total anthocyanin enriched in EH while pelargonidin-3-O-galactoside (14.56 mg•g-1) accounted for 2.14% (Figure 3E).

Transcriptome sequencing data assembly and annotation
Transcriptome sequencing analysis was carried out on mature JFZ and EH fruit peel samples.A total of 29.47 Gb raw reads with sequencing error rate lower than 0.03% were obtained from the sequencing of all six libraries, and the biological replicates of different samples were clustered together to show high reproducibility (Figure S1A).A total of 28.30 Gb clean reads were obtained with Q20>97.68%,Q30>93.42% after filtering and the GC content ranged from 46.87% to 47.26% (Table S5).More than 91% of the clean reads from JFZ could be unique mapped to reference genome, while only about 77% for that of the wild EH (Table S6).Differentially expressed gene (DEG) analysis identified 12,888 DEGs between the two ripen fruit samples, among these 6113 were down-regulated in JFZ and 6775 were up-regulated (Figure S1B).KEGG, and GO databases annotation showed that lots of DEGs were significantly enriched in pigment metabolism related pathways like 'pigment metabolic process', 'pigment biosynthetic process', 'phenylpropanoid biosynthesis', 'isoflavonoid biosynthesis' and 'terpenoid biosynthesis' (Figures S1C, D).To further explore the mechanism of carotenoids and flavonoids accumulating in loquat fruits, the expression patterns of enzyme encoding genes in the carotenoid and flavonoid pathways were analyzed.

qRT-PCR validated key carotenoid metabolic structural genes
DNA genotyping showed that JFZ is an EjPSY2A-EjPSY2A d heterozygous plant while EH is EjPSY2A-EjPSY2A homozygous species (Figure 5A).This result indicated that the weak carotenoid contents in EH were not caused by the loss of PSY enzyme function as white-fleshed cultivars performed.Instead, it may be induced by the difference in gene expression levels.To further confirm whether the above-mentioned significantly expressed genes contributed to the carotenoid accumulation divergence of different loquat species, we then collected fruit samples from orange-, red-and purple-coloring varieties at green-, color breaker-and ripen stages for gene expression analyses (Figure 5B).Gene expression assays showed that DXR, GGPS, PSY, ZDS and ZEP were significantly upregulated from green to color breaker and/or ripen stages, and expressed highest in JFZ (Figure 5C).While DXR, GGPS and ZDS were obviously down-regulated in EH as fruits ripening.On the contrary, the expression levels of both of the active NCED/CCDs (Ej00040698 and Ej00005370) were increased during fruit maturation in both of the two varieties, and they expressed at higher levels in EH than in JFZ.In addition, the transcript levels of PSY (Ej00015134), ZDS (Ej00041636) and ZEP (Ej00004893) in JFZ were more than 5.8 folds to that of EH.Meanwhile, the transcript levels of NCED/CCDs (Ej00040698, Ej00005370 and Ej00034109, function in carotenoids degradation) in EH were 7.88~460.77folds higher to that in JFZ during fruit coloring.Expression analysis of anthocyanin biosynthesis enzyme encoding genes 55 enzyme encoding genes (six PAL, four C4H, three 4CL, six CHS, ten CHI, three F3H, six F3'H, three DFR, two ANS, six UGT78, three FNS, one F3'5'H and three LAR) throughout the anthocyanin and flavonoid biosynthesis pathway were identified (Figure 6, Table S7).Transcriptome data showed that the expression patterns of UGT78 (Ej00006885 and Ej00057656), ANS (Ej00061364), DFR (Ej00081751 and Ej00054205), F3'H (Ej00076015 and Ej00065084), F3H (Ej00042569 and Ej00026228), CHI (Ej00071798 and Ej00070948), CHS (Ej00014264, Ej00014720, Ej00054582, Ej00014465 and Ej00054946), 4CL (Ej00013378 and Ej00005091), PAL (Ej00021556, Ej00051389 and Ej00064002) were notably associated with the higher anthocyanin accumulation in peel of EH (Figure 6, Figure S2).Among these, the higher transcript levels of UGT78, DFR, F3'H, F3H, CHI, and CHS were positively associated with high cyanidin-3-O-galactoside and pelargonidin-3-O-galactoside in the red-colored EH (Figure 6).

qRT-PCR validated key anthocyanin biosynthesis enzyme encoding genes
To further investigate whether the above identified genes contributed to anthocyanin accumulation in loquat fruits, qRT-PCR was carried out to verify their expression patterns of genes in the last seven steps of anthocyanin biosynthesis pathway as formerly performed for carotenoid metabolic genes.The expression data showed that CHS (Ej00054582 and Ej00014720), CHI (Ej00071798), F3H (Ej00026228 and Ej00042569), F3'H (Ej00065084), DFR (Ej00081751), ANS (Ej00061364), and UGT78 (Ej00006885) were significantly up-regulated in peel of both redcolored EH and purple-colored ES while down-regulated in JFZ as fruits ripen.Another F3'H (Ej00076015) was sharply downregulated from green stage to ripen stage in JFZ, while gently in EH.Moreover, the transcript levels of all these genes were significantly higher in EH than in JFZ (Figure 7).The expression levels of ANS (Ej00061364), F3H (Ej00042569), F3'H (Ej00065084), CHS (Ej00054582) and CHI (Ej00071798) in EH were notably 7.4~8.9,4.4~7.0,4.4~4.9,7.5~27.0and 55.0~73.1 folds to that in JFZ as fruits start coloring.In addition, the expression level of UGT78 (Ej00057656) in EH was more than 4.6 folds to that in JFZ.

Discussion
Evolution of anthocyanin and carotenoid biosynthesis underlies fruit color variation from ancient loquat to modern cultivar Commonly, cultivated loquat fruits are classified into white-, yellow-, orange-, and orange-red fleshed groups, due to variations in carotenoid accumulation capabilities of their fruits.Sadana (1949) first revealed that b-carotene is the predominant pigment positively associated with fruit color of cultivated loquats.Then, 23 (Zhou et al., 2007), 25 (De Faria et al., 2009)  violaxanthin-myristate-caprate, b-cryptoxanthin myristate and lutein dipalmitate newly identified here, we identified the most carotenoid constituents (38 molecules) from loquat fruit via UPLC-MS/MS (Figures 2A, B, Table S1).In accordance with former researches in loquat (Zhou et al., 2007;De Faria et al., 2009;Fu et al., 2012), bcarotene was found to be the most abundant compound in both JFZ and EH fruits (Figure 2).b-Carotene is also the most abundant carotenoids of apple (Ampomah-Dwamena et al., 2012) and apricot (Zhou et al., 2020), which greatly contributed to their flesh pigmentation.In contrast, lutein and b-cryptoxanthin were the main carotenoid components of peach (Wu et al., 2022) and cherry (Demir, 2013).b-carotene, violaxanthin palmitate and rubixanthin are the top three carotenoids detected in the cultivated JFZ, while lutein is the second most carotenoid of EH (Figure 2E).These results suggest that Rosaceae fruit trees are conserved in carotenoid production.Different accumulation capacity of each component, and fold changes of the major carotene constitutes are responsible for flesh color variation of these fruits.The variations in contents of major carotenoid, b-carotene and b-cryptoxanthin, were discovered to underlie the flesh color difference between white and orange-red fleshed cultivars (Zhou et al., 2007;De Faria et al., 2009;Fu et al., 2012).The 10-fold variation of b-carotene and total carotenoid results in white-fleshed wild loquat fruits and orange-fleshed JFZ fruits (Figure 2).
As b-carotene and cyanidin-3-O-galactoside themselves show orange or red colors (Tanaka et al., 2008), the metabolic analysis here reveals that b-carotene and its derivatives contribute to orange flesh of cultivated loquat (Figure 4), while cyanidin-3-O-galactoside underlies red pigmentation of the wild species (Figure 6).The red appearance of wild loquat with high concentrations of anthocyanins is promising to meet current consumer expectations for novel color.

Key carotenoid biosynthesis steps in loquat fruits
Carotenoids are derived from IPP and its allylic isomer DMAPP.The primary metabolic pathways of carotenoids have been widely studied in fruit crop and other horticulture plants (Nisar et al., 2015;Hermanns et al., 2020).Among dozens of carotenoid metabolic steps, PSY is regarded as a main ratelimiting enzyme, and variations in Psy-A1, MePSY2 and ClPSY1 resulted in greatly changes of total carotenoids content in wheat (He et al., 2007), cassava (Welsch et al., 2010) and watermelon (Liu et al., 2021).In addition, transcript levels of PSY homologues were also positively associated with fruit carotenoid content in citrus (Peng et al., 2013) and apple (Ampomah-Dwamena et al., 2015).In loquat, a segment deletion in C-terminal of EjPSY2A was discovered to cause lower carotenoid accumulation in fruits of white-fleshed loquat varieties (Fu et al., 2014).In this study, both JFZ and EH showed dominant genotype in EjPSY2A locus (Figure 5A), while the white EjPSY2A transcript level in JFZ was more than 11-fold higher than that in EH (Figure 5C).This suggests that low transcript level of key carotenoid biosynthesis gene acts as another candidate mechanism for weak carotenoid-pigmentation of wild loquat fruit.
Compared to EH, ZDS (z-Carotene desaturase) and ZEP (zeaxanthin epoxidase) expression levels in JFZ were more than 7fold higher during fruits coloration (Figures 4, 5B, C).As a key enzyme in the carotenoid biosynthesis pathway, ZDS can catalyze zcarotene to form lycopene.EjZDS was also up-regulated during fruit pigmentation of the orange-colored 'Obusa' loquat (Hadjipieri et al., 2017).Overexpression of apple MdZDS notably improved both carotenoid biosynthesis and saline-alkali stress tolerance in transgenic plants (Wang et al., 2023).In addition, ZEP paralog in yellow−fleshed sweet potato promoted carotenoid accumulation through the epoxidation of b-carotene and b-cryptoxanthin (Suematsu et al., 2020).While mutation in CaZEP contributes to orange coloration by improving carotenoid contents in pepper fruit (Lee et al., 2021).
On the other hand, NCED/CCD family proteins include NCEDs, CCD7, CCD8, CCD4, and CCD1 function in degradation of carotenoids into apocarotenoids.Carotenoid levels were negatively correlated with NCED/CCD expression, and natural variations in PpCCD4, CrCCD4b and SiCCD1 strongly enhanced carotenoid content in peach (Falchi et al., 2013), citrus (Zheng et al., 2019) and millet (He et al., 2022).Corresponding to the very low carotenoid levels in white-fleshed fruit (Figure 2), transcript levels of NCED/CCDs in white-fleshed EH were 7.88 to 460.77 folds higher to that in orange-fleshed JFZ during fruit coloring (Figure 5C).Collectively, the strongly positive correlations of key biosynthesis gene expression patterns and negative correlations of metabolic gene expression patterns with carotenoid level changes in the loquat fruits suggest that PSY, ZDS, ZEP and NCED/CCD may be key biosynthesis/metabolic genes in loquat carotenoid accumulation.

Key steps for anthocyanin biosynthesis in wild loquat fruits
Generally, anthocyanins biosynthesis can be divided into the early general phenylpropanoid pathway and the late flavonoid pathway (Figure 6).The key enzymes in the phenylpropanoid pathway, PAL, C4H and 4CL, convert phenylalanine to 4coumaroyl-CoA (Tanaka et al., 2008).Then, 4-coumaroyl-CoA combines malonyl-CoA are catalyzed by a series of enzymes (CHS, CHI, F3H, F3'H, F3'5'H, DFR, ANS, UGT, etc.) to synthesize anthocyanin and other flavonoid compounds (Tanaka et al., 2008).UGT78 is the last and key enzyme for the biosynthesis of cyanidin-3-O-galactoside and pelargonidin-3-O-galactoside (Figure 6).VvGT6 (Ono et al., 2010), CsUGT78A15 (He et al., 2021), MrUGT78W1 (Ren et al., 2022) andMdUGT78T2 (Clayton-Cuch et al., 2023) all function in transferring galactosyl from UDP-Gal to flavonols to produce quercetin 3-O-galactoside and cyanidin-3-O-galactoside in grape, tea, waxberry and apple.EH accumulated a large amount of cyanidin-3-O-galactoside in its fruits (Figure 3).Correspondingly, notably high UGT78 transcription was detected in this species (Figures 6,7).ANS is a key enzyme at the end of the plant anthocyanin biosynthetic pathway that catalyzing the colorless leucoanthocyanidins into red-colored cyanidins.In this study, we identified two ANS homologues in loquat genome, and one of them (Ej00061364) highly expressed in red-colored EH, while it was undetectable in JFZ during fruit coloring (Figures 6, 7).Mutations in the coding region of RiANS and SmeFAS resulted in loss of function of ANS protein and leads to loss of anthocyanin pigments in raspberry fruit (Rafique et al., 2016) and eggplant flower (Chen et al., 2018).On the other hand, overexpression of ANS dramatically elevated anthocyanin concentration in strawberry fruit (Giampieri et al., 2018) and silencing of SlANS expression significantly decreased anthocyanin accumulation in tomato (Sun et al., 2023).
Moreover, the expression levels of many enzyme-encoding genes upstream of ANS were also notably upregulated in EH as fruits initiated coloring (Figures 6, 7).Among these, CHS and CHI were the mostly upregulated at both color breaker and ripen stages (7.5~27.0 and 8.6~105.7 folds compared to JFZ, see in Figure 7).CHS is the first committed step of flavonoid biosynthesis.Tobacco plants constitutively expressing McCHS (isolated from crabapple) displayed a higher anthocyanins accumulation and a deeper red petal color (Tai et al., 2014).Fruit collected from CHS-silenced apple line lacked flavonoids in the skin and flesh (Dare et al., 2013).DcCHI1 (Zhu et al., 2021) or CnCHI4 (Yu et al., 2022) overexpression significantly increased flavonoid production in tobacco.Furthermore, the red-pigmented EH also increased F3H and F3'H transcript levels to 4.4~9.9 or 4.4~22.6folds higher than that in JFZ (Figure 7).Up-regulating the expression of CitF3H improves anthocyanin accumulation in blood orange (Ma et al., 2023), oppositely, mutation in FvF3H blocks anthocyanin biosynthesis and results in pink strawberry fruits (Xu et al., 2023).DlF3′H plays important role in selecting which anthocyanins component to be accumulated in red longan pericarp (Yi et al., 2021), loss of function of the F3'H (tt7) in Arabidopsis restricts catalyzation from dihydrokaempferol to dihydroquercetin, and leads to overaccumulation of kaempferol-3-rhamnoside in seed coat to compromise seed longevity (Niñoles et al., 2023).Totally, gene expression assays here demonstrate that UGT78 (Ej00057656), ANS (Ej00061364), F3H (Ej00042569), F3'H (Ej00065084), CHS (Ej00054582) and CHI (Ej00071798) play crucial roles in anthocyanin biosynthesis flux determining in the red-colored wild loquat.

Frontiers 2
FIGURE 1Trees and fruits of cultivated and wild loquats.(A) Large tree and orange-colored fruits of the 'Jiefangzhong' cultivar.(B) Small tree and red-colored fruits of Eriobotrya henryi.(C) Fruit weight and fruit diameter of JFZ and EH.
FIGURE 3 Anthocyanin components in peel of cultivated and wild loquat fruits.(A) Chromatograms of cyanidin-3-O-galactoside and other anthocyanins in JFZ and EH by UPLC-MS/MS.(B) Heat map of flavonoids metabolite clusters.(C) Total flavonoids metabolite content in JFZ and EH.(D) Cyanidin, pelargonidin, petunidin, delphinidin, peonidin, procyanidin and flavonoid contain of the two species.(E) Detail anthocyanin compound levels of the two species.*** indicates P < 0.001 and NS indicates no significant difference by Student's t-test.
FIGURE 5Genotyping and gene expression analyses of carotenoid metabolism genes in peel of cultivated and wild loquat.(A) EjPSY2A genotyping.(B) Fruit samples from green-, color breaker-and ripen developmental stages of JFZ and EH.(C) Expression patterns of significantly expressed genes, identified by RNA sequencing, at three key fruit development stages.