Integrated Metabolomic and Transcriptomic Profiles Provide Insights into the Mechanisms of Anthocyanin and Carotenoid Biosynthesis in Petals of Medicago sativa ssp. sativa and Medicago sativa ssp. falcata

The petals of Medicago sativa ssp. sativa and M. sativa ssp. falcata are purple and yellow, respectively. Free hybridization between M. sativa ssp. sativa and M. sativa ssp. falcata has created hybrids with various flower colors in nature. Moreover, the flower colors of alfalfa are closely correlated with yield, nutritional quality, stress tolerance and other agronomic characteristics. To elucidate the underlying mechanisms of flower color formation in M. sativa ssp. sativa and M. sativa ssp. falcata, we conducted an integrative analysis of the transcriptome and metabolome of alfalfa with three different petal colors (purple, yellow and cream). The metabolic profiles suggested that anthocyanins and carotenoids are the crucial pigments in purple and yellow flowers, respectively. A quantitative exploration of the anthocyanin and carotenoid components indicated that the accumulations of cyanidin, delphinidin, peonidin, malvidin, pelargonidin and petunidin derivatives are significantly higher in purple flowers than in cream flowers. In addition, the content of carotenes (phytoene, α-carotene and β-carotene) and xanthophylls (α-cryptoxanthin, lutein, β-cryptoxanthin, zeaxanthin, antheraxanthin and violaxanthin derivatives) was markedly higher in yellow flowers than in cream flowers. Furthermore, we found that delphinidin-3,5-O-diglucoside and lutein were the predominant pigments accumulated in purple and yellow flowers, respectively. The transcriptomic results revealed that twenty-five upregulated structural genes (one C4H, three 4CL, twelve CHS, two CHI, one F3H, one F3′H, one F3′5′H and four DFR) are involved in the accumulation of anthocyanins in purple flowers, and nine structural genes (two PSY, one ZDS, two CRTISO, two BCH, one ZEP and one ECH) exert an effect on the carotenoid biosynthesis pathway in yellow flowers. The findings of this study reveal the underlying mechanisms of anthocyanin and carotenoid biosynthesis in alfalfa with three classic flower colors.


Introduction
Alfalfa (Medicago sativa) is an important forage, food and ingredient in Chinese herbal medicine, and it is widely planted around the world [1].It is the most abundantly planted forage, which has excellent traits such as high yield, outstanding nutritional quality, strong hardiness and drought resistance [2,3].Three alfalfa subspecies show the most typical flower colors, including M. sativa ssp.sativa with purple flowers, M. sativa ssp.falcata with yellow flowers and M. sativa ssp.varia (Martyn) with cream flowers [4].The flower colors of alfalfa are diverse as M. sativa ssp.sativa and M. sativa ssp.falcata can be freely crossed without reproductive isolation, and the agronomic traits of alfalfa with different flower colors are significantly distinct, such as plant height, leaf area, nutritional quality and stress resistance [5].Therefore, understanding the potential mechanism underlying the pigment biosynthesis of alfalfa with petals of different colors would be informative with regard to the utilization of and nutritive research on these germplasms.
In this study, an analysis of the metabolomes and transcriptomes of the petals of the above-mentioned alfalfa with purple flowers, yellow flowers and cream flowers (hereafter, abbreviated as P, Y and C, respectively) was conducted.Through a comparative analysis of the metabolomes and transcriptomes, we studied the anthocyanin and carotenoid biosynthesis pathways, the anthocyanin and carotenoid contents, and transcription factors that are potentially associated with flower color formation.This work provides insights into the molecular regulatory mechanisms of flower color formation of alfalfa with three different flower colors.

Anthocyanin Components Accumulated in Purple Flowers and Cream Flowers
To elucidate which anthocyanins might play a major role in the color formation of purple flowers, we investigated the results of the quantitative analysis and the DAE heatmap between the purple flowers and cream flowers.The DAE heatmap showed that the accumulations of malvidins  3F) exhibited higher content level in the P group compared to the C group.These results illustrate that these malvidin, pelargonidin, petunidin, peonidin, cyanidin and delphinidin derivatives might participate in the color formation of purple flowers.

Targeted Carotenoid Metabolite Assays of Flowers of Three Different Colors
To further explore whether carotenoids contribute to the color formation of the three alfalfa germplasms with different flower colors, targeted carotenoid metabolomics was performed on their petals.As shown in Figure 4A, the PCA chart displays evident separation of the samples based on flower color, suggesting that the experiment was reliable Plants 2024, 13, 700 6 of 18 Among these anthocyanin derivatives, a quantitative analysis showed that some metabolites maintain a high level in the P group and are significantly up-accumulated in P vs. C (Table S2) S2).These results indicate that these metabolites might be the key anthocyanins involved in the color formation of purple flowers.Interestingly, we discovered that the content of delphinidin-3,5-O-diglucoside accounted for more than 91.63% of the total anthocyanins in purple flowers (Figure S1), suggesting that delphinidin-3,5-O-diglucoside plays a more essential role in the color formation of purple flowers than other anthocyanins.
Additionally, we identified that four flavonoids (kaempferol-  S2), respectively, suggesting that these flavonoids might be closely associated with anthocyanin accumulation in purple flowers.

Targeted Carotenoid Metabolite Assays of Flowers of Three Different Colors
To further explore whether carotenoids contribute to the color formation of the three alfalfa germplasms with different flower colors, targeted carotenoid metabolomics was performed on their petals.As shown in Figure 4A, the PCA chart displays evident separation of the samples based on flower color, suggesting that the experiment was reliable and reproducible.
The compound class heatmap of carotenoids indicates that a total of 49 differentially accumulated metabolites (DAMs) were identified from the three petal samples (Figure 4B) and classified into two categories: carotenes (5) and xanthophylls (44) (Figure 4C).Among these DAMs, we detected 11 upregulated and 9 downregulated DAMs, 33 upregulated and 5 downregulated DAMs, and 32 upregulated and 7 downregulated DAMs in the P vs. C comparison, the Y vs. C comparison and the P vs. Y comparison (Table 2), and the hierarchical clustering of carotenoid DAMs clearly showed that most metabolites maintain a higher content in the petals of the yellow flowers compared to that in the purple and cream flowers (Figure 4D).Moreover, a quantitative analysis of the carotenoids content indicated that the accumulation of some metabolites maintains significantly high levels in the Y group, such as lutein myristate (9.01 µg•g −1 ), lutein dimyristate (8.35 µg•g −1 ), lutein (60.76 µg•g −1 ), lutein dilaurate (5.15 µg•g −1 ), violaxanthin palmitate (4.00 µg•g −1 ) and zeaxanthin dipalmitate (7.89 µg•g −1 ), while the content of these metabolites was low in the P and C groups (Table S3).In addition to the six carotenoids, the content of remaining carotenoids was also very low in the P and C groups, ranging from 0 to 2.41 µg•g −1 (Table S3).Additionally, the content of five down-accumulated carotenoids (β-cryptoxanthin myristate, canthaxanthin, rubixanthin caprate, antheraxanthin dipalmitate and β-cryptoxanthin laurate) in Y vs. C Plants 2024, 13, 700 7 of 18 was very low in the C group, ranging from 0 to 0.059 µg•g −1 (Table S3).Simultaneously, the accumulation of seven down-accumulated carotenoids (β-cryptoxanthin oleate, γ-carotene, β-cryptoxanthin myristate, canthaxanthin, 5,6epoxy-lutein-caprate-palmitate, lutein distearate and β-cryptoxanthin laurate) in Y vs. P was very low in the P group, ranging from 0.00023 to 0.060 µg•g −1 (Table S3).These results revealed that carotenoids likely facilitate the yellow pigmentation of petals.

Transcriptome Analysis of Flower Petals of the Three Different Colors
To uncover the molecular mechanism of color formation in the three samples of petals, an analysis of the transcriptome of purple flowers, yellow flowers and cream flowers was performed.Then, we conducted a comparative analysis of the purple flowers vs. yellow flowers, the purple flowers vs. cream flowers and the yellow flowers vs. cream flowers.After cleaning and quality checking, the RNA-seq obtained 59.21 Gb of clean reads, with no less than 5 Gb of clean reads per library.The average percentage of the Q30 bases was 94%.More than 73% of the clean reads from the nine biological replicates of the three petal samples could be uniquely mapped to the reference genome (Table S5).These tran-  S4).

Transcriptome Analysis of Flower Petals of the Three Different Colors
To uncover the molecular mechanism of color formation in the three samples of petals, an analysis of the transcriptome of purple flowers, yellow flowers and cream flowers was performed.Then, we conducted a comparative analysis of the purple flowers vs. yellow flowers, the purple flowers vs. cream flowers and the yellow flowers vs. cream flowers.After cleaning and quality checking, the RNA-seq obtained 59.21 Gb of clean reads, with no less than 5 Gb of clean reads per library.The average percentage of the Q30 bases was 94%.More than 73% of the clean reads from the nine biological replicates of the three petal samples could be uniquely mapped to the reference genome (Table S5).These transcriptome results suggested that the RNA-sequencing datasets were reliable for further research.According to the FPKM values, the hierarchical clustering of DEGs revealed that the gene expression patterns were different among the three alfalfa species (Figure S3).The volcano maps showed that 4816 upregulated and 5045 downregulated genes, 5686 upregulated and 5908 downregulated genes, and 4816 upregulated and 5045 downregulated genes were detected in the P vs. Y comparison, P vs. C comparison and Y vs. C comparison, respectively (Figure S4).The KEGG analysis indicated that many DEGs were enriched in anthocyanin accumulation-related pathways in the P vs. C comparison, such as phenylpropanoid biosynthesis (ko00940), flavonoid biosynthesis (ko00941), isoflavonoid biosynthesis (ko00943), and flavone and flavonol biosynthesis (ko00944), and some DEGs were significantly enriched in carotenoid biosynthesis (ko00906) in the Y vs. C comparison (Figure S5).
To confirm the reliability of the RNA-Seq results, we selected ten crucial genes participating in the anthocyanin and carotenoid biosynthetic pathways to conduct qRT-PCR.The relative expression levels of the genes were similar to the RNA-seq results, which suggested that the RNA-Seq data were reliable in this work (Figure S6).

Expression Analysis of Carotenoid Metabolic Pathway Genes
Simultaneously, we established a carotenoid biosynthesis pathway to explore the molecular mechanism of carotenoid accumulation in the yellow flowers (Figure 7).By analyzing the differences in gene expression levels between the yellow flowers and cream flowers, we detected a total of 16 DEGs related to carotenoid biosynthesis, including PSY (4), ZDS (1), CRTISO (2), BCH (2), ZEP (6) and ECH (1). Among

Expression Analysis of Carotenoid Metabolic Pathway Genes
Simultaneously, we established a carotenoid biosynthesis pathway to explore the molecular mechanism of carotenoid accumulation in the yellow flowers (Figure 7).By analyzing the differences in gene expression levels between the yellow flowers and cream flowers, we detected a total of 16 DEGs related to carotenoid biosynthesis, including PSY (4), ZDS (1), CRTISO (2), BCH (2), ZEP (6) and ECH (1).

Identification of Transcription Factors Related to Anthocyanin and Carotenoid Biosynthesis
Transcription factors (TFs), also known as trans-acting factors, regulate plant development, abiotic stress response and the biosynthesis of secondary metabolites by changing gene expression [25,26,27].In our studies, a total of 584 differentially expressed TFs were identified in the purple flowers vs. cream flowers.Anthocyanin biosynthesis was primarily regulated by MYB, bHLH and WRKY TFs [17].Among these, we identified MYB (49), bHLH (24) and WRKY (27) differentially expressed TFs, which might play an essential role in the regulation of candidate genes related to anthocyanin biosynthesis in purple flowers.
A previous study suggested that MYB, bHLH, AP2/ERF and WRKY TFs are the essential transcription factors related to flower color involved in regulating the carotenoid anabolic pathway [23].By comparing the yellow flowers and the cream flowers, we identified a total of 582 differentially expressed TFs, including MYB (52), bHLH (24), AP2/ERF (44), WRKY (23) and other TFs.These TFs may participate in carotenoid biosynthesis in yellow flowers.

Discussion
The colors of alfalfa flowers are spectacularly diverse in nature, such as purple, yellow, red, blue, green, pink, gray and cream [5,28].Alfalfa varieties with three representative flower colors-purple, yellow and cream-were selected to study the mechanisms of flower color formation in this work.By analyzing metabolomic data, we discovered that anthocyanins and carotenoids are involved in the color formation of purple and yellow flowers, respectively.Using transcriptomic analysis, we uncovered some DEGs associated with anthocyanin biosynthesis and carotenoid biosynthesis that probably participate in pigment accumulation.Furthermore, an integrative transcriptome and metabolome analysis was performed to reveal the mechanism of alfalfa flower coloration.

Identification of Transcription Factors Related to Anthocyanin and Carotenoid Biosynthesis
Transcription factors (TFs), also known as trans-acting factors, regulate plant development, abiotic stress response and the biosynthesis of secondary metabolites by changing gene expression [25][26][27].In our studies, a total of 584 differentially expressed TFs were identified in the purple flowers vs. cream flowers.Anthocyanin biosynthesis was primarily regulated by MYB, bHLH and WRKY TFs [17].Among these, we identified MYB (49), bHLH (24) and WRKY (27) differentially expressed TFs, which might play an essential role in the regulation of candidate genes related to anthocyanin biosynthesis in purple flowers.
A previous study suggested that MYB, bHLH, AP2/ERF and WRKY TFs are the essential transcription factors related to flower color involved in regulating the carotenoid anabolic pathway [23].By comparing the yellow flowers and the cream flowers, we identified a total of 582 differentially expressed TFs, including MYB (52), bHLH (24), AP2/ERF (44), WRKY (23) and other TFs.These TFs may participate in carotenoid biosynthesis in yellow flowers.

Discussion
The colors of alfalfa flowers are spectacularly diverse in nature, such as purple, yellow, red, blue, green, pink, gray and cream [5,28].Alfalfa varieties with three representative flower colors-purple, yellow and cream-were selected to study the mechanisms of flower color formation in this work.By analyzing metabolomic data, we discovered that anthocyanins and carotenoids are involved in the color formation of purple and yellow flowers, respectively.Using transcriptomic analysis, we uncovered some DEGs associated with anthocyanin biosynthesis and carotenoid biosynthesis that probably participate in pigment accumulation.Furthermore, an integrative transcriptome and metabolome analysis was performed to reveal the mechanism of alfalfa flower coloration.

Component and Content of Anthocyanins and Carotenoids in Petals of the Three Different Colors
Anthocyanins are the crucial pigments that endow petals with various colors, and commonly include cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin [29].A previous study suggested that malvidin and petunidin derivatives are the major anthocyanins found in the purple flowers of alfalfa [30].In our present work, we observed that in addition to malvidin and petunidin derivatives, cyanidin, pelargonidin, peonidin and delphinidin derivatives are also the essential anthocyanins in the petals of purple flowers.
In recent years, many studies have reported that carotenoids are closely correlated with yellow color formation in plant tissues [31][32][33].In agreement with previous findings, we uncovered that most carotenoids are highly accumulated in yellow flowers compared to cream flowers, including α-carotene, β-carotene, (E/Z)-phytoene, α-cryptoxanthin, lutein and its derivatives, β-cryptoxanthin and its derivatives, zeaxanthin derivatives, violaxanthin and its derivatives, antheraxanthin and neoxanthin.The results reveal that these carotenoids are involved in the color formation of yellow flowers.

Key Genes That Participate in Anthocyanin Biosynthesis in Purple Flowers
Key genes participating in anthocyanin biosynthesis in petals have been studied [34,35].Flavonoids are synthesized by using phenylalanine as a precursor, and PAL, C4H and 4CL can convert phenylalanine to 4-coumaroyl-CoA.In this work, we found that the genes, C4H (MsG0180005984.01)and 4CL (MsG0480020729.01,MsG0480021654.01 and MsG0480021856.01), are highly expressed in purple flowers in comparison to cream flowers.These four genes are associated with the synthesis of 4-coumaroyl-CoA.Subsequently, 4-coumaroyl-CoA is catalyzed by three enzymes (CHS, CHI and F3H) to synthesize dihydrokaempferol.The CHS enzyme is the first committed step in the biosynthesis of anthocyanins, which involves the conversion of 4-coumaroyl-CoA to naringenin chalcone.Deng et al. used virus-induced gene silencing (VIGS) to knock out GCHS1 in gerbera and obtained transgenic strains with lighter flowers [36].In the present study, we identified twelve genes of CHS that were highly expressed in purple flowers more than in cream flowers; therefore, the low expression levels of CHS might be the primary reason for the loss of six anthocyanins in cream flowers.Afterwards, 4-coumaroyl-CoA is converted to naringenin by CHI.In this work, we found that two CHI genes (MsG0280009867.01 and MsG0180006194.01) were significantly upregulated in purple flowers, suggesting that CHI might be involved in anthocyanin biosynthesis in purple flowers.Moreover, the high expression of F3H supplied an adequate amount of dihydrokaempferol.The metabolomic analysis showed that dihydrokaempferol accumulation in purple flowers (32.63 µg•g −1 ) is about four times higher than in cream flowers (8.31 µg•g −1 ).Concurrently, we detected that the gene, F3H (MsG0880045644.01,Log 2 fold-change value = 1.59), was significantly upregulated in purple flowers compared to cream flowers.These results indicate that the gene, F3H (MsG0880045644.01),plays a critical role in dihydrokaempferol accumulation in purple flowers.
In the next step, F3 ′ H catalyzes dihydrokaempferol to dihydroquercetin, which is a precursor in the synthesis of cyanidin and peonidin.In the present study, we detected that the gene, F3 ′ H (MsG0780039504.01, Log 2 fold-change value = 2.20), is highly expressed in purple flowers compared to cream flowers.The results indicate that F3 ′ H may participate in the synthesis of cyanidin and peonidin derivatives.Dihydrokaempferol is also catalyzed to dihydromyricetin by F3 ′ 5 ′ H. Dihydromyricetin is a precursor in the synthesis of delphinidin, petunidin and malvidin.Previous research has suggested that delphinidin, petunidin and malvidin can endow plant tissues and organs with a purple color, and the gene, F3 ′ 5 ′ H, is frequently associated with purple color formation [37,38].In the present work, we detected that the gene, F3 ′ 5 ′ H (MsG0380013382.01, Log 2 fold-change value = 1.80), is significantly upregulated in purple flowers compared to cream flowers.At the same time, the content of delphinidin, petunidin and malvidin derivatives was found to be significantly higher in purple flowers than in cream flowers.These results reveal that the gene, F3 ′ 5 ′ H, plays an essential role in the accumulations of delphinidin, petunidin and malvidin.Additionally, the metabolic analysis indicated that delphinidin-3,5-O-diglucoside is the most essential anthocyanin involved in the color formation of purple flowers.Based on the above results, it is reasonable to speculate that F3 ′ 5 ′ H is the most critical gene associated with the color formation of purple flowers.
DFR is a key enzyme and can catalyze dihydrokaempferol, dihydroquercetin and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin, respectively.Most studies have demonstrated that DFR is the critical gene correlated with anthocyanin biosynthesis in plant tissues and organs [35,39].In this work, we discovered four DFR genes that were highly expressed in purple flowers compared to cream flowers.These results indicate that the highly expressed levels of DFR are closely associated with the accumulations of various anthocyanins in purple flowers.The high expression levels of the gene, FLS, increased flavone and flavonol accumulations and negatively regulated anthocyanin biosynthesis [40,41].In this study, the high expression of two FLS genes was identified in cream flowers compared to purple flowers; however, the quantitative analysis of metabolites showed that flavone and flavonol (kaempferol-3-O-rutinoside, rutin and afzelin) are significantly up-accumulated in purple flowers compared to cream flowers.Therefore, we concluded that FLS does not contribute significantly to anthocyanin biosynthesis in cream flowers.

Key Genes That Participate in Carotenoid Biosynthesis in Yellow Flowers
PSY is the first enzyme involved in carotenoid biosynthesis, and it catalyzes geranylgeranyl pyrophosphate to phytoene [42].Eschscholzia californica is known for its bright golden-orange flowers, and the deletion of PSY prevents the synthesis of carotenoids in white cultivars [43].In this study, two PSY genes (MsG0380013530.01,Log 2 fold-change value = 7.91; novel.10662,Log 2 fold-change value = 3.30) were significantly upregulated in yellow flowers compared to cream flowers, and phytoene maintained a high content in yellow flowers compared to cream flowers.These results indicate that PSY plays a critical role in phytoene synthesis in yellow flowers.After phytoene is catalyzed to ζ-carotene by PDS, ZDS and CRTISO can catalyze ζ-carotene to lycopene.Li [44] confirmed that high expression levels of the genes, CRTISO and ZDS, can increase carotenoid accumulation for golden-leaf coloration in the mutant G. biloba.In this study, we detected that one ZDS gene (MsG0180004504.01,Log 2 fold-change value = 1.50) and two CRTISO genes (MsG0880045723.01,Log 2 fold-change value = 2.32; MsG0180003155.01,Log 2 fold-change value = 1.73) are significantly upregulated in yellow flowers compared to cream flowers.The results suggest that higher expression levels of the ZDS and CRTISO genes may facilitate carotenoid accumulations in yellow flowers.During carotenoid biosynthesis, BCH, an important enzyme involved in carotenoid biosynthesis, can catalyze α-carotene, β-carotene and β-cryptoxanthin to zeinoxanthin, β-cryptoxanthin and zeaxanthin, respectively.Moreover, a previous study revealed that a high expression level of BCH is associated with the yellow color formation of petals [45].In the present work, we observed that two BCH genes (MsG0780039795.01,Log2 fold-change value = 1.25;MsG0880047112.01,Log2 fold-change value = 1.53) are significantly upregulated in yellow flowers compared to cream flowers.Combined with the results of the metabolic measurements, we found that zeinoxanthin, β-cryptoxanthin and zeaxanthin maintain high levels in yellow flowers in comparison to cream flowers.These results suggest that BCH genes positively participate in the accumulations of β-cryptoxanthin and zeaxanthin.Subsequently, zeinoxanthin is catalyzed to lutein by ECH.Our metabolic analysis showed that lutein is significantly upaccumulated in yellow flowers compared to cream flowers, and the expression of one ECH gene (MsG0180003453.01,Log 2 fold-change value = 2.08) showed a higher expression level in yellow flowers than in cream flowers.Therefore, we concluded that higher expression levels of the ECH gene may contribute to lutein accumulations and eventually lead to the color formation of yellow flowers.

TFs Related to Anthocyanin and Carotenoid Biosynthesis
TFs of the MYB family play a vital role in the regulation of anthocyanin biosynthesis [46].For example, Zhou et al. observed that CmMYB3-like negatively regulated anthocyanin biosynthesis in Chrysanthemum morifolium [47].bHLH TFs are associated with MYB and WD to regulate anthocyanins' biosynthesis [48].In addition, WRKY TFs responsible for regulating anthocyanin biosynthesis have been reported [49].Among all the differentially expressed genes, we identified 49 MYB, 24 bHLH and 27 WRKY TFs in the comparison of purple flowers vs. cream flowers.These results will enable the clarification of the functions of TFs during anthocyanin biosynthesis in purple flowers.
The transcription factor, AP2.2, interacts with its partner SINAT2 to negatively regulate the expression level of the gene, PSY, and decrease the carotenoid content in Arabidopsis leaves [50].Moreover, previous studies suggested TFs of the R2R3-MYB, bHLH and WRKY families are also involved in regulating carotenoid biosynthesis [51][52][53].In the current study, 52 MYB, 24 bHLH, 44 AP2/ERF, 23 WRKY TFs with differential expression were identified in the comparison of yellow flowers vs. cream flowers.These TFs may have essential functions in controlling carotenoid biosynthesis in yellow flowers.

Plant Materials
The three samples (alfalfa with purple flowers, yellow flowers and cream flowers) were cultivated at the Institute of grassland research of the Chinese Academy of Agricultural Sciences in Hohhot (40 • 58 ′ N, 111 • 78 ′ E).All samples were planted in the same environment and under the same management practices.We collected petals of each color from the same stem when the plants reached the blooming stage.We named the petals of the purple flowers 'P', those of the yellow flowers 'Y' and those of the cream flowers 'C'.Three biological replicates were obtained for the three samples, and the petals of each color were taken from 10 flowers and pooled for each biological replicate.

Transcriptome Sequencing and Data Analysis
The total RNA of the petals (P, Y and C) was extracted using ethanol precipitation and CTAB-PBIOZO.The quality and preliminary quantification of mRNAs were detected using an Agilent 2100 Bioanalyzer (Agilent Technologies, Baden-Württemberg, Germany) and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively.Nine cDNA libraries were sequenced by utilizing the Illumina sequencing 6000 platform.Clean reads were acquired after low-quality sequences were withdrawn and then assembled using the Fastp v0.19.3.All non-redundant transcripts were mapped to the M. sativa reference genome (https://figshare.com/articles/dataset/M_sativa_genome_and_annotation_files/12623960 (accessed on 20 October 2023)).Differentially expressed genes (DEGs) were determined by using DESeq2.Moreover, we established criteria to screen significantly differential expression (False discovery rate (FDR < 0.01) and fold change (FC ≥ 2)).Enrichment analysis was carried out according to the hypergeometric test, with the pathway-based hypergeometric distribution being checked against the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) term-based profiles.

Extraction, Separation, Identification and Quantification of Anthocyanins
In terms of the AB Sciex QTRAP 6500 LC-MS/MS platform, anthocyanin accumulations were detected by MetWare Biotechnology Company (Wuhan, China) (http://www.metware.cn/(accessed on 10 July 2023)).First, about 0.05 g of powder was extracted with 0.5 mL of methanol/water/hydrochloric acid (500:500:1, v/v/v).Afterwards, the extract was vortexed for 300 s, subjected to ultrasound for 300 s and centrifuged at 12,000 r/60 s for 180 s at 4 • C; these steps were then repeated one more time.Afterwards, the supernatants were obtained and filtrated by utilizing a 0.22 µm membrane filter.
By utilizing an UPLC-APCI-MS/MS system (UPLC, ExionLC™ AD, https://sciex.com.cn/ (accessed on 12 July 2023) and MS, Applied Biosystems 6500 Triple Quadrupole, https://sciex.com.cn/(accessed on 12 July 2023)), the sample extracts of anthocyanins were examined.Linear ion trap (LIT) and triple quadrupole (QQQ) scans were performed using a triple QTRAP, QTRAP ® 6500+ LC-MS/MS System equipped with an ESI Turbo Ion-Spray interface, which was run in the positive ion mode and managed by the Analyst 1.6.3software (Sciex).By using scheduled multiple reaction monitoring (MRM), the content of anthocyanins was investigated.Data acquisitions were conducted through the Analyst 1.6.3software (Sciex).Subsequently, we quantitatively evaluated metabolites by employing the Multiquant 3.0.3software (Sciex).Mass spectrometric parameters, including declustering potentials (DPs) and collision energies (CEs) for individual MRM transitions, were generated via DP and CE optimization.Then, we detected the specific set of MRM transitions for each period.Notably, anthocyanin test results include both quantitative and semi-quantitative substances.Among them, the substances with standards were qualitatively quantified using standards, and the substances without standards were qualitatively quantified according to the mass spectrometry cleavage law, and the quantification was quantified via the calibration of analogs (anthocyanins were selected as delphinidin-3,5-O-diglucoside).

Extraction, Separation, Identification and Quantification of Carotenoids
Similarly, we extracted a total of 0.05 g of material by utilizing 0.5 mL of a solution that was formed by mixing N-Hexane, acetone and ethanol in equal proportions.Subsequently, we vortexed the extract for 20 min at about 20 • C.After being centrifuged at 12,000 r/min for 300 s at 4 • C, we picked up the liquid, and the above steps were repeated.Subsequently, the liquid was vaporized to dryness in a vacuum and rebuilt in a solution that was formed by mixing MeOH/MTBE in equal proportions, and the liquid was filtered.
In addition to the QTRAP ® 6500+ LC-MS/MS System equipped with an APCI Heated Nebulizer, the next steps and methods used were the same as those used for analyzing anthocyanins.Similarly, carotenoid test results also included both quantitative and semiquantitative substances.Among them, the substances with standards were qualitatively quantified using standards, and the substances without standards were qualitatively quantified according to the mass spectrometry cleavage law, and the quantification was quantified by the calibration of analogs (carotenoids were selected as neoxanthin).

qRT-PCR Analysis
RNA was extracted and reverse-transcribed using a RNA pure plant kit (Tb Green ® Premix Ex Taq™ II (TAKARA, Beijing, China)) and Monad first-strand cDNA Synthesis Kit (SuperScript, Shanghai, China), respectively.Moreover, by utilizing the PRIMER-BLAST, we designed the primers (Table S1).In addition, we selected the gene (MsG0380015289.01)as the reference gene due to its high and steady expression levels in all samples based on the transcriptome data.Samples were placed in a real-time PCR machine and assays were set up on ABI Quant Studio 1 software.All results were obtained from three biological replicates.The primers are listed in Table S6.

Conclusions
In this work, we revealed the molecular mechanism of flower color formation in M. sativa ssp.falcata (purple flowers) and M. sativa ssp.falcata (yellow flowers) via a comparative analysis of the metabolome and transcriptome of three types of petals (purple, yellow and cream).The metabolome results demonstrated that anthocyanins (cyanidin, delphinidin, peonidin malvidin, pelargonidin and petunidin derivatives) are the critical pigments involved in the color formation of purple flowers, and the content of delphinidin-3,5-Odiglucoside and lutein was highest in purple and yellow flowers, respectively.Moreover,

Figure 1 .
Figure 1.The phenotypes of alfalfa with different flower colors: Alfalfa with (A) purple flowers, (B) yellow flowers and (C) cream flowers.Figure 1.The phenotypes of alfalfa with different flower colors: Alfalfa with (A) purple flowers, (B) yellow flowers and (C) cream flowers.

Figure 1 .
Figure 1.The phenotypes of alfalfa with different flower colors: Alfalfa with (A) purple flowers, (B) yellow flowers and (C) cream flowers.Figure 1.The phenotypes of alfalfa with different flower colors: Alfalfa with (A) purple flowers, (B) yellow flowers and (C) cream flowers.

Figure 2 .
Figure 2. Targeted metabolomic analysis of anthocyanins in the three samples of petals.P1, P2 and P3 represent the three replicates of the P group; Y1, Y2 and Y3 represent the three replicates of the Y group; C1, C2 and C3 represent the three replicates of the C group; Scale: color changes (green to red) represent low to high accumulation.(A) PCA plot analysis of anthocyanin metabolome.The xaxis represents principal component 1 (PC1); the y-axis represents principal component 2 (PC2); (B) Venn diagram of DAEs among the three groups.(C) Compound class heatmap of anthocyanin DAMs.(D) Hierarchical clustering of anthocyanin DAMs.

Figure 2 .
Figure 2. Targeted metabolomic analysis of anthocyanins in the three samples of petals.P1, P2 and P3 represent the three replicates of the P group; Y1, Y2 and Y3 represent the three replicates of the Y group; C1, C2 and C3 represent the three replicates of the C group; Scale: color changes (green to red) represent low to high accumulation.(A) PCA plot analysis of anthocyanin metabolome.The x-axis represents principal component 1 (PC1); the y-axis represents principal component 2 (PC2); (B) Venn diagram of DAEs among the three groups.(C) Compound class heatmap of anthocyanin DAMs.(D) Hierarchical clustering of anthocyanin DAMs.

20 Figure 3 .
Figure 3.The DAE heatmap of malvidin (A), pelargonidin (B), petunidin (C), peonidin (D), cyanidin (E), delphinidin (F) and flavonoid (G) in purple flowers vs. cream flowers.P1, P2 and P3 represent the three replicates of the P group; C1, C2 and C3 represent the three replicates of the C group; low to high accumulation is suggested by color changes (green to red).

Figure 3 .
Figure 3.The DAE heatmap of malvidin (A), pelargonidin (B), petunidin (C), peonidin (D), cyanidin (E), delphinidin (F) and flavonoid (G) in purple flowers vs. cream flowers.P1, P2 and P3 represent the three replicates of the P group; C1, C2 and C3 represent the three replicates of the C group; low to high accumulation is suggested by color changes (green to red).

Figure 4 .
Figure 4. Targeted metabolomic analysis of carotenoids in the three samples of petals.P1, P2 and P3 represent the three replicates of the P group; Y1, Y2 and Y3 represent the three replicates of the Y group; C1, C2 and C3 represent the three replicates of the C group; Scale: color changes (green to red) represent low to high accumulation.(A) PCA plot analysis of carotenoid metabolome.The xaxis represents principal component 1 (PC1); the y-axis represents principal component 2 (PC2); the three samples (P, Y and C) are distinguished by different colors.(B) Venn diagram of DAEs among

Figure 4 .
Figure 4. Targeted metabolomic analysis of carotenoids in the three samples of petals.P1, P2 and P3 represent the three replicates of the P group; Y1, Y2 and Y3 represent the three replicates of the Y group; C1, C2 and C3 represent the three replicates of the C group; Scale: color changes (green to red) represent low to high accumulation.(A) PCA plot analysis of carotenoid metabolome.The x-axis represents principal component 1 (PC1); the y-axis represents principal component 2 (PC2); the three samples (P, Y and C) are distinguished by different colors.(B) Venn diagram of DAEs among the three groups.(C) Compound class heatmap of carotenoid DAMs.(D) Hierarchical clustering of carotenoid DAMs.

2. 4 .
Identification of Carotenoid Components Involved in the Color Formation of Yellow Flowers Subsequently, we investigated the results of the quantitative analysis and the DAE heatmap of the yellow flowers and cream flowers to explore which carotenoids might give rise to the color formation of the yellow flowers.Then, we detected that the accumulation of three carotenes (α-carotene, Log 2 fold change values = 1.13; β-carotene, Log 2 fold change values = 1.15; (E/Z)-phytoene, Log 2 fold change values = 1.77) was significantly higher in the Y group than in the C group (Figure 5, Table

20 Figure 5 .
Figure 5. DAE heatmap of carotenes and xanthophylls in yellow flowers vs. cream flowers.Y1, Y2 and Y3 represent the three replicates of the Y group; C1, C2 and C3 represent the three replicates of the C group; low to high accumulation is suggested by color changes (green to red).

Table 1 .
DAMs of anthocyanins in the three comparison groups.

Table 2 .
DAMs of carotenoids in the three comparison groups.