Integrated analyses of the mechanism of flower color formation in alfalfa (Medicago sativa)

Integrated analyses of the mechanism of flower color formation in alfalfa (Medicago sativa) | 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 Integrated analyses of the mechanism of flower color formation in alfalfa (Medicago sativa) Zhaozhu Wen, Huancheng Liu, Qian Zhang, Xuran Lu, Kai Jiang, Qinyan Bao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4239305/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 Alfalfa ( Medicago sativa ) is one of the most valuable forages in the world. As an outcrossing species, it needs bright flowers to attract pollinators to deal with self-incompatibility. Although various flower colors have been observed and described in alfalfa a long time ago, the biochemical and molecular mechanism of its color formation is still unclear. By analyzing alfalfa lines with five contrasting flower colors including white (cream-colored), yellow, lavender (purple), dark purple and dark blue, various kinds and levels of anthocyanins, carotenoids and other flavonoids were detected in different colored petals, and their roles in color formation were revealed. Notably, the content of delphinidin-3,5- O -diglucoside in lines 3, 4 and 5 was 58.88, 100.80 and 94.07 times that of line 1, respectively. Delphinidin-3,5- O -diglucoside was the key factor for purple and blue color formation. Lutein and β-carotene were the main factors for the yellow color formation. By analyzing differentially expressed genes responsible for specific biochemical pathways and compounds, 27 genes were found to be associated with purple and blue color formation, and 14 genes were found to play an important role in yellow color formation. These findings provide a basis for understanding the biochemical and molecular mechanism of alfalfa flower color formation. alfalfa Medicago sativa flower color anthocyanins carotenoids delphinidin-3 5-O-diglucoside Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Flower color is an important trait for plants as visual signaling to attract pollinators and ensure their reproductive success [ 1 , 2 ] . Some floral species, such as orchids, are characterized by their fantastic floral morphology and high levels of corolla color polymorphism and variability, which have played an important role in pollination [ 1 , 3 ] . As one of the most important forages in the world, alfalfa ( Medicago sativa L., 2n = 4x = 32) is a typical cross-pollinated plant with a high degree of self-incompatibility, attracting insect pollinators using its bright flowers [ 4 – 6 ] . Flavonoids and carotenoids are the two major floral pigments, and anthocyanins constitute the largest group of flavonoids. The biosynthesis pathway of anthocyanins, which affect plant flower colors, is regulated by many genes. 4CL ( 4-coumarate: CoA ligase ), CHS ( chalcone synthase ), CHI ( chalcone isomerase ), F3'H ( flavonoid 3'-hydroxylase ), F3'5'H ( flavonoid 3', 5'-hydroxylase ) and DFR ( dihydroflavonol reductase ) are important enzymatic genes in the upstream anthocyanin synthesis pathway. Among them, 4CL not only participates in the biosynthesis of anthocyanin but also is involved in lignin formation and the production of other phenolic compounds [ 7 ] . F3'H and F3'5'H can change the type of flavonoids, transforming naringenin/dihydrokaempferol into eriodictyol/dihydroquercetin and dihydrotricetin/ dihydromyricetin [ 8 ] . Blocking the synthesis of F3'5'H affects the accumulation and reduction of anthocyanins, leading to a light flower color in pea ( Pisum sativum ) [ 9 ] . Arabidopsis DFR mutant prevents dihydroplavonones from being reduced to leucopelargonidin, making the seed coat white, similar to the f3'h mutant phenotype [ 10 , 11 ] . At the downstream of the anthocyanin biosynthesis pathway, anthocyanidins can form anthocyanins through glycosylation, methylation and acylation reactions under the action of various UDP-glycosyltransferases ( UGTs ). This process makes anthocyanins more stable in property, more abundant in color and more complex in structure. Genes in the downstream of anthocyanin biosynthesis are regulated by the ternary complexes of transcription factors, named MYB-bHLH-WD40 (MBW). These MBW complexes include MYB (such as PAP1 and TT2), bHLH (such as TT8, GL3 and EGL3) and WD40 proteins (such as TTG1) [ 12 – 15 ] . The conservatism of the interaction of MYB, bHLH and WD40 in controlling proanthocyanidins (PAs) and anthocyanin biosynthesis has been verified in many plants [ 16 ] . MBW usually targets with the promoters of structural genes to activate the transcription of these genes in plants [ 12 ] . MtTT8 interacts with MYB and MtWD40-1 to form a regulatory complex that regulates the anthocyanin biosynthesis genes and affects the seed coat and flower color in Medicago truncatula [ 17 ] . Allelic variations in the promoters between GhTT19 LW and GhTT19 LR, rather than genic regions, are the genetic cause of petal color variations in cotton [ 18 ] . Flavonoids, especially anthocyanins, help to produce a variety of flower colors ranging from light yellow to orange, red, blue, purple and dark purple, whereas carotenoids provide yellow, orange and bright red colors to flowers. With C6-C3-C6 as the carbon skeleton, anthocyanins form the basic structures of delphinidin, peonidin, petudinin, malvidin, pelargonidin and cyanidin through different combinations, which react with different sugar, methyl and acyl groups to produce more than 600 anthocyanins [ 19 ] . The main forms of anthocyanins vary among plant species and in different organs. Cyanidins and pelargonidins are the main pigments in red plant organs, while delphinidins, petudinins and malvidins are important chromogenic substances in many blue/purple plant organs [ 20 ] . To understand the key metabolic compounds and the associated gene expression networks controlling flower color formation in alfalfa, multiple lines with contrasting flower colors were analyzed by metabolic profiling and RNA-seq. Combined analysis revealed specific compounds and genes affecting flower color formation in alfalfa. This study provides new insights into the biochemical and molecular mechanisms associated with the biosynthesis and regulation of anthocyanins and carotenoids in alfalfa flowers. Materials and Methods Plant materials and growth conditions Alfalfa lines of the “Wudi” population used in this study were grown at Wudi county (117.97, 37.92), Jiaozhou Experimental Station (120.10, 36.31) and Qingdao Agricultural University (120.40, 36.32), Shandong province, China. Petals were collected from fully opened petals, and 3.0 g of the collected petals per sample was used for metabolic analysis and RNA-seq analysis. Metabolic analysis The petal samples were freeze-dried and ground into powder (30 Hz, 1.5 min). Then, 50 mg of the powdered sample was weighed, used for extraction with 0.5 mL methanol/water/hydrochloric acid (500:500:1, V/V/V), and vortexed and ultrasonicated for 5 min. The samples were then centrifuged at 12,000 g for 3 min at 4°C. The supernatant of each sample was filtered through a membrane filter (0.22 µm, Anpel). The sample extracts of anthocyanins and flavonoids were analyzed using an UPLC-ESI-MS/MS system, and carotenoids were analyzed using an UPLC-APCI-MS/MS system (UPLC, ExionLC™ AD, https://sciex.com.cn/ ; MS, Applied Biosystems 6500 Triple Quadrupole, https://sciex.com.cn/ ). The anthocyanin, flavonoid and carotenoid contents were detected using MetWare ( http://www.metware.cn/ ) based on the AB Sciex QTRAP 6500 LC-MS/MS platform. RNA-seq analysis Total RNA was extracted from the alfalfa petals using the TRIzol® Reagent Kit according to the manufacturer’s instructions (Invitrogen). RNA quality was determined by 2100 Bioanalyzer (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). RNA-seq transcriptome libraries were sequenced using the Illumina HiSeq xten/NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA). The clean reads were mapped and annotated following the reference genome with orientation mode using the HISAT2 ( http://ccb.jhu.edu/software/hisat2/index.shtml ) software [ 4 , 21 ] . Essentially, differential expression analysis was performed using the DESeq2/DEGseq/EdgeR with Q value ≤ 0.05, and DEGs with |log2FC| > 1 and Q value ≤ 0.05 (DESeq2 or EdgeR)/Q value ≤ 0.001 (DEGseq) were significant DEGs. KEGG functional enrichment and pathway analyses were carried out using Goatools ( https://github.com/tanghaibao/Goatools ) and KOBAS ( http://kobas.cbi.pku.edu.cn/home.do ) [ 22 ] . Statistical analysis Cluster analysis and variance analysis were performed using SPSS. Principal component analysis was performed using MetWare. Results Characterization of alfalfa lines with different flower colors In contrast to the common “lavender” flowers in alfalfa, the “Wudi” population has amazing color polymorphism, including white (cream-colored), yellow, lavender (purple), dark purple, dark blue and some intermediate colors. Growing the materials in distant locations did not affect the flower color, and the coloration remained consistent throughout the perennial life cycle of the plants. Thus, variation in floral color is likely genetically determined rather than a result of, for example, soil chemistry. Lines 1 (white/cream-colored), 2 (yellow), 3 (lavender/purple), 4 (dark purple) and 5 (dark blue) with obvious color differences were selected from the “Wudi” population (Fig. 1 a, b). The colors of the style, stigma, ovary, filament and anther were indistinguishable after removing the petals (Supplementary Fig. 1). Therefore, the pigment difference in alfalfa flowers lays on the petal rather than other organs. To identify the petal color more clearly, the petal colors of lines 1–5 were measured with a getcolor. The petal colors of fully opened flowers were calibrated on the red, green and blue (RGB) scale, and the specific RGB values of petals 1–5 were determined as #eae9f7, #f9f27a, #8c5d98, #82217c and #4e4370, respectively (Fig. 1 c). According to the cyan, magenta, yellow and black (CMYK) color scale ( https://www.colorhexa.com ), #eae9f7 (line 1) is composed of 5.3% cyan, 5.7% magenta, 0% yellow and 3.1% black; #f9f27a (line 2) is 0% cyan, 2.8% magenta, 51% yellow and 2.4% black; #8c5d98 (line 3) is 7.9% cyan, 38.8% magenta, 0% yellow and 40.4% black; #82217c (line 4) consists of 0% cyan, 74.6% magenta, 4.6% yellow and 49% black; and #4e4370 (line 5) is 30.4% cyan, 40.2% magenta, 0% yellow and 56.1% black (Fig. 1 d). This result suggests that the lavender, dark purple and dark blue petal colors of lines 3–5 are caused by the addition of different degrees of purple obtained from the blending of magenta and black to the white base of line 1 (Fig. 1 c, d). Cell morphology of the deepest colored regions of the petals was observed by scanning electron microscope (SEM) to determine the influence of epidermal cells on petal color. No significant difference was found in petal epidermal cell structures between the five lines (Supplementary Fig. 2). Anthocyanin content is much higher in purple and blue petals Metabolic analysis of flavonoid, anthocyanin and carotenoid was conducted using petals collected from different colored alfalfa lines. The total ion flow map (TIC) obtained from QC samples indicated the sum of all ion strengths at different time points (Supplementary Fig. 3). In the detection multipeak map (XIC), the chromatographic peaks of different colors corresponded to different carotenoid, flavonoid and anthocyanin metabolites (Supplementary Fig. 4). The superposition of the TIC map of the QC samples showed that the response intensity and retention time of each peak strongly overlapped, indicating that the data were reliable (Supplementary Fig. 5). Principal component analysis (PCA) of all pigment metabolites and flavonoids did not place the samples into groups consistent with petal colors (Fig. 2 a, b). However, PCA of anthocyanins placed the samples into three groups consistent with petal colors. Lines 1 and 2 were in the light color group, and line 3 was in the normal color group, while lines 4 and 5 were in the dark color group, suggesting that anthocyanins play a critical role in purple and blue color determination (Fig. 2 c). Contents of different pigments were analyzed in the petals. The results showed that anthocyanin content in purple (lines 3 and 4) and blue petals (line 5) was significantly higher than petals of other colors (lines 1 and 2) (Fig. 2 d, Table 1 ); flavones and flavonols content in purple and blue petals was significantly lower than white color petals (Fig. 2 d, Table 1 ). The content of anthocyanins accounted for 6.37%, 8.79%, 86.05%, 93.90% and 93.82% of the total pigment content in lines 1, 2, 3, 4 and 5, respectively. No significant difference was observed in anthocyanin content between lines 1 and 2 (Fig. 2 d, Table 1 ). The content of anthocyanins in lines 3, 4 and 5 was 73.53, 122.83 and 117.66 times that of line 1, respectively (Table 1 ). The content of anthocyanins in lines 4 and 5 was 1.67 and 1.60 times that of line 3, respectively (Table 1 ). The results indicated that the difference in petal color between white and purple/blue was specifically caused by the difference in anthocyanin content. Thus, anthocyanin biosynthesis is crucial for color heterogeneity in alfalfa. Table 1 Pigment content in alfalfa lines (1–5) with different petal colors Anthocyanidins Flavones Flavonols Carotenoids Flavanols Procyanidins Xanthophylls Phenonic acids Isoflavanones Chalcones Total carotenoids 1 64.33 C d 482.82 A a 433.98 A a 3.09 E e 0.38 C c 1.63E-02 A a 22.02 C c 0.17 C d 1.02 AB a 2.54 C c 25.11 C e 2 79.99 C d 404.08 B b 363.68 B b 14.81 A a 0.17 D d 9.56E-03 C cd 44.13 A a 0.30 A a 0.54 C d 2.17 D d 58.94 A a 3 4730.31 B c 394.62 B b 339.02 B b 3.52 D d 1.02 B b 1.26E-02 AB b 24.04 C c 0.18 C d 0.91 B b 3.42 B b 27.55 C d 4 7902.30 A a 206.55 C c 252.62 C c 5.72 C c 1.95 A a 7.44E-03 C d 41.27 AB b 0.25 B b 1.07 A a 4.28 A a 46.99 B c 5 7569.34 A b 167.05 D d 275.39 C c 13.41 B b 0.99 B b 1.12E-02 BC bc 39.26 B b 0.22 B c 0.65 C c 1.46 E e 52.68 AB b Delphinidin-3,5- O -diglucoside is the key pigment metabolite affecting the purple and blue color of alfalfa flowers According to the basic structure of anthocyanins, a total of 52 anthocyanins were identified in alfalfa petals, including 14 cyanidins, 11 delphinidins, 7 malvidins, 8 pelargonidins, 6 peonidins and 7 petunidins (Supplementary Table 1). The content of delphinidins accounted for 5.51%, 6.84%, 58.58%, 66.76% and 64.04% of the total pigment content in lines 1, 2, 3, 4 and 5, respectively (Supplementary Fig. 6a). According to the functional groups, 9 types of anthocyanins were detected, including anthocyanidin-3,5- O -diglucoside, anthocyanidin-3- O -glucoside, anthocyanidin-3- O -(6- O -p-coumaroyl)-glucoside, anthocyanidin-3- O -rutinoside, anthocyanidin-3- O -sophoroside, anthocyanidin-3- O -sambubioside, anthocyanidin-3- O -(6- O -malonyl-beta-D-glucoside), delphinidin-3- O -rutinoside-5- O -glucoside and cyanidin-3,5,3'- O -triglucoside. Anthocyanidin-3,5- O -diglucoside catalyzed by UGT75C1 had the most significant difference between line 1 and lines 3/4/5. The content of anthocyanidin-3,5- O -diglucoside accounted for 5.61%, 7.28%, 78.79%, 84.04% and 86.74% of the total pigment content in lines 1, 2, 3, 4 and 5, respectively (Fig. 3 a, Supplementary Fig. 6b). As one of anthocyanidin-3,5- O -diglucosides, delphinidin-3,5- O -diglucoside content in lines 1 and 2 was only about 5.26% and 6.38% of total pigment, while in lines 3, 4 and 5, the delphinidin-3,5- O -diglucoside content was 56.90%, 63.62% and 61.94% of total pigment, respectively (Fig. 3 b). The content of delphinidin-3,5- O -diglucoside in lines 3, 4 and 5 was 58.88, 100.80 and 94.07 times that of line 1, respectively. Cluster analysis of all anthocyanins and total pigments showed that delphinidin-3,5- O -diglucoside was closest to the total pigments (Fig. 3 c). Therefore, delphinidin-3,5- O -diglucoside was the key pigment causing the purple or bule petal color in alfalfa. Alfalfa petal color is affected by the expression of flavonoid biosynthesis-related pathway genes RNA-seq analysis was conducted using petals collected from different colored alfalfa lines. More than 40 million raw reads were generated from each library, and 88–93% of the trimmed reads were uniquely mapped to the alfalfa (cultivar “XinJiangDaYe”) reference genome (Supplementary Table 2). The differentially expressed genes (DEGs) were analyzed by DESeq software, and the selected DEGs (|log2FC| ≥ 1 and FDR < 0.05) were then compared and analyzed. Compared to the line 1 control, 11,300, 15,137, 10,414 and 21,186 genes were differentially expressed in lines 2, 3, 4 and 5, respectively, of which 6486, 6942, 5950 and 10,965, respectively, were upregulated and 4814, 8195, 4464 and 10221, respectively, were downregulated (Fig. 4 a). In general, there were more upregulated DEGs than downregulated DEGs. The upregulated DEGs identified in the 1 vs 2–5 comparison groups were involved in 120–122 KEGG metabolic pathways, and 24, 24, 15 and 23 significantly enriched metabolic pathways were identified in the 1 vs 2, 1 vs 3, 1 vs 4 and 1 vs 5 groups, respectively. Among them, flavone and flavonol biosynthesis (ko00944) and carotenoid biosynthesis (ko00906) were significantly enriched in 1 vs 2 (Fig. 4 b). Flavone and flavonol biosynthesis (ko00944) and flavonoid biosynthesis (ko00941) were all significantly enriched in 1 vs 3 and 1 vs 4 (Fig. 4 c–d). Thus, flavonoid biosynthesis-related pathways may play an important role in petal color. Analysis of DEGs related to flavonoid and anthocyanin biosynthesis pathway Compared to line 1, 1754 genes were expressed in pathways related to flavonoid biosynthesis. Among them, 212 genes were involved in anthocyanin biosynthesis, 778 genes belonged to UGT family, and 764 genes related to MBW proteins (439 MYB and MYB -related genes, 194 bHLH and 131 WD40 ) (Supplementary Fig. 7). Based on KEGG enrichment analysis and petal color differences, 27 DEGs (every sample FPKM > 1, |log2FC| ≥ 1 in 1 vs 3, 1 vs 4 and 1 vs 5 groups) were significantly enriched in anthocyanin biosynthesis-related pathways. These 27 DEGs include 2 4CL , 3 F3'H , 6 DFR , 6 UGT , 2 bHLH and 8 MYB genes, which are considered candidate genes regulating purple petal color in alfalfa (Fig. 5 , Supplementary Table 3). Lutein and β-carotene are the main pigment metabolites affecting the yellow petal color in alfalfa Finally, we analyzed the cause of the formation of yellow petals in alfalfa. According to RNA-seq analysis, upregulated DGEs were enriched in carotenoid biosynthesis pathway and flavone and flavonol biosynthesis pathway in the 1 vs 2 group (Fig. 4 ). Compared to white colored line 1, the content of flavones and flavonols was significantly lower (16.31% and 16.20%, respectively) in yellow colored line 2, while the content of total carotenoids was 2.35 times that of line 1 (Table 1 ). Therefore, carotenoids play a key role in determining the yellow petal color in alfalfa. Compared to line 1, the top seven carotenoids (lutein, β-carotene, zeaxanthin, (E/Z)-phytoene, violaxanthin, neoxanthin and β-cryptoxanthin) were significantly higher in line 2 (Fig. 6 a, b). In the carotene category, the content of β-carotene was the highest, accounted for 18.05% of total carotenoids (total carotenes + total xanthophylls) in line 2, and it was 4.29 times that of line 1 (Fig. 6 a). Among the xanthophylls, lutein content was the highest, accounted for 38.04% of total carotenoids in line 2, and it was 1.89 times that of line 1 (Fig. 6 b). Therefore, lutein and β-carotene were the main metabolites leading to the yellow petal color in alfalfa. Compared to line 1, 6 PSY , 1 LcyE , 1 LcyB and 6 ZEP genes were significantly upregulated in line 2 and likely to be involved in regulating the formation of yellow petals in alfalfa (Fig. 6 c). Discussions Variation in flower color plays an important ecological role by attracting pollinators and influencing reproductive success in flowering plants, particularly for plants with a high degree of self-incompatibility, such as alfalfa. Alfalfa is a widely cultivated forage crop around the world due to its remarkable adaptability, high biomass yield, exceptional nutritive value and notable capacity for nitrogen fixation [ 23 – 25 ] . Although various flower colors have been observed and described in alfalfa a long time ago [ 26 ] , the biochemical and molecular mechanism of its color formation is still unclear. By analyzing alfalfa lines with five contrasting flower colors, various kinds and levels of anthocyanins, carotenoids and other flavonoids were detected in different colored petals, and their roles in color formation were revealed. Genes responsible for specific biochemical pathways and compounds associated with color formation were also revealed. This study provides an in-depth interpretation of the flower coloration mechanism in alfalfa. Key metabolic compounds affecting petal color formation in alfalfa The presence of specific types of anthocyanins affects flower colors. For example, cyanidin 3-glucoside and cyanidin 3-(6-malonyglucoside) are dominant pigments in the black-colored flowers in Alpine orchid Gymnadenia rhellicani [ 27 ] ; maintaining both high relative and absolute content of cyanidin 3,5- O -diglucoside may be a prerequisite for the formation of red petals in Rosa rugosa [ 28 ] . Various types of anthocyanins have reported in the formation of purple and blue flowers. Cyanidin 3,5- O -diglucoside, malvidin 3,5-diglucoside and cyanidin 3- O -galactoside were found to be responsible for the purple flower color in Salvia miltiorrhiza [ 29 ] . High levels of malvidin-3,5-di- O -glucoside were detected in purple flowers in Lagerstroemia indica [ 30 ] . Significantly higher content of delphinidin-3,5- O -diglucoside was detected in 8–16 deep purple–red-flowered germplasm resources with somewhat unique and visible blue hue in R. rugosa [ 28 ] . In the current study, we found that delphinidin-3,5- O -diglucoside is the primary anthocyanin responsible for the formation of both purple and blue colored petals in alfalfa. This conclusion is consistent with work of Wang et al. (2022) in R. rugosa [ 28 ] , but different from that of Duan et al. (2020) in alfalfa [ 31 ] , which used only two different colored lines. The formation of yellow flowers is considered to come from flavonoids or carotenoids. The yellow-colored tree peony flower displayed relatively higher contents of tetrahydroxychalcone (THC), flavones, and flavonols compared to purple–red flowers, but no anthocyanin production was detected [ 32 ] . More flavonoid metabolic compounds were detected in yellow and white flowers of safflower compared to red flowers [ 33 ] . All- trans -violaxanthin and total carotenoid were the main factors affecting yellow flower color in Narcissus [ 34 ] . In the current study, we found that lutein and β-carotene are the main factors contributing the formation of yellow colored petals in alfalfa. Key candidate genes responsible for petal color formation in alfalfa The flavonoid synthesis pathway is relatively conserved, and it has been well studied in Arabidopsis and other model plants [ 16 , 35 ] . However, the transcription levels of structural genes and transcription factors involved in the anthocyanin biosynthesis pathway differ among plants or in the same plant at different developmental stages or in different tissues, thus affecting the formation of flower colors. In the current study, genes involved in the anthocyanin biosynthesis pathway were identified in alfalfa, including enzymatic genes 4CL (2), F3'H (3), DFR (6) and UGT (6), and transcription factor genes bHLH (2) and MYB (8). Differential expression of an ANS gene regulated by MYB1 affects the morphs that differ solely in cyanidin pigments, making black, red and white floral morphs in the Alpine orchid [ 27 ] . CgsMYB12 is an R2R3-MYB transcription factor responsible for anthocyanin pigmentation of the basal region (‘cup’) in the petal of C. gracilis ssp. sonomensis [ 36 ] . In Chrysanthemum morifolium , the difference in petal color is caused by the expression variation of the anthocyanin biosynthesis gene CmMYB6 ; after demethylation of the CmMYB6 promoter using the dCas9-TET1cd system, the flower color returns from yellow to pink [ 37 ] . Over-expression of CsMYB5-1 and CsMYB5-2 slightly lighten the color of the flowers in alfalfa [ 38 ] . MYBs are also involved in carotenoid accumulation. WHITE PETAL1 (WP1) is an anthocyanin-related R2R3-MYB protein that plays a critical role in regulating floral carotenoid pigmentation in Medicago truncatula [ 39 ] . In the current study, eight MYBs ( MS.gene67361 , MS.gene84326 , MS.gene61126 , MS.gene48572 , MS.gene26169 , MS.gene32128 , MS.gene61428 and MS.gene005866 ) were identified to be involved in the formation of purple petals. These MYB genes together with the enzymatic genes and the bHLH genes play important roles in color formation in alfalfa flowers, but their specific functions need to be studied further. Taken together, we propose a possible model explaining the formation of heterochromatic flowers in alfalfa. The difference in petal color between white (line 1), purple (line 3 and 4) and blue (line 5) petals was mainly caused by the difference in 4CL , F3’H , DFR , UGT , bHLH and MYB expression levels and consequently the accumulation of delphinidin-3,5-O-diglucoside (Fig. 7 ). The difference in petal color between white (line 1) and yellow (line 2) petals was mainly affected by the genes PSY , LcyE , lcyB and ZEP , and consequently the production of lutein and β-carotene, resulting in the change of flower color from white to yellow (Fig. 7 ). Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are included in this article and available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National Natural Science Foundation of China (U1906201). Author contributions W.ZY., Y.G. and W.Z. designed and planned the experiments, W.Z., L.H., Z.Q., L.X., J.K. and B.Q. performed research, W.Z. analyzed the data, W.Z. wrote—original draft preparation and W.ZY., Y.G., Z.Z., W.Z. wrote—review and editing. All authors have read and agreed to the published version of the manuscript. Acknowledgments We would like to express special gratitude to all the personnel who supported or helped with this study. References Gigord LD, Macnair MR, Smithson A. Negative frequency-dependent selection maintains a dramatic flower color polymorphism in the rewardless orchid Dactylorhiza sambucina (L.) Soo. Proc Natl Acad Sci U S A. 2001;98(11):6253–5. Guitián JA, Sobral M, Veiga T, Losada M, Guitián P, Guitián JM. Differences in pollination success between local and foreign flower color phenotypes: a translocation experiment with Gentiana lutea (Gentianaceae). PeerJ. 2017;5:e2882. 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Identification of the regulatory networks and hub genes controlling alfalfa floral pigmentation variation using RNA-sequencing analysis. BMC Plant Biol 2020, 20(1). Luo X, Sun D, Wang S, Luo S, Fu Y, Niu L, Shi Q, Zhang Y. Integrating full-length transcriptomics and metabolomics reveals the regulatory mechanisms underlying yellow pigmentation in tree peony ( Paeonia suffruticosa Andr.) flowers. Hortic Res 2021, 8(1). Wang R, Ren C, Dong S, Chen C, Xian B, Wu Q, Wang J, Pei J, Chen J. Integrated metabolomics and transcriptome analysis of flavonoid biosynthesis in safflower ( Carthamus tinctorius L.) with different colors. Front Plant Sci 2021, 12. Gómez-Gómez L, Li X, Lu M, Tang D, Shi Y. Composition of carotenoids and flavonoids in Narcissus cultivars and their relationship with flower color. PLoS ONE 2015, 10(11). Jun JH, Xiao X, Rao X, Dixon RA. Proanthocyanidin subunit composition determined by functionally diverged dioxygenases. Nat Plants. 2018;4(12):1034–43. Lin RC, Rausher MD. R2R3-MYB genes control petal pigmentation patterning in Clarkia gracilis ssp. sonomensis (Onagraceae). New Phytol. 2021;229(2):1147–62. Tang M, Xue W, Li X, Wang L, Wang M, Wang W, Yin X, Chen B, Qu X, Li J, et al. Mitotically heritable epigenetic modifications of CmMYB6 control anthocyanin biosynthesis in chrysanthemum. New Phytol. 2022;236(3):1075–88. Zhang H, Zheng G, Fan C, Di S, Wang X, Gao L, Dzyubenko N, Chapurin V, Pang Y. Ectopic expression of tea MYB genes alter spatial flavonoid accumulation in alfalfa ( Medicago sativa ). PLoS ONE 2019, 14(7). Meng Y, Wang Z, Wang Y, Wang C, Zhu B, Liu H, Ji W, Wen J, Chu C, Tadege M, et al. The MYB activator WHITE PETAL1 associates with MtTT8 and MtWD40-1 to regulate carotenoid-derived flower pigmentation in Medicago truncatula . Plant Cell. 2019;31(11):2751–67. Additional Declarations No competing interests reported. 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20:31:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1816206,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4239305/v1/a17152f1-e21d-45eb-943f-f539e189a67b.pdf"},{"id":55013623,"identity":"71b10091-139c-48ae-90af-558f4bbd7f2c","added_by":"auto","created_at":"2024-04-19 21:15:11","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":86486182,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4239305/v1/fe97bd2a82071c2a669c243d.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated analyses of the mechanism of flower color formation in alfalfa (Medicago sativa)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFlower color is an important trait for plants as visual signaling to attract pollinators and ensure their reproductive success\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Some floral species, such as orchids, are characterized by their fantastic floral morphology and high levels of corolla color polymorphism and variability, which have played an important role in pollination\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. As one of the most important forages in the world, alfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e L., 2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;32) is a typical cross-pollinated plant with a high degree of self-incompatibility, attracting insect pollinators using its bright flowers\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFlavonoids and carotenoids are the two major floral pigments, and anthocyanins constitute the largest group of flavonoids. The biosynthesis pathway of anthocyanins, which affect plant flower colors, is regulated by many genes. \u003cem\u003e4CL\u003c/em\u003e (\u003cem\u003e4-coumarate: CoA ligase\u003c/em\u003e), \u003cem\u003eCHS\u003c/em\u003e (\u003cem\u003echalcone synthase\u003c/em\u003e), \u003cem\u003eCHI\u003c/em\u003e (\u003cem\u003echalcone isomerase\u003c/em\u003e), \u003cem\u003eF3'H\u003c/em\u003e (\u003cem\u003eflavonoid 3'-hydroxylase\u003c/em\u003e), \u003cem\u003eF3'5'H\u003c/em\u003e (\u003cem\u003eflavonoid 3', 5'-hydroxylase\u003c/em\u003e) and \u003cem\u003eDFR\u003c/em\u003e (\u003cem\u003edihydroflavonol reductase\u003c/em\u003e) are important enzymatic genes in the upstream anthocyanin synthesis pathway. Among them, \u003cem\u003e4CL\u003c/em\u003e not only participates in the biosynthesis of anthocyanin but also is involved in lignin formation and the production of other phenolic compounds\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eF3'H\u003c/em\u003e and \u003cem\u003eF3'5'H\u003c/em\u003e can change the type of flavonoids, transforming naringenin/dihydrokaempferol into eriodictyol/dihydroquercetin and dihydrotricetin/ dihydromyricetin\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Blocking the synthesis of \u003cem\u003eF3'5'H\u003c/em\u003e affects the accumulation and reduction of anthocyanins, leading to a light flower color in pea (\u003cem\u003ePisum sativum\u003c/em\u003e)\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Arabidopsis \u003cem\u003eDFR\u003c/em\u003e mutant prevents dihydroplavonones from being reduced to leucopelargonidin, making the seed coat white, similar to the \u003cem\u003ef3'h\u003c/em\u003e mutant phenotype\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. At the downstream of the anthocyanin biosynthesis pathway, anthocyanidins can form anthocyanins through glycosylation, methylation and acylation reactions under the action of various UDP-glycosyltransferases (\u003cem\u003eUGTs\u003c/em\u003e). This process makes anthocyanins more stable in property, more abundant in color and more complex in structure.\u003c/p\u003e \u003cp\u003eGenes in the downstream of anthocyanin biosynthesis are regulated by the ternary complexes of transcription factors, named MYB-bHLH-WD40 (MBW). These MBW complexes include MYB (such as PAP1 and TT2), bHLH (such as TT8, GL3 and EGL3) and WD40 proteins (such as TTG1)\u003csup\u003e[\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The conservatism of the interaction of MYB, bHLH and WD40 in controlling proanthocyanidins (PAs) and anthocyanin biosynthesis has been verified in many plants\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. MBW usually targets with the promoters of structural genes to activate the transcription of these genes in plants\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. MtTT8 interacts with MYB and MtWD40-1 to form a regulatory complex that regulates the anthocyanin biosynthesis genes and affects the seed coat and flower color in \u003cem\u003eMedicago truncatula\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Allelic variations in the promoters between \u003cem\u003eGhTT19\u003c/em\u003eLW and \u003cem\u003eGhTT19\u003c/em\u003eLR, rather than genic regions, are the genetic cause of petal color variations in cotton\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFlavonoids, especially anthocyanins, help to produce a variety of flower colors ranging from light yellow to orange, red, blue, purple and dark purple, whereas carotenoids provide yellow, orange and bright red colors to flowers. With C6-C3-C6 as the carbon skeleton, anthocyanins form the basic structures of delphinidin, peonidin, petudinin, malvidin, pelargonidin and cyanidin through different combinations, which react with different sugar, methyl and acyl groups to produce more than 600 anthocyanins\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The main forms of anthocyanins vary among plant species and in different organs. Cyanidins and pelargonidins are the main pigments in red plant organs, while delphinidins, petudinins and malvidins are important chromogenic substances in many blue/purple plant organs\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo understand the key metabolic compounds and the associated gene expression networks controlling flower color formation in alfalfa, multiple lines with contrasting flower colors were analyzed by metabolic profiling and RNA-seq.\u0026nbsp;Combined analysis revealed specific compounds and genes affecting flower color formation in alfalfa. This study provides new insights into the biochemical and molecular mechanisms associated with the biosynthesis and regulation of anthocyanins and carotenoids in alfalfa flowers.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003eAlfalfa lines of the \u0026ldquo;Wudi\u0026rdquo; population used in this study were grown at Wudi county (117.97, 37.92), Jiaozhou Experimental Station (120.10, 36.31) and Qingdao Agricultural University (120.40, 36.32), Shandong province, China. Petals were collected from fully opened petals, and 3.0 g of the collected petals per sample was used for metabolic analysis and RNA-seq analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMetabolic analysis\u003c/h2\u003e \u003cp\u003eThe petal samples were freeze-dried and ground into powder (30 Hz, 1.5 min). Then, 50 mg of the powdered sample was weighed, used for extraction with 0.5 mL methanol/water/hydrochloric acid (500:500:1, V/V/V), and vortexed and ultrasonicated for 5 min. The samples were then centrifuged at 12,000 g for 3 min at 4\u0026deg;C. The supernatant of each sample was filtered through a membrane filter (0.22 \u0026micro;m, Anpel). The sample extracts of anthocyanins and flavonoids were analyzed using an UPLC-ESI-MS/MS system, and carotenoids were analyzed using an UPLC-APCI-MS/MS system (UPLC, ExionLC\u0026trade; AD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sciex.com.cn/\u003c/span\u003e\u003cspan address=\"https://sciex.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; MS, Applied Biosystems 6500 Triple Quadrupole, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sciex.com.cn/\u003c/span\u003e\u003cspan address=\"https://sciex.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The anthocyanin, flavonoid and carotenoid contents were detected using MetWare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.metware.cn/\u003c/span\u003e\u003cspan address=\"http://www.metware.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) based on the AB Sciex QTRAP 6500 LC-MS/MS platform.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the alfalfa petals using the TRIzol\u0026reg; Reagent Kit according to the manufacturer\u0026rsquo;s instructions (Invitrogen). RNA quality was determined by 2100 Bioanalyzer (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). RNA-seq transcriptome libraries were sequenced using the Illumina HiSeq xten/NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA). The clean reads were mapped and annotated following the reference genome with orientation mode using the HISAT2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ccb.jhu.edu/software/hisat2/index.shtml\u003c/span\u003e\u003cspan address=\"http://ccb.jhu.edu/software/hisat2/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) software\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Essentially, differential expression analysis was performed using the DESeq2/DEGseq/EdgeR with Q value\u0026thinsp;\u0026le;\u0026thinsp;0.05, and DEGs with |log2FC| \u0026gt; 1 and Q value\u0026thinsp;\u0026le;\u0026thinsp;0.05 (DESeq2 or EdgeR)/Q value\u0026thinsp;\u0026le;\u0026thinsp;0.001 (DEGseq) were significant DEGs. KEGG functional enrichment and pathway analyses were carried out using Goatools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tanghaibao/Goatools\u003c/span\u003e\u003cspan address=\"https://github.com/tanghaibao/Goatools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and KOBAS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://kobas.cbi.pku.edu.cn/home.do\u003c/span\u003e\u003cspan address=\"http://kobas.cbi.pku.edu.cn/home.do\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eCluster analysis and variance analysis were performed using SPSS. Principal component analysis was performed using MetWare.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of alfalfa lines with different flower colors\u003c/h2\u003e \u003cp\u003eIn contrast to the common \u0026ldquo;lavender\u0026rdquo; flowers in alfalfa, the \u0026ldquo;Wudi\u0026rdquo; population has amazing color polymorphism, including white (cream-colored), yellow, lavender (purple), dark purple, dark blue and some intermediate colors. Growing the materials in distant locations did not affect the flower color, and the coloration remained consistent throughout the perennial life cycle of the plants. Thus, variation in floral color is likely genetically determined rather than a result of, for example, soil chemistry. Lines 1 (white/cream-colored), 2 (yellow), 3 (lavender/purple), 4 (dark purple) and 5 (dark blue) with obvious color differences were selected from the \u0026ldquo;Wudi\u0026rdquo; population (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). The colors of the style, stigma, ovary, filament and anther were indistinguishable after removing the petals (Supplementary Fig.\u0026nbsp;1). Therefore, the pigment difference in alfalfa flowers lays on the petal rather than other organs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the petal color more clearly, the petal colors of lines 1\u0026ndash;5 were measured with a getcolor. The petal colors of fully opened flowers were calibrated on the red, green and blue (RGB) scale, and the specific RGB values of petals 1\u0026ndash;5 were determined as #eae9f7, #f9f27a, #8c5d98, #82217c and #4e4370, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). According to the cyan, magenta, yellow and black (CMYK) color scale (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.colorhexa.com\u003c/span\u003e\u003cspan address=\"https://www.colorhexa.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), #eae9f7 (line 1) is composed of 5.3% cyan, 5.7% magenta, 0% yellow and 3.1% black; #f9f27a (line 2) is 0% cyan, 2.8% magenta, 51% yellow and 2.4% black; #8c5d98 (line 3) is 7.9% cyan, 38.8% magenta, 0% yellow and 40.4% black; #82217c (line 4) consists of 0% cyan, 74.6% magenta, 4.6% yellow and 49% black; and #4e4370 (line 5) is 30.4% cyan, 40.2% magenta, 0% yellow and 56.1% black (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). This result suggests that the lavender, dark purple and dark blue petal colors of lines 3\u0026ndash;5 are caused by the addition of different degrees of purple obtained from the blending of magenta and black to the white base of line 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003eCell morphology of the deepest colored regions of the petals was observed by scanning electron microscope (SEM) to determine the influence of epidermal cells on petal color. No significant difference was found in petal epidermal cell structures between the five lines (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnthocyanin content is much higher in purple and blue petals\u003c/h2\u003e \u003cp\u003eMetabolic analysis of flavonoid, anthocyanin and carotenoid was conducted using petals collected from different colored alfalfa lines. The total ion flow map (TIC) obtained from QC samples indicated the sum of all ion strengths at different time points (Supplementary Fig.\u0026nbsp;3). In the detection multipeak map (XIC), the chromatographic peaks of different colors corresponded to different carotenoid, flavonoid and anthocyanin metabolites (Supplementary Fig.\u0026nbsp;4). The superposition of the TIC map of the QC samples showed that the response intensity and retention time of each peak strongly overlapped, indicating that the data were reliable (Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003ePrincipal component analysis (PCA) of all pigment metabolites and flavonoids did not place the samples into groups consistent with petal colors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). However, PCA of anthocyanins placed the samples into three groups consistent with petal colors. Lines 1 and 2 were in the light color group, and line 3 was in the normal color group, while lines 4 and 5 were in the dark color group, suggesting that anthocyanins play a critical role in purple and blue color determination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eContents of different pigments were analyzed in the petals. The results showed that anthocyanin content in purple (lines 3 and 4) and blue petals (line 5) was significantly higher than petals of other colors (lines 1 and 2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); flavones and flavonols content in purple and blue petals was significantly lower than white color petals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The content of anthocyanins accounted for 6.37%, 8.79%, 86.05%, 93.90% and 93.82% of the total pigment content in lines 1, 2, 3, 4 and 5, respectively. No significant difference was observed in anthocyanin content between lines 1 and 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The content of anthocyanins in lines 3, 4 and 5 was 73.53, 122.83 and 117.66 times that of line 1, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The content of anthocyanins in lines 4 and 5 was 1.67 and 1.60 times that of line 3, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results indicated that the difference in petal color between white and purple/blue was specifically caused by the difference in anthocyanin content. Thus, anthocyanin biosynthesis is crucial for color heterogeneity in alfalfa.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePigment content in alfalfa lines (1\u0026ndash;5) with different petal colors\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnthocyanidins\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavones\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFlavonols\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCarotenoids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFlavanols\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProcyanidins\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eXanthophylls\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePhenonic acids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eIsoflavanones\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eChalcones\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTotal carotenoids\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.33 C d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e482.82 A a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e433.98 A a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.09 E e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.38 C c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.63E-02 A a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22.02 C c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.17 C d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.02 AB a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.54 C c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e25.11 C e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.99 C d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e404.08 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e363.68 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.81 A a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17 D d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.56E-03 C cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e44.13 A a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.30 A a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.54 C d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.17 D d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e58.94 A a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4730.31 B c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e394.62 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e339.02 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.52 D d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.02 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.26E-02 AB b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24.04 C c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.18 C d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.91 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3.42 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e27.55 C d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7902.30 A a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e206.55 C c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e252.62 C c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.72 C c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.95 A a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.44E-03 C d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e41.27 AB b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.25 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.07 A a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e4.28 A a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e46.99 B c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7569.34 A b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e167.05 D d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e275.39 C c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.41 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.99 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.12E-02 BC bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e39.26 B b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.22 B c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.65 C c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.46 E e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e52.68 AB b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDelphinidin-3,5-\u003c/b\u003e \u003cb\u003eO\u003c/b\u003e \u003cb\u003e-diglucoside is the key pigment metabolite affecting the purple and blue color of alfalfa flowers\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAccording to the basic structure of anthocyanins, a total of 52 anthocyanins were identified in alfalfa petals, including 14 cyanidins, 11 delphinidins, 7 malvidins, 8 pelargonidins, 6 peonidins and 7 petunidins (Supplementary Table\u0026nbsp;1). The content of delphinidins accounted for 5.51%, 6.84%, 58.58%, 66.76% and 64.04% of the total pigment content in lines 1, 2, 3, 4 and 5, respectively (Supplementary Fig.\u0026nbsp;6a). According to the functional groups, 9 types of anthocyanins were detected, including anthocyanidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside, anthocyanidin-3-\u003cem\u003eO\u003c/em\u003e-glucoside, anthocyanidin-3-\u003cem\u003eO\u003c/em\u003e-(6-\u003cem\u003eO\u003c/em\u003e-p-coumaroyl)-glucoside, anthocyanidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside, anthocyanidin-3-\u003cem\u003eO\u003c/em\u003e-sophoroside, anthocyanidin-3-\u003cem\u003eO\u003c/em\u003e-sambubioside, anthocyanidin-3-\u003cem\u003eO\u003c/em\u003e-(6-\u003cem\u003eO\u003c/em\u003e-malonyl-beta-D-glucoside), delphinidin-3-\u003cem\u003eO\u003c/em\u003e-rutinoside-5-\u003cem\u003eO\u003c/em\u003e-glucoside and cyanidin-3,5,3'-\u003cem\u003eO\u003c/em\u003e-triglucoside. Anthocyanidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside catalyzed by UGT75C1 had the most significant difference between line 1 and lines 3/4/5. The content of anthocyanidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside accounted for 5.61%, 7.28%, 78.79%, 84.04% and 86.74% of the total pigment content in lines 1, 2, 3, 4 and 5, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, Supplementary Fig.\u0026nbsp;6b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs one of anthocyanidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucosides, delphinidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside content in lines 1 and 2 was only about 5.26% and 6.38% of total pigment, while in lines 3, 4 and 5, the delphinidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside content was 56.90%, 63.62% and 61.94% of total pigment, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The content of delphinidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside in lines 3, 4 and 5 was 58.88, 100.80 and 94.07 times that of line 1, respectively. Cluster analysis of all anthocyanins and total pigments showed that delphinidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside was closest to the total pigments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Therefore, delphinidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside was the key pigment causing the purple or bule petal color in alfalfa.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAlfalfa petal color is affected by the expression of flavonoid biosynthesis-related pathway genes\u003c/h2\u003e \u003cp\u003eRNA-seq analysis was conducted using petals collected from different colored alfalfa lines. More than 40\u0026nbsp;million raw reads were generated from each library, and 88\u0026ndash;93% of the trimmed reads were uniquely mapped to the alfalfa (cultivar \u0026ldquo;XinJiangDaYe\u0026rdquo;) reference genome (Supplementary Table\u0026nbsp;2). The differentially expressed genes (DEGs) were analyzed by DESeq software, and the selected DEGs (|log2FC| \u0026ge; 1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were then compared and analyzed. Compared to the line 1 control, 11,300, 15,137, 10,414 and 21,186 genes were differentially expressed in lines 2, 3, 4 and 5, respectively, of which 6486, 6942, 5950 and 10,965, respectively, were upregulated and 4814, 8195, 4464 and 10221, respectively, were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In general, there were more upregulated DEGs than downregulated DEGs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe upregulated DEGs identified in the 1 vs 2\u0026ndash;5 comparison groups were involved in 120\u0026ndash;122 KEGG metabolic pathways, and 24, 24, 15 and 23 significantly enriched metabolic pathways were identified in the 1 vs 2, 1 vs 3, 1 vs 4 and 1 vs 5 groups, respectively. Among them, flavone and flavonol biosynthesis (ko00944) and carotenoid biosynthesis (ko00906) were significantly enriched in 1 vs 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Flavone and flavonol biosynthesis (ko00944) and flavonoid biosynthesis (ko00941) were all significantly enriched in 1 vs 3 and 1 vs 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u0026ndash;d). Thus, flavonoid biosynthesis-related pathways may play an important role in petal color.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of DEGs related to flavonoid and anthocyanin biosynthesis pathway\u003c/h2\u003e \u003cp\u003eCompared to line 1, 1754 genes were expressed in pathways related to flavonoid biosynthesis. Among them, 212 genes were involved in anthocyanin biosynthesis, 778 genes belonged to UGT family, and 764 genes related to MBW proteins (439 \u003cem\u003eMYB\u003c/em\u003e and \u003cem\u003eMYB\u003c/em\u003e-related genes, 194 \u003cem\u003ebHLH\u003c/em\u003e and 131 \u003cem\u003eWD40\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;7).\u003c/p\u003e \u003cp\u003eBased on KEGG enrichment analysis and petal color differences, 27 DEGs (every sample FPKM\u0026thinsp;\u0026gt;\u0026thinsp;1, |log2FC| \u0026ge; 1 in 1 vs 3, 1 vs 4 and 1 vs 5 groups) were significantly enriched in anthocyanin biosynthesis-related pathways. These 27 DEGs include 2 \u003cem\u003e4CL\u003c/em\u003e, 3 \u003cem\u003eF3'H\u003c/em\u003e, 6 \u003cem\u003eDFR\u003c/em\u003e, 6 \u003cem\u003eUGT\u003c/em\u003e, 2 \u003cem\u003ebHLH\u003c/em\u003e and 8 \u003cem\u003eMYB\u003c/em\u003e genes, which are considered candidate genes regulating purple petal color in alfalfa (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLutein and β-carotene are the main pigment metabolites affecting the yellow petal color in alfalfa\u003c/h2\u003e \u003cp\u003eFinally, we analyzed the cause of the formation of yellow petals in alfalfa. According to RNA-seq analysis, upregulated DGEs were enriched in carotenoid biosynthesis pathway and flavone and flavonol biosynthesis pathway in the 1 vs 2 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Compared to white colored line 1, the content of flavones and flavonols was significantly lower (16.31% and 16.20%, respectively) in yellow colored line 2, while the content of total carotenoids was 2.35 times that of line 1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, carotenoids play a key role in determining the yellow petal color in alfalfa.\u003c/p\u003e \u003cp\u003eCompared to line 1, the top seven carotenoids (lutein, β-carotene, zeaxanthin, (E/Z)-phytoene, violaxanthin, neoxanthin and β-cryptoxanthin) were significantly higher in line 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). In the carotene category, the content of β-carotene was the highest, accounted for 18.05% of total carotenoids (total carotenes\u0026thinsp;+\u0026thinsp;total xanthophylls) in line 2, and it was 4.29 times that of line 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Among the xanthophylls, lutein content was the highest, accounted for 38.04% of total carotenoids in line 2, and it was 1.89 times that of line 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Therefore, lutein and β-carotene were the main metabolites leading to the yellow petal color in alfalfa. Compared to line 1, 6 \u003cem\u003ePSY\u003c/em\u003e, 1 \u003cem\u003eLcyE\u003c/em\u003e, 1 \u003cem\u003eLcyB\u003c/em\u003e and 6 \u003cem\u003eZEP\u003c/em\u003e genes were significantly upregulated in line 2 and likely to be involved in regulating the formation of yellow petals in alfalfa (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussions","content":"\u003cp\u003eVariation in flower color plays an important ecological role by attracting pollinators and influencing reproductive success in flowering plants, particularly for plants with a high degree of self-incompatibility, such as alfalfa. Alfalfa is a widely cultivated forage crop around the world due to its remarkable adaptability, high biomass yield, exceptional nutritive value and notable capacity for nitrogen fixation\u003csup\u003e[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Although various flower colors have been observed and described in alfalfa a long time ago\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, the biochemical and molecular mechanism of its color formation is still unclear. By analyzing alfalfa lines with five contrasting flower colors, various kinds and levels of anthocyanins, carotenoids and other flavonoids were detected in different colored petals, and their roles in color formation were revealed. Genes responsible for specific biochemical pathways and compounds associated with color formation were also revealed. This study provides an in-depth interpretation of the flower coloration mechanism in alfalfa.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eKey metabolic compounds affecting petal color formation in alfalfa\u003c/h2\u003e \u003cp\u003eThe presence of specific types of anthocyanins affects flower colors. For example, cyanidin 3-glucoside and cyanidin 3-(6-malonyglucoside) are dominant pigments in the black-colored flowers in Alpine orchid \u003cem\u003eGymnadenia rhellicani\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e; maintaining both high relative and absolute content of cyanidin 3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside may be a prerequisite for the formation of red petals in \u003cem\u003eRosa rugosa\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVarious types of anthocyanins have reported in the formation of purple and blue flowers. Cyanidin 3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside, malvidin 3,5-diglucoside and cyanidin 3-\u003cem\u003eO\u003c/em\u003e-galactoside were found to be responsible for the purple flower color in \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. High levels of malvidin-3,5-di-\u003cem\u003eO\u003c/em\u003e-glucoside were detected in purple flowers in \u003cem\u003eLagerstroemia indica\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Significantly higher content of delphinidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside was detected in 8\u0026ndash;16 deep purple\u0026ndash;red-flowered germplasm resources with somewhat unique and visible blue hue in \u003cem\u003eR. rugosa\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the current study, we found that delphinidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside is the primary anthocyanin responsible for the formation of both purple and blue colored petals in alfalfa. This conclusion is consistent with work of Wang et al. (2022) in \u003cem\u003eR. rugosa\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, but different from that of Duan et al. (2020) in alfalfa\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, which used only two different colored lines.\u003c/p\u003e \u003cp\u003eThe formation of yellow flowers is considered to come from flavonoids or carotenoids. The yellow-colored tree peony flower displayed relatively higher contents of tetrahydroxychalcone (THC), flavones, and flavonols compared to purple\u0026ndash;red flowers, but no anthocyanin production was detected\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. More flavonoid metabolic compounds were detected in yellow and white flowers of safflower compared to red flowers\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. All-\u003cem\u003etrans\u003c/em\u003e-violaxanthin and total carotenoid were the main factors affecting yellow flower color in Narcissus\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. In the current study, we found that lutein and β-carotene are the main factors contributing the formation of yellow colored petals in alfalfa.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eKey candidate genes responsible for petal color formation in alfalfa\u003c/h2\u003e \u003cp\u003eThe flavonoid synthesis pathway is relatively conserved, and it has been well studied in \u003cem\u003eArabidopsis\u003c/em\u003e and other model plants\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. However, the transcription levels of structural genes and transcription factors involved in the anthocyanin biosynthesis pathway differ among plants or in the same plant at different developmental stages or in different tissues, thus affecting the formation of flower colors. In the current study, genes involved in the anthocyanin biosynthesis pathway were identified in alfalfa, including enzymatic genes \u003cem\u003e4CL\u003c/em\u003e (2), \u003cem\u003eF3'H\u003c/em\u003e (3), \u003cem\u003eDFR\u003c/em\u003e (6) and \u003cem\u003eUGT\u003c/em\u003e (6), and transcription factor genes \u003cem\u003ebHLH\u003c/em\u003e (2) and \u003cem\u003eMYB\u003c/em\u003e (8).\u003c/p\u003e \u003cp\u003eDifferential expression of an \u003cem\u003eANS\u003c/em\u003e gene regulated by \u003cem\u003eMYB1\u003c/em\u003e affects the morphs that differ solely in cyanidin pigments, making black, red and white floral morphs in the Alpine orchid \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eCgsMYB12\u003c/em\u003e is an R2R3-MYB transcription factor responsible for anthocyanin pigmentation of the basal region (\u0026lsquo;cup\u0026rsquo;) in the petal of \u003cem\u003eC. gracilis ssp. sonomensis\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eChrysanthemum morifolium\u003c/em\u003e, the difference in petal color is caused by the expression variation of the anthocyanin biosynthesis gene \u003cem\u003eCmMYB6\u003c/em\u003e; after demethylation of the \u003cem\u003eCmMYB6\u003c/em\u003e promoter using the dCas9-TET1cd system, the flower color returns from yellow to pink\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Over-expression of \u003cem\u003eCsMYB5-1\u003c/em\u003e and \u003cem\u003eCsMYB5-2\u003c/em\u003e slightly lighten the color of the flowers in alfalfa\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. MYBs are also involved in carotenoid accumulation. WHITE PETAL1 (WP1) is an anthocyanin-related R2R3-MYB protein that plays a critical role in regulating floral carotenoid pigmentation in \u003cem\u003eMedicago truncatula\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. In the current study, eight \u003cem\u003eMYBs\u003c/em\u003e (\u003cem\u003eMS.gene67361\u003c/em\u003e, \u003cem\u003eMS.gene84326\u003c/em\u003e, \u003cem\u003eMS.gene61126\u003c/em\u003e, \u003cem\u003eMS.gene48572\u003c/em\u003e, \u003cem\u003eMS.gene26169\u003c/em\u003e, \u003cem\u003eMS.gene32128\u003c/em\u003e, \u003cem\u003eMS.gene61428\u003c/em\u003e and \u003cem\u003eMS.gene005866\u003c/em\u003e) were identified to be involved in the formation of purple petals. These \u003cem\u003eMYB\u003c/em\u003e genes together with the enzymatic genes and the \u003cem\u003ebHLH\u003c/em\u003e genes play important roles in color formation in alfalfa flowers, but their specific functions need to be studied further.\u003c/p\u003e \u003cp\u003eTaken together, we propose a possible model explaining the formation of heterochromatic flowers in alfalfa. The difference in petal color between white (line 1), purple (line 3 and 4) and blue (line 5) petals was mainly caused by the difference in \u003cem\u003e4CL\u003c/em\u003e, \u003cem\u003eF3\u0026rsquo;H\u003c/em\u003e, \u003cem\u003eDFR\u003c/em\u003e, \u003cem\u003eUGT\u003c/em\u003e, \u003cem\u003ebHLH\u003c/em\u003e and \u003cem\u003eMYB\u003c/em\u003e expression levels and consequently the accumulation of delphinidin-3,5-O-diglucoside (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The difference in petal color between white (line 1) and yellow (line 2) petals was mainly affected by the genes \u003cem\u003ePSY\u003c/em\u003e, \u003cem\u003eLcyE\u003c/em\u003e, \u003cem\u003elcyB\u003c/em\u003e and \u003cem\u003eZEP\u003c/em\u003e, and consequently the production of lutein and β-carotene, resulting in the change of flower color from white to yellow (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are included in this article and available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (U1906201).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eW.ZY., Y.G. and W.Z. designed and planned the experiments, W.Z., L.H., Z.Q., L.X., J.K. and B.Q. performed research, W.Z. analyzed the data, W.Z. wrote\u0026mdash;original draft preparation and W.ZY., Y.G., Z.Z., W.Z. wrote\u0026mdash;review and editing.\u0026nbsp;All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express special gratitude to all the personnel who supported or helped with this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGigord LD, Macnair MR, Smithson A. 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Plant Cell. 2019;31(11):2751\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"alfalfa, Medicago sativa, flower color, anthocyanins, carotenoids, delphinidin-3,5-O-diglucoside","lastPublishedDoi":"10.21203/rs.3.rs-4239305/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4239305/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e) is one of the most valuable forages in the world. As an outcrossing species, it needs bright flowers to attract pollinators to deal with self-incompatibility. Although various flower colors have been observed and described in alfalfa a long time ago, the biochemical and molecular mechanism of its color formation is still unclear. By analyzing alfalfa lines with five contrasting flower colors including white (cream-colored), yellow, lavender (purple), dark purple and dark blue, various kinds and levels of anthocyanins, carotenoids and other flavonoids were detected in different colored petals, and their roles in color formation were revealed. Notably, the content of delphinidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside in lines 3, 4 and 5 was 58.88, 100.80 and 94.07 times that of line 1, respectively. Delphinidin-3,5-\u003cem\u003eO\u003c/em\u003e-diglucoside was the key factor for purple and blue color formation. Lutein and β-carotene were the main factors for the yellow color formation. By analyzing differentially expressed genes responsible for specific biochemical pathways and compounds, 27 genes were found to be associated with purple and blue color formation, and 14 genes were found to play an important role in yellow color formation. These findings provide a basis for understanding the biochemical and molecular mechanism of alfalfa flower color formation.\u003c/p\u003e","manuscriptTitle":"Integrated analyses of the mechanism of flower color formation in alfalfa (Medicago sativa)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-19 21:15:01","doi":"10.21203/rs.3.rs-4239305/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":"bf63ecf6-47b1-4d37-a370-e5d1c7c38ff7","owner":[],"postedDate":"April 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-17T20:23:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-19 21:15:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4239305","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4239305","identity":"rs-4239305","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}