Elucidation of the Core Betalain Biosynthesis Pathway in Amaranthus tricolor

Elucidation of the Core Betalain Biosynthesis Pathway in Amaranthus tricolor | 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 Elucidation of the Core Betalain Biosynthesis Pathway in Amaranthus tricolor Yu-Cheng Chang, Yi-Ching Chiu, Nai-Wen Tsao, Yuan-Lin Chou, Choon-Meng Tan, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-110761/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2021 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Amaranthus tricolor L., a vegetable Amaranthus species, is an economically important crop containing large amounts of betalains. Betalains are natural antioxidants and can be classified into betacyanins and betaxanthins, with red and yellow colors, respectively. A. tricolor cultivars with varying betalain contents, leading to striking red to green coloration, have been commercially produced. However, the molecular differences underlying betalain biosynthesis in various cultivars of A. tricolor remain largely unknown. In this study, A. tricolor cultivars with different colors were chosen for comparative transcriptome analysis. The elevated expression of AmCYP76AD1 in a red-leaf cultivar of A. tricolor was proposed to play a key role in producing red betalain pigments. The functions of AmCYP76AD1, AmDODAa1, AmDODAa2, and AmcDOPA5GT were also characterized through the heterologous engineering of betalain pigments in Nicotiana benthamiana. Moreover, high and low L-DOPA 4,5-dioxygenase activities of AmDODAa1 and AmDODAa2, respectively, were confirmed through in vitro enzymatic assays. Thus, comparative transcriptome analysis combined with functional and enzymatic studies allowed the construction of a core betalain biosynthesis pathway of A. tricolor. These results not only provide novel insights into betalain biosynthesis and evolution in A. tricolor but also provide a basal framework for examining genes related to betalain biosynthesis among different species of Amaranthaceae. Accession numbers: The nucleotide sequences reported in this article have been submitted to [GenBank] under accession numbers [MT740230, MT741954, MT741955, MT741956]. General Biochemistry Epigenetics & Genomics Bioinformatics Amaranthus tricolor betalain CYP76AD1 DODA cDOPA5GT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Betalains are classified into betacyanins and betaxanthins, which provide red-violet and yellow coloration, respectively 1 . Similar to anthocyanins, betalains exhibit antioxidant activity in the form of free-radical scavenging and accumulate in response to different stresses, such as UV-B radiation, high-intensity light, salinity, heat, and drought 2,3,4,5 . In addition to their potential roles in protecting plants against abiotic stresses, betalains play a role in defense against pathogenic fungi 6 . Moreover, as water-soluble natural pigments, betalains are widely used as food additives because of their health-promoting properties and color stability over a wide range of pH values 7,8 . Betalains occur only in Caryophyllales and have never been detected in anthocyanin-producing plants 9,10 . Although the molecular basis for the mutual exclusion of betalains and anthocyanins is still unclear, breakthroughs in the identification of genes involved in betalain biosynthesis have shed light on the evolution of betalain pigmentation in Caryophyllales 9,10,11,12 . Unlike anthocyanins, which are derived from L-phenylalanine, betalains are synthesized from L-tyrosine 13,14 . Initially, L-tyrosine is hydroxylated to produce L-DOPA by tyrosinases encoded by CYP76AD1 and its orthologs 15,16 . L-DOPA can be converted into betalamic acid by L-DOPA 4,5-dioxygenase encoded by DODA , and betalamic acid can then spontaneously condense with amino acids to form betaxanthins 1,12,17 . Alternatively, L-DOPA can be converted into cyclo -DOPA through the oxidase activity of CYP76AD1, and cyclo -DOPA can then spontaneously condense with betalamic acid to form betanidin 9,18 . Betanidin is further glycosylated by betanidin 5- O -glucosyl-transferase encoded by B5GT to form betanin, the most common betacyanin. Glycosylation can also occur on cyclo -DOPA, catalyzed by cyclo -DOPA 5- O -glucosyltransferase encoded by cDOPA5GT , to produce cyclo -DOPA-glucoside, which then spontaneously condenses with betalamic acid to form betanin 10,19 . Phylogenetic analyses revealed that the CYP76AD and DODA genes, encoding key enzymes in the core biosynthetic pathway of betalains, are highly duplicated in Caryophyllales 10,20 . The CYP76AD gene lineage has undergone at least three duplication events, giving rise to three clades: CYP76ADa, CYP76ADb and CYP76ADg 9 . The CYP76ADa clade includes the CYP76AD1 and CYP76AD3 genes, whose products possess both the tyrosine hydroxylase and L-DOPA 4,5-dioxygenase activities required for L-DOPA and cyclo -DOPA formation, respectively 16 . The CYP76ADb clade includes the CYP76AD5 , CYP76AD6 , and CYP76AD15 genes, which possess only the tyrosine hydroxylase activity required for L-DOPA formation 16 . However, the functions of the genes in the CYP76ADg clade in betalain biosynthesis have not been determined. Duplication events in the DODA gene lineage also gave rise to two major clades: DODAa and DODAb. The function of DODAb is unknown, but the evolution of L-DOPA 4,5-dioxygenase activity in betalain-producing plants was proposed to be led by DODAa 9 . Nevertheless, only one paralogous gene from the DODAa clade shows high L-DOPA 4,5-dioxygenase activity in each species, and others exhibit barely detectable activity 12,21 . An increased understanding of the betalain biosynthesis pathway has facilitated the metabolic engineering of betalains, providing new sources for basic research studies and commercial applications 22,23 . For example, the fluorescent betaxanthins produced by the expression of MjDODA a 1 in yeast were used as chemical biosensors to reveal the tyrosine hydroxylase activity of BvCYP76AD1 15 . The production of semisynthetic betaxanthins by spontaneous condensation between fluorescent betalamic acid and the amino groups of proteins provides an alternative method for labeling proteins for multiple biological applications 24 . Fine-turning the content ratio of betacyanins and betaxanthins via the differential expression of BvCYP76AD1 and BvCYP76AD6 in non-Caryophyllales makes it possible to create a range of color patterns in the background of anthocyanin-producing plants 6 . The heterologous production of betalains via the coexpression of BvCYP76AD1 , BvDODA a 1 , and MjcDOPA5GT in tobacco enhances the resistance of transgenic plants to Botrytis cinereal infection 6 . In addition, betanin rice generated through the coexpression of meloS , BvCYP76AD1 , and BvDODA a 1 in rice endosperm shows higher antioxidant activity and provides a functional food 25 . Amaranth species are economically important crops containing large amounts of betalains. They can be classified into three categories: vegetable Amaranthus , grain Amaranthus , and weed Amaranthus species 26 . Amaranthus tricolor L., a vegetable Amaranthus species, is widely distributed in warm and tropical regions and is cultivated as a leafy vegetable. A. tricolor is rich in natural antioxidants and is able to tolerate abiotic stresses; it has been used as a traditional Chinese medicinal herb for the treatment of eruptive fever, pain, sore throat, dysentery, anemia, bronchitis, colic, etc. 27 . A. tricolor cultivars with various contents of betacyanins and betaxanthins, resulting in striking colors ranging from red to green, have been commercially produced 27,28 . However, the molecular basis underlying betalain biosynthesis in A. tricolor remains largely unknown. Recently, virus-induced gene silencing was applied to elucidate the function of CYP76AD1 in producing betalain pigments in A. tricolor 29 . In addition, a comparative analysis of a transcriptome database constructed from different leaf samples of A. tricolor cv. Dahong was performed to construct a putative metabolic pathway of betalains in A. tricolor 30 . Candidate genes encoding enzymes catalyzing the formation of L-DOPA, cyclo -DOPA, betalamic acid, cyclo -DOPA-glucoside, and betanin were obtained from a transcriptome database and showed higher expression levels in red areas of A. tricolor leaves than in in green areas 30 . However, functional characterization and enzyme activity analyses are still needed to elucidate the roles of these candidate genes in the betalain biosynthesis pathway of A. tricolor . In this study, A. tricolor cultivars with different colors were chosen for comparative transcriptome analysis. The key gene showing elevated expression in a red-leaf cultivar of A. tricolor was identified, and the results indicated that the dual activities of tyrosine hydroxylase and L-DOPA oxidase are important for producing red betalain pigments in A. tricolor . The core betalain biosynthesis pathway of A. tricolor was further constructed based on the functional characterization of betalain biosynthesis genes through the heterologous engineering of betalain pigments in Nicotiana benthamiana and in vitro enzymatic assays of L-DOPA 4,5-dioxygenase activities. These results provide novel insights into betalain biosynthesis and evolution in A. tricolor . Results AmCYP76AD1 is highly expressed in a red-leaf cultivar of A. tricolor A. tricolor cultivars are important leafy vegetables that display leaf colors ranging from red to green depending on the betalain content 28 . To elucidate the genetic factors that influence betalain pigment accumulation in red- and green-leaf cultivars of A. tricolor (hereafter referred to as AMR and AMG, respectively) (Fig. 1a, b, Supplementary Fig. S1a, b), specific primer pairs were designed based on available sequence information from the NCBI database or previously published studies to selectively examine the transcript levels of genes related to the betalain biosynthesis pathway by qRT-PCR (Supplementary Table S1). In 3-week-old A. tricolor , AmCYP76AD1 and AmPPO showed higher expression levels in AMR than in AMG (Fig. 1c). Notably, only AmCYP76AD1 exhibited a highly differential expression pattern, showing an ~200-fold difference between AMR and AMG. In contrast, AmDODA , AmcDOPA5GT , AmB5GT , AmUGT79B30-like 4 , AmMYB1 , AmADH , AmCATPO , and AmTyDC did not show a significant differential expression pattern between AMR and AMG (Fig. 1c). The highly differential expression pattern of AmCYP76AD1 between AMR and AMG was also observed in 4-week-old A. tricolor (Supplementary Fig. S1c). Moreover, as a key element in the initiation of the betalain biosynthesis pathway, AmCYP76AD1 transcript levels displayed a high correlation with betalain pigment contents (Fig. 2a, b). Further phylogenetic reconstruction and LOGO analysis revealed that AmCYP76AD1 belongs to the CYP76ADa clade (Fig. 2c, d), whose members possesses both the tyrosine hydroxylase and L-DOPA oxidase activities required for L-DOPA and cyclo -DOPA formation, respectively (Fig. 1d). These results suggest that the elevated expression of AmCYP76AD1 is necessary for betalain pigment accumulation, which leads to an obvious red-violet color in the leaves and stems of AMR, but not in those of AMG. AmDODA exhibits a marginal level of L-DOPA 4,5-dioxygenase activity Although candidate transcripts related to betalain biosynthesis were identified previously in A. tricolor 30,31 , their functional and enzymatic activities have not yet been characterized. To functionally characterize the enzyme activities of AmCYP76AD1, AmDODA, and AmcDOPA5GT in the core pathway of betalain biosynthesis (Fig. 1d), 35S promoter-driven cDNAs encoding C-terminal YFP- or FLAG (SFP)-tagged AmCYP76AD1, AmDODA, and AmcDOPA5GT were transiently coexpressed in N. benthamiana leaves by agroinfiltration. Upon expression, only a small amount of betalain pigment was produced in N. benthamiana leaves, which was barely detectable (Fig. 3a). In contrast, as a positive control, high production of betalain pigments with red-violet color was observed when the Beta vulgaris tyrosinase gene ( BvCYP76AD1 ), the B. vulgaris L-DOPA 4,5-dioxygenase gene ( BvDODA a 1 ), and the Mirabilis jalapa cyclo -DOPA 5- O -glucosyltransferase gene ( MjcDOPA5GT ), were coexpressed in N. benthamiana leaves (Fig. 3a). To elucidate the A. tricolor genes responsible for the negligible activity of betalain synthesis in transient analysis, a series of coinfiltration assays were carried out by replacing the positive control genes individually with AmCYP76AD1 , AmDODA , and AmcDOPA5GT . The replacements of BvCYP76AD1 and MjcDOPA5GT by AmCYP76AD1 and AmcDOPA5GT , respectively, resulted in high amounts of betalain pigment production in N. benthamiana leaves (Fig. 3b). However, AmDODA failed to replace the function of BvDODA a 1 . The coexpression of BvCYP76AD1 , AmDODA , and MjcDOPA5GT only produced marginal levels of betalain pigments, which were barely detectable (Fig. 3b). Together with the comparable levels of proteins detected by western blotting (Fig. 3c), these results suggest that the L-DOPA 4,5-dioxygenase activity of AmDODA is very low compared to that of BvDODAa1. Two DODA a homologues are present in A. tricolor Recently, a phylogenetic study of Caryophyllales suggested that at least two DODA a genes are present in betalain-pigmented species, including Amaranthus hypochondriacus 12 . To identify the DODAa homologue exhibiting a high level of L-DOPA 4,5-dioxygenase activity in A. tricolor , the RNA sequencing of aerial tissues derived from AMR and AMG plants was performed on the Illumina HiSeq 4000 platform. Two transcript libraries of AMR and AMG were built from the high-quality reads through de novo assembly and functional annotation (Supplementary Table S2, S3). The relative abundance of transcripts between AMR and AMG was illustrated in an MA plot (Fig. 4a). In addition, the relevant genes involved in the synthesis of betalain pigments were identified through in silico analysis and further highlighted in the MA plot (Fig. 4a, Supplementary Table S4). As expected, only AmCYP76AD1 was expressed at a significantly higher level in AMR than in AMG (Fig. 4a). These results suggest that AmCYP76AD1 is the key enzyme responsible for betalain pigment accumulation in AMR and that the loss of AmCYP76AD1 expression in AMG results in the green color phenotype. Additionally, two DODA a homologues, AmDODA a 1 and AmDODA a 2 (referred to as AmDODA ), were recovered through in silico analysis (Supplementary Fig. S2, Table S4). This indicated that gene duplication has occurred at least once in the DODA a lineage of A. tricolor . A reduced phylogenetic tree of DODAa was further generated using AmDODAa1, AmDODAa2, and previously characterized DODAa homologues from B. vulgaris , Carnegiea gigantea , Chenopodium quinoa, Mesembryanthemum crystallinum , M. jalap a, Parakeelya mirabilis , and Stegnosperma halimifolium (Fig. 4b). Two clades, DODAa1 and DODAa2, were obtained, and each of them presented seven previously identified conserved residues that are functionally important for high and marginal activities of L-DOPA 4,5-dioxygenase, respectively (Fig. 4c). Among these sequences, AmDODAa1 belongs to the DODAa1 clade and contains seven residues (DDYNDEI) associated with high L-DOPA 4,5-dioxygenase activity; AmDODAa2 (AmDODA) belongs to the DODAa2 clade and contains seven residues (YGFKNNT) associated with marginal L-DOPA 4,5-dioxygenase activity. These results suggest that AmDODAa1 may exhibit the high level of L-DOPA 4,5-dioxygenase activity required for betalain pigment production in A. tricolor . AmDODA a 1, but not AmDODA a 2, exhibits a high level of L-DOPA 4,5-dioxygenase activity As a key step in betalain biosynthesis, L-DOPA 4,5-dioxygenase can convert L-DOPA into betalamic acid, the basic structural unit of all betalains 1,32 . To functionally characterize the L-DOPA 4,5-dioxygenase activity of AmDODAa1, AmDODA a 1 was coexpressed with BvCYP76AD1 and MjcDOPA5GT by agroinfiltration. As a result, high production of betalain pigments was observed when comparable amounts of proteins were expressed in N. benthamiana leaves. (Fig. 3b, c). These results indicate that AmDODAa1, but not AmDODAa2, exhibits a high level of L-DOPA 4,5-dioxygenase activity, similar to that of BvDODAa1. To verify enzyme activity in vitro , AmDODAa1 and AmDODAa2 were expressed as SUMO-fused recombinant proteins in an Escherichia coli expression system (Fig. 5a). Enzymatic reactions were conducted following the method described by Sasaki et al. (2009) 32 , in which crude extracts prepared from E. coli were used. After incubation for 5 min at 30°C, a bright yellow color derived from betalamic acid was observed in the reaction mixture containing L-DOPA, ascorbic acid, and a crude extract prepared from E. coli harboring AmDODA a 1 or BvDODA a 1 , but not AmDODA a 2 (Fig. 5b). However, only a very weak yellow color was observed when the reaction mixture contained twofold crude extract prepared from E. coli harboring AmDODA a 2 (Fig. 5b). As a control, a reaction mixture containing the crude extract was prepared from E. coli harboring only the vector, and no color was observed (Fig. 5b). The reaction products were then subjected to LC-MS/MS analysis and revealed that the clear peak at a retention time of 7.5 min was betalamic acid (Fig. 5c). These results confirm that AmDODAa2 exhibits marginal levels of L-DOPA 4,5-dioxygenase activity. Reconstruction of the core betalain biosynthesis pathway of A. tricolor in N. benthamiana In this study, we also attempted to use TRV-based virus-induced gene silencing (VIGS) to examine the functional activities of genes involved in betalain biosynthesis in A. tricolor . However, the transient silencing of AmCYP76AD1 in A. tricolor was particularly challenging and failed in our hands. In addition, the attempted overexpression of AmCYP76AD1 to complement the betalain pigments in the leaves of AMG was unsuccessful using an agroinfiltration system. These differences might have resulted from the different varieties and low transformation efficiency of A. tricolor 33 . To reconstruct the core betalain biosynthesis pathway of A. tricolor , AmCYP76AD1 , AmDODA a 1 , and AmcDOPA5GT were transiently overexpressed in N. benthamiana leaves by agroinfiltration for the heterologous engineering of betalain pigments. Similar to the vector-only control, the heterologous expression of single AmCYP76AD1 , AmDODA a 1 , or AmcDOPA5GT was not sufficient to produce any betalain pigment in N. benthamiana (Fig. 6a). However, low production of betalain pigments was observed when AmCYP76AD1 and AmDODA a 1 were coexpressed in N. benthamiana (Fig. 6a). In contrast, no betalain pigment was observed when AmCYP76AD1 and AmcDOPA5GT or AmDODA a 1 and AmcDOPA5GT were coexpressed in N. benthamiana (Fig. 6a). Only the coexpression of AmCYP76AD1 , AmDODA a 1 , and AmcDOPA5GT together was sufficient to produce high amounts of betalain pigments in N. benthamiana , which resulted in a strong red-violet color (Fig. 6a). The strong red-violet color was similar to that in the positive control in which BvCYP76AD1 , BvDODA a 1 , and MjcDOPA5GT were coexpressed in N. benthamiana (Fig. 6a). As expected, the coexpression of AmCYP76AD1 , AmDODA a 2 , and AmcDOPA5GT only produced marginal levels of betalain pigments, which were barely detectable (Fig. 6a). Together with the comparable amount of proteins detected by western blotting (Fig. 6b), our results suggest that the enzyme activities of AmCYP76AD1, AmDODAa1, and AmDOPA5GT are sufficient to construct the core betalain biosynthesis pathway of A. tricolor . Discussion Molecular genetics have shed light on the betalain biosynthesis pathway and its evolutionary significance in Caryophyllales. Based on phylogenetic analysis, CYP76AD homologues can be classified into α, β, and γ clades 9 . To date, only the functions of CYP76ADα and CYP76ADβ clade homologues, such as CYP76AD1 and CYP76AD6 , have been reported 10 . For example, the cosilencing of CYP76AD1 and CYP76AD6 represses the production of betacyanins and betaxanthins in B. vulgaris , causing a green leaf phenotype 16 . In this study, a CYP76AD6 -like ( AmCYP76AD6 ) gene, belonging to the CYP76ADβ clade according to phylogenetic construction and LOGO analysis (Fig. 2c, d), was also identified in A. tricolor through transcriptome analysis (Supplementary Fig. S3). However, the expression of AmCYP76AD6 was extremely low and was difficult to detect in AMR and AMG. As a result, it is difficult to functionally connect AmCYP76AD6 with the production of betalains in A. tricolor . In addition, although PPO , a polyphenol oxidase gene, and CATPO , a catalase-phenol oxidase gene, were previously proposed to be involved in betalain biosynthesis via monophenolase activity 34,35 , their transcripts did not show highly differential expression patterns between AMR and AMG (Fig. 1c, Supplementary Fig. S1c). As a result, we propose that the elevated expression of AmCYP76AD1 is necessary for the occurrence of a red-violet color phenotype in A. tricolor ; in contrast, the loss of AmCYP76AD1 expression results in a green color phenotype in A. tricolor (Fig. 1a-c). The existence of the AmCYP76AD1 gene in AMG examined by PCR using genomic DNA as a template confirmed the loss of AmCYP76AD1 expression in AMG (Supplementary Fig. S4). Together with the functional characterization of the enzymatic activity of AmCYP76AD1 through the heterologous engineering of betalain pigments in N. benthamiana (Fig. 3b, 6a), we conclude that AmCYP76AD1, a CYP76ADα homologue required for the initiation of the betalain biosynthesis pathway, plays a key role in betalain pigment accumulation in A. tricolor . Accordingly, the transcript levels of AmCYP76AD1 displayed a high correlation with betalain pigment contents (Fig. 2a, b). In recent years, with the elucidation of the central committed steps of the betalain biosynthesis pathway, comparative transcriptome analyses have been intensively applied to identify genes involved in regulating betalain biosynthesis in Caryophyllales 16,20,30,36,37 . However, numerous duplication events have led to difficulty in elucidating the functional activities of key enzymes in betalain-pigmented species through annotation 10 . For example, duplication events gave rise to two major clades of DODA homologues, DODAa and DODAb, but only one gene paralog in the DODAa clade of each species exhibits high levels of L-DOPA 4,5-dioxygenase activity 12,21 . Thus, it is necessary to examine the possible involvement of annotated genes in betalain biosynthesis on the basis of experimental evidence. In this study, the AmDODA a 1 and AmDODA a 2 genes, which belong to the DODAa clade according to phylogenetic construction and LOGO analysis (Fig. 4b, c), were identified in A. tricolor through transcriptome analysis (Supplementary Fig. S2, Table S4). Based on the heterologous engineering of betalain pigments in N. benthamiana and in vitro biochemical studies (Fig. 3b, 5b), we report that AmDODAa1 displayed a high level of L-DOPA 4,5-dioxygenase activity to produce betalamic acid, but such activity was barely detectable for AmDODAa2. These results indicate that at least one duplication event has occurred in the DODAa lineage of A. tricolor , and the primary function of AmDODAa2 remains to be further studied. Betalains are composed of betacyanins and betaxanthins. In contrast to betaxanthins, which are derived from betalamic acid via spontaneous condensation with amino acids or other amines, a large number of betacyanins are composed of betanidin conjugated with glycosyl moieties 9,10 . We characterized the function of AmcDOPA5GT , a cyclo -DOPA 5- O -glucosyltransferase gene, through the heterologous engineering of betalain pigments in N. benthamiana . The coexpression of AmCYP76AD1 , AmDODA a 1 , and AmcDOPA5GT enabled the production of high levels of betalain pigments with a dark red color (Fig. 6a). In contrast, low production of betalain pigments was observed when AmCYP76AD1 and AmDODA a 1 were coexpressed (Fig. 6a). Our results suggest the importance of AmcDOPA5GT in the glycosylation reaction during betalain biosynthesis in A. tricolor . In fact, the metabolic pathway of betalain biosynthesis is very complex due to multiple glycosylation steps, and different betacyanins have been identified 10,38 . For example, betanin, the most common betacyanin, is not only produced by cyclo -DOPA 5- O -glucosyltransferase but is also produced by betanidin 5- O -glucosyl-transferase through the glycosylation of betanidin 39,40 . In this study, AmB5GT , a betanidin 5- O -glucosyl-transferase gene, was also identified through comparative transcriptome analyses (Supplementary Table S4). Although AmcDOPA5GT showed higher expression levels than AmB5GT in both AMR and AMG (Supplementary Table S4), it remains to be determined which of the two glycosylation routes is more important for the formation of betanin in A. tricolor . Recently, betalain biosynthesis in different pitaya species, such as Hylocereus polyrhizus , Hylocereus costaricensis , Hylocereus undatus , and Hylocereus megalanthus , has been intensively studied through comparative transcriptome analysis 36,37,41,42 . However, further studies remain to be conducted to provide experimental evidence and strengthen the understanding of the roles of candidate genes in betalain biosynthesis. Here, complementation assays conducted through the heterologous engineering of betalain pigments in nonbetalain-producing plants provided a solution for the easy and rapid comparison of the functional activities of genes involved in the core betalain biosynthesis pathway between betalain-pigmented species of Caryophyllales. Using the coexpression of BvCYP76AD1 , BvDODA a 1 , and MjcDOPA5GT in N. benthamiana as a positive control, the functional activities of A. tricolor genes responsible for betalain synthesis could be compared through a series of complementation assays (Fig. 3b, c). We showed that comparable amounts of betalain pigments were observed when the functional activities of positive genes were individually replaced with AmCYP76AD1 , AmDODA a 1 , and AmcDOPA5GT in transient coexpression assays (Fig. 3b, c). Our results indicate that AmCYP76AD1 , AmDODA a 1 , and AmcDOPA5GT exhibit high tyrosinase, L-DOPA 4,5-dioxygenase, and cyclo -DOPA 5- O -glucosyltransferase activities, respectively, which are similar to those in B. vulgaris and M. jalap a. Accordingly, in vitro biochemical studies demonstrated that AmDODAa1 displayed comparable L-DOPA 4,5-dioxygenase activity to BvDODAa1 in producing betalamic acid (Fig. 5b). These results provide novel insights into betalain biosynthesis and evolution in A. tricolor . In conclusion, a comparative transcriptome analysis combined with functional and enzymatic studies were performed to reveal the core betalain biosynthesis pathway of A. tricolor. The heterologous engineering of betalain pigments through the coexpression of AmCYP76AD1 , AmDODA a 1 , and AmcDOPA5GT in N. benthamiana enabled the production of high amounts of betalain pigments with a red-violet color similar to those in the red-leaf cultivar of A. tricolor . Although the metabolic pathway of betalain biosynthesis is very complex, the core betalain biosynthesis pathway of A. tricolor constructed here not only provides a basal framework for examining genes related to betalain biosynthesis within the species of Amaranthaceae but also sheds light on the evolution of the betalain biosynthesis pathway in Caryophyllales. Methods Plant materials and growth conditions A. tricolor , B. vulgaris , M. jalapa , and N. benthamiana plants were grown at 26°C in a semicontrolled walk-in chamber under a 16:8-h light:dark photoperiod. Soil (Jiffy) mixed with vermiculite and pearlstone was used. Seeds of A. tricolor cv. Hung Hsien (red-leaf cultivar) and A. tricolor cv. Pai Hsien (green-leaf cultivar) were purchased from KNOWN-YOU SEED CO., LTD. Betalain pigment extraction and measurement For betalain pigment measurement, betalain contents were determined as described previously with some modification 43 . Briefly, leaves of seedlings were collected and ground into powder in liquid nitrogen. Betalain pigments were extracted with extraction buffer (methanol:chloroform:H 2 O [1:2:1]). After centrifugation, the upper (hydrophilic) layer was collected to measure the absorbance at 538 nm and 476 nm for betacyanins and betaxanthins, respectively. The relative betalain content was calculated with the following equation: ( A 538 + A 476 )/gram). Plasmid construction All plasmid constructs were generated using standard restriction site reconstruction methods and confirmed by DNA sequencing. Am CYP76AD1 , Am DODA a 1 , Am DODA a 2 , Am cDOPA5GT , Bv CYP76AD1 , Bv DODA a 1 , and Mj cDOPA5GT were amplified from A. tricolor , B. vulgaris , or M. jalapa cDNA libraries using AccuPrime pfx DNA polymerase (Invitrogen). For the transient expression of C-terminal YFP- or FLAG (SFP)-tagged proteins in N. benthamiana , PCR products encoding Am CYP76AD1 , Am DODA a 1 , Am DODA a 2 , Am cDOPA5GT , Bv CYP76AD1 , Bv DODA a 1 , and Mj cDOPA5GT were subcloned into pBA-C-SFP or pBA-C-YFP vectors under the control of a Cauliflower mosaic virus ( CaMV ) 35S promoter 44 . To produce N-terminal SUMO-tagged recombinant proteins, PCR products encoding Am DODA a 1 , Am DODA a 2 , and Bv DODA a 1 were subcloned into the pET-SUMO (Invitrogen) vector 45 . For the VIGS assay, a cDNA fragment of Am CYP76AD1 was amplified and subcloned into the pTRV2 vector 46 . The primer sequences used for plasmid construction are listed in Supplementary Table S5. Quantitative real-time polymerase chain reaction (qRT-PCR) and statistical analysis TRIzol TM (Invitrogen)-extracted total RNA was reverse transcribed using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) according to the manufacturer’s instructions. Briefly, each sample was prepared from the leaves of three biologically distinct 3-week-old or 4-week-old A. tricolor plants. Then, cDNA was synthesized from 1 μg of total RNA using a mixture of random hexamers and oligo(dT) 20 under the following conditions: 25°C for 10 min, followed by 50°C for 40 min. The cDNA was employed as a template for qRT-PCR using the KAPA SYBR Fast qPCR Kit (Kapa Biosystems). Three technical replicates were performed on a CFX96 TM Real-time System (Bio-Rad) under the following conditions: 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 55°C for 30 s. The expression levels of selected genes were determined by normalization to the reference gene Actin . Statistically significant differences were determined using Student’s t -test in SPSS version 20.0. The primer sequences employed for qRT-PCR analyses are listed in Supplementary Table S1. PCR analyses using genomic DNA extracted from AMR and AMG as a template were performed to confirm the specificity of the primers (Supplementary Fig. S4). Transient coexpression assay and western blotting Plasmids for the transient expression of AmCYP76AD1-YFP, AmDODAa1-SFP, AmDODAa2-SFP, AmcDOPA5GT-SFP, BvCYP76AD1-YFP, BvDODAa1-SFP, or MjcDOPA5GT-SFP were transformed into the Agrobacterium tumefaciens strain ABI. C-terminal tagged proteins were coexpressed using a mixture of A. tumefaciens carrying the desired constructs in N. benthamiana leaves by agroinfiltration following the method described previously 47 . After three days, the infiltrated leaves were photographed and ground into a powder in liquid nitrogen for total cell extract preparation. Briefly, 0.1 g of sample powder was added to 0.2 ml of 2.5× SDS sample buffer (5 mM EDTA, 5% SDS, 0.3 M Tris–HCl, pH 6.8, 20% glycerol, 1% β-mercaptoethanol, and bromophenol blue), which was then heated at 95°C in a dry bath for 10 min. After centrifugation at 13,000× g for 10 min, the supernatant was obtained, and total proteins were separated by SDS-PAGE. Western blotting assays were performed to monitor protein levels using specific polyclonal and monoclonal antibodies against YFP- and FLAG-tag, respectively. Chemiluminescence signals generated by ECL reagents (PerkinElmer) were captured with an ImageQuant LAS 4000 mini imager (GE Healthcare). All experiments were repeated at least three times using biologically distinct samples prepared from two infiltrated leaves. In vitro L-DOPA 4,5-dioxygenase activity assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis An in vitro L-DOPA 4,5-dioxygenase activity assay was performed according to the method described previously with some modifications 32 . Briefly, plasmids for the expression of N-terminal SUMO-tagged Am DODA a 1 , Am DODA a 2 and Bv DODA a 1 were transformed into Escherichia coli strain BL21 (DE3). The transformants were grown in 50 ml LB medium, and the recombinant proteins were induced with 0.2 mM IPTG at 22°C for 16 hours. Harvested cells were washed, resuspended, and disrupted by sonication in 50 mM sodium phosphate buffer (pH 7.0). The crude extract (supernatant) was used for the enzyme activity assay after centrifugation at 14,000x g for 15 min. The amount of recombinant protein was quantified with Protein Assay Reagent (Bio-Rad) and via Coomassie blue staining SDS-PAGE with BSA as the standard. Basically, the reaction (100 μl) was performed with the crude extract containing 8 μg DODA protein, 27 mM ascorbic acid, and 6.75 mM L-DOPA at 30°C for 5 min. LC-MS/MS was performed using a Dionex UltiMate 3000 system (Thermo Fisher Scientific) linked with an amaZon speed-ion trap mass spectrometer (Bruker). Betalamic acid was detected on a Waters BEH shield RP18 column with two eluting solvent systems: (A) H 2 O with 0.1% formic acid, (B) 100% acetonitrile. The gradient elution program was set as follows: 0-3 min (100% A), 9 min (55% A and 45% B), 12-13 min (100% B). The flow rate was 0.3 ml min -1 , and the detector wavelength was 424 nm. The electrospray ionization mass parameters were set as follows: 4.5 kV capillary, 500 V end plate offset voltage, 40.0 psi nebulizer pressure, 8.0 l min -1 dry gas, and 230°C dry temperature. The measurement was operated in multiple reaction-monitoring (MRM) with the positive ion mode. Next-generation sequencing and MA plot To perform next-generation sequencing, aerial tissues derived from three biologically distinct 3-week-old A. tricolor plants were collected. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA quality was examined via 1.2% (wt/vol) formaldehyde gel electrophoresis and with an Experion RNA analysis kit (Bio-Rad, Munich). Only high-quality RNA was used for next-generation sequencing performed on the Illumina HiSeq 4000 platform with 150 paired-end reads. For each dataset (AMR and AMG), 100 million reads were generated, and de novo assembly was performed with the Trinity tool. The assembled transcripts were annotated with BlastX in UniProt. Gene expression levels were normalized as FPKM values, and differentially expressed genes were identified according to an FDR 2 or < -2 (Supplementary Table S2, S3). An MA plot was generated based on the average concentration (logCPM) and fold-change (logFC) values to show the relative abundances of transcripts between AMR and AMG. Phylogenetic tree reconstruction and LOGO analysis Phylogenetic trees were reconstructed using MEGA-X software based on the protein sequence comparisons of CYP76AD and DODA homologues from different betalain-producing species. Multiple sequence alignments were performed using the MUSCLE program and were processed to generate a maximum likelihood phylogenetic tree via the Jones-Taylor-Thornton (JTT) model with bootstrapping to perform molecular evolutionary analysis. The numbers at the branch points are bootstrap values representing the percentages of replicate trees based on 1000 repeats. LOGO analyses were performed via WebLogo (http://weblogo.berkeley.edu/logo.cgi) based on selected conserved amino acids of CYP76AD and DODA homologues reported previously 9,12,21,48 . The species, families, and accession numbers of CYP76AD and DODAa homologues are available in Supplementary Table S6. Declarations Acknowledgements This work was financially supported (in part) by grants-in-aid from the Ministry of Science and Technology (MOST-109-2628-B-005-006) and the Advanced Plant Biotechnology Center from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. Author contributions Y.-C.C. carried out most of the experiments; Y.-C.C. and Y.-L.C. designed and carried out the enzymatic assays. N.-W.T. performed the LC-MS analysis. C.-M.T., Y.-H.C., and P.-C.L. performed RNA-seq and plasmid constructions. Y.-C.L., L.-C.H. and S.-Y.W. contributed to the interpretation of data. J.-Y.Y. conceived and wrote the manuscript. Competing interests The authors declare no competing interests. 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Supplementary Files Changetal.Supplementaryfile.docx Changetal.FigS1.tif Changetal.FigS2.tif Changetal.FigS3.tif Changetal.FigS4.tif Changetal.TableS1.xlsx Changetal.TableS2.xlsx Changetal.TableS3.xlsx Changetal.TableS4.xlsx Changetal.TableS5.xlsx Changetal.TableS6.xlsx Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2021 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 30 Dec, 2020 Reviews received at journal 04 Dec, 2020 Reviewers agreed at journal 26 Nov, 2020 Reviewers agreed at journal 22 Nov, 2020 Reviewers invited by journal 22 Nov, 2020 Editor assigned by journal 21 Nov, 2020 Editor invited by journal 20 Nov, 2020 Submission checks completed at journal 20 Nov, 2020 First submitted to journal 18 Nov, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-110761","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":5073330,"identity":"3c899a17-0d4f-4a49-aa33-c325938345bc","order_by":0,"name":"Yu-Cheng Chang","email":"","orcid":"","institution":"Institute of Biochemistry, National Chung Hsing University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-Cheng","middleName":"","lastName":"Chang","suffix":""},{"id":5073331,"identity":"757871dd-7926-41d9-9ef8-1b3e853f62d1","order_by":1,"name":"Yi-Ching Chiu","email":"","orcid":"","institution":"Institute 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Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACxgYQWcHAYACieYjXcgahRYJIq9pI0cI8I/nZw6/z7sibSyQwPnjbxlBncICQBTPSzI1ltz0z3DkjgdlwbhuDBBFaEsykJbcdTjC4kcAmzQvUYkZYS/o3ack5YC3sv4nUkmMm+bEBYgszcVp63pRJMxw7bLjhzMNmyTnnJCT3E9Ji2J6+TfJHzWF5g+PJBz+8KbPhl2wgpGVCAgMzJNLBsUpETMrzH2Bg/EFY3SgYBaNgFIxkAAD56kFJmmup6wAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Biochemistry, National Chung Hsing University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jun-Yi","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2020-11-18 06:59:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-110761/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-110761/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-021-85486-x","type":"published","date":"2021-03-17T19:02:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":3831056,"identity":"668c446e-3f9c-45fb-adf0-de3842b19c76","added_by":"auto","created_at":"2020-11-25 19:38:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2515397,"visible":true,"origin":"","legend":"Identification of AmCYP76AD1 as a key element required for betalain pigment production in Amaranthus tricolor. (a) The leaf-color phenotypes of the red-leaf cultivar (AMR) and green-leaf cultivar (AMG) of three-week-old A. tricolor. (b) Extraction of chlorophyll pigments (hydrophobic layer) and betalain pigments (hydrophilic layer) from three-week-old leaves of AMR and AMG (left panel). Absorbance spectra of the extracted betalain pigments from AMR and AMG (right panel). The absorbance at 538 nm for betacyanins is indicated with a red dashed line, and the absorbance at 476 nm for betaxanthins is indicated with a yellow dashed line. (c) Expression levels of genes related to the betalain biosynthesis pathway in AMR and AMG analyzed by qRT-PCR. Statistically significant differences were determined using Student’s t-test (*P \u003c 0.01 for AMR vs. AMG). (d) Putative core betalain biosynthesis pathway in A. tricolor.","description":"","filename":"Changetal.Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/4c5c7c7924a5ecb9541202f7.jpg"},{"id":3831058,"identity":"e4540c7a-441c-49ce-b850-0b767431ff8a","added_by":"auto","created_at":"2020-11-25 19:38:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1868632,"visible":true,"origin":"","legend":"Expression pattern, phylogenetic reconstruction, and LOGO analysis of AmCYP76AD1. (a) The leaf color phenotypes and betalain absorbance spectra of the upper and lower leaves of a four-week-old plants of a red-leaf cultivar (AMR) of A. tricolor. (b) Expression levels of core betalain biosynthesis genes in the upper and lower leaves of four-week-old AMR plants analyzed by qRT-PCR. Statistically significant differences were determined using Student’s t-test (*P \u003c 0.01 for the upper leaves of AMR vs. the lower leaves of AMR). (c) Phylogenetic tree of CYP76AD homologues. The species, families, and accession numbers of CYP76AD homologues are available in Supplementary Table S6. (d) Proportional LOGO plots of selected amino acids identified by Brockington et al. (2015)9 were generated based on the CYP76AD and CYP76AD homologues listed in (c). Positions are numbered according to the residues of AmCYP76AD1 and AmCYP76AD6.","description":"","filename":"Changetal.Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/4a222321e4f05731922c0afe.jpg"},{"id":3831060,"identity":"d31f997f-3313-4c5c-9ac2-031fb8ba0ee0","added_by":"auto","created_at":"2020-11-25 19:38:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108769,"visible":true,"origin":"","legend":"Functional characterization of the enzyme activities of AmCYP76AD1, AmDODA, and AmcDOPA5GT by agroinfiltration for the heterologous engineering of betalain pigments in Nicotiana benthamiana. (a, b) N. benthamiana leaves coinfiltrated with Agrobacterium harboring plasmids for the expression of BvCYP76AD1-YFP (BvAD1), BvDODA1-SFP (BvDA1), MjcDOPA5GT-SFP (Mj5GT), AmCYP76AD1-YFP (AmAD1), AmDODA-SFP (AmDA), and AmcDOPA5GT-SFP (Am5GT). Both the adaxial (left) and abaxial (right) sides of leaves are presented in each panel. (c) Western blotting assays conducted with antibodies against the YFP- or FLAG (SFP)-tag to examine the expression levels of YFP-tagged CYP76AD1 (upper panel), SFP-tagged DODA (middle panel), and SFP-tagged cDOPA5GT (middle panel). As a loading control, the large subunit of Rubisco visualized by Coomassie brilliant blue staining is indicated by the arrowhead (lower panel).","description":"","filename":"Changetal.Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/80d2ad8eab7153dc76216cc1.jpg"},{"id":3831062,"identity":"c24c9316-4d5c-4b0c-b78f-b4c64538c500","added_by":"auto","created_at":"2020-11-25 19:38:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79619,"visible":true,"origin":"","legend":"In silico analysis of relevant genes involved in the betalain biosynthesis pathway of A. tricolor. (a) The relative abundance of transcripts between AMR and AMG is presented in the MA plot (logCPM vs. logFC). Each dot presents a gene, and the relevant genes associated with betalain pigment synthesis are highlighted. (b) Phylogenetic tree of functionally characterized DODA homologues. The species, families, and accession numbers of the DODA homologues are available in Supplementary Table S6. (c) Proportional LOGO plots of seven functionally important residues identified by Bean et al. (2018)21 were generated based on the DODA1 and DODA2 homologues listed in (b). Positions are numbered according to the residues of AmDODA1 and AmDODA2.","description":"","filename":"Changetal.Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/7b1a796442dcddfa6d8ba72d.jpg"},{"id":3831064,"identity":"ffb685cd-a97b-4752-adc1-00e546d5467a","added_by":"auto","created_at":"2020-11-25 19:38:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74842,"visible":true,"origin":"","legend":"Examination of the L-DOPA 4,5-dioxygenase activity of AmDODA1 and AmDODA2 in vitro. (a) Crude extracts prepared from E. coli expressing SUMO-fused BvDODA1, AmDODA1, or AmDODA2 were examined by Coomassie brilliant blue staining. Recombinant proteins were expressed in similar amounts. (b) Enzymatic reactions were conducted following the method described by Sasaki et al. (2009)32 with some modifications. The reaction mixtures contained L-DOPA, ascorbic acid, and the crude extract prepared from E. coli harboring the BvDODA1, AmDODA1, or AmDODA2 gene. Yellow color derived from betalamic acid was examined to evaluate the enzymatic activity of L-DOPA 4,5-dioxygenase. (c) Elution profiles of the in vitro reactions were performed by LC-MS/MS. Betalamic acid was confirmed by a mass fragmentation profile of the peak at a retention time of 7.5 min.","description":"","filename":"Changetal.Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/d7f160cbf14cdcbec5999581.jpg"},{"id":3831066,"identity":"dd433612-4057-4dfc-bd2c-957ddf257ed7","added_by":"auto","created_at":"2020-11-25 19:38:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2229430,"visible":true,"origin":"","legend":"Reconstruction of the core betalain biosynthesis pathway of A. tricolor in N. benthamiana by agroinfiltration for the heterologous engineering of betalain pigments. (a) N. benthamiana leaves coinfiltrated with Agrobacterium harboring plasmids for the expression of BvCYP76AD1-YFP (BvAD1), BvDODA1-SFP (BvDA1), MjcDOPA5GT-SFP (Mj5GT), AmCYP76AD1-YFP (AmAD1), AmDODA1-SFP (AmDA1), AmDODA2-SFP (AmDA2), and AmcDOPA5GT-SFP (Am5GT). Both the adaxial (left) and abaxial (right) sides of leaves are presented in each panel. (b) Western blotting assays were conducted to examine the expression levels of YFP-tagged CYP76AD1 (upper panel), SFP-tagged DODA (middle panel), and SFP-tagged cDOPA5GT (middle panel) using antibodies against the YFP- or FLAG (SFP)-tag. As a loading control, the large subunit of Rubisco visualized with Coomassie brilliant blue staining is indicated by the arrowhead (lower panel).","description":"","filename":"Changetal.Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/3f5e53b8c5e3756124619005.jpg"},{"id":15670071,"identity":"a23f816d-8720-4618-8ce6-a1162bed2da3","added_by":"auto","created_at":"2021-11-18 13:56:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1076030,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/c998655d-230a-44f5-a7b3-dbd8d323483c.pdf"},{"id":3831057,"identity":"4d05f6d0-3ba0-4bc2-8036-e0acab1f91f8","added_by":"auto","created_at":"2020-11-25 19:38:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12674943,"visible":true,"origin":"","legend":"","description":"","filename":"Changetal.Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/a4b3439908409024822b3ee7.docx"},{"id":3831059,"identity":"4ebb3e84-7d94-4914-bc3b-5afb22c44206","added_by":"auto","created_at":"2020-11-25 19:38:37","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5305464,"visible":true,"origin":"","legend":"","description":"","filename":"Changetal.FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/9670379af859a2e260661845.tif"},{"id":3831061,"identity":"7a6bad6c-f89b-423d-911e-c63104c50784","added_by":"auto","created_at":"2020-11-25 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19:38:39","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10537744,"visible":true,"origin":"","legend":"","description":"","filename":"Changetal.FigS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/939ae5e00fd58220401ad7c2.tif"},{"id":3831067,"identity":"e368e943-3d59-45de-af25-5d4dd354770e","added_by":"auto","created_at":"2020-11-25 19:38:39","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":10777,"visible":true,"origin":"","legend":"","description":"","filename":"Changetal.TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/7d1486cca06c53f4c08a88c0.xlsx"},{"id":3831068,"identity":"766e5181-7c30-4935-afdd-3ad7eab357a7","added_by":"auto","created_at":"2020-11-25 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19:38:40","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":11144,"visible":true,"origin":"","legend":"","description":"","filename":"Changetal.TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-110761/v1/0d2ebeabc1062a5b10bdca15.xlsx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eElucidation of the Core Betalain Biosynthesis Pathway in \u003cem\u003eAmaranthus tricolor\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBetalains are classified into betacyanins and betaxanthins, which provide red-violet and yellow coloration, respectively\u003csup\u003e1\u003c/sup\u003e. Similar to anthocyanins, betalains exhibit antioxidant activity in the form of free-radical scavenging and accumulate in response to different stresses, such as UV-B radiation, high-intensity light, salinity, heat, and drought\u003csup\u003e2,3,4,5\u003c/sup\u003e. In addition to their potential roles in protecting plants against abiotic stresses, betalains play a role in defense against pathogenic fungi\u003csup\u003e6\u003c/sup\u003e. Moreover, as water-soluble natural pigments, betalains are widely used as food additives because of their health-promoting properties and color stability over a wide range of pH values\u003csup\u003e7,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBetalains occur only in Caryophyllales and have never been detected in anthocyanin-producing plants\u003csup\u003e9,10\u003c/sup\u003e. Although the molecular basis for the\u0026nbsp;mutual exclusion\u0026nbsp;of\u0026nbsp;betalains\u0026nbsp;and anthocyanins is still unclear, breakthroughs in the identification of genes involved in betalain biosynthesis have shed light on the evolution of betalain pigmentation in Caryophyllales\u003csup\u003e9,10,11,12\u003c/sup\u003e. Unlike anthocyanins, which are derived from L-phenylalanine, betalains are synthesized from L-tyrosine\u003csup\u003e13,14\u003c/sup\u003e. Initially, L-tyrosine is hydroxylated to produce L-DOPA by tyrosinases encoded by \u003cem\u003eCYP76AD1\u003c/em\u003e and its orthologs\u003csup\u003e15,16\u003c/sup\u003e. L-DOPA can be converted into betalamic acid by L-DOPA 4,5-dioxygenase encoded by \u003cem\u003eDODA\u003c/em\u003e\u003cu\u003e,\u003c/u\u003e and betalamic acid can then spontaneously condense with amino acids to form betaxanthins\u003csup\u003e1,12,17\u003c/sup\u003e. Alternatively, L-DOPA can be converted into \u003cem\u003ecyclo\u003c/em\u003e-DOPA through the oxidase activity of CYP76AD1, and \u003cem\u003ecyclo\u003c/em\u003e-DOPA can then spontaneously condense with betalamic acid to form betanidin\u003csup\u003e9,18\u003c/sup\u003e. Betanidin is further glycosylated by betanidin 5-\u003cem\u003eO\u003c/em\u003e-glucosyl-transferase encoded by \u003cem\u003eB5GT\u003c/em\u003e to form betanin, the most common betacyanin. Glycosylation can also occur on \u003cem\u003ecyclo\u003c/em\u003e-DOPA, catalyzed by \u003cem\u003ecyclo\u003c/em\u003e-DOPA 5-\u003cem\u003eO\u003c/em\u003e-glucosyltransferase encoded by \u003cem\u003ecDOPA5GT\u003c/em\u003e, to produce \u003cem\u003ecyclo\u003c/em\u003e-DOPA-glucoside, which then spontaneously condenses with betalamic acid to form betanin\u003csup\u003e10,19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePhylogenetic analyses revealed that the \u003cem\u003eCYP76AD\u003c/em\u003e and \u003cem\u003eDODA\u003c/em\u003e genes, encoding key enzymes in the core biosynthetic pathway of betalains, are highly duplicated in Caryophyllales\u003csup\u003e10,20\u003c/sup\u003e. The \u003cem\u003eCYP76AD\u003c/em\u003e gene lineage has undergone at least three duplication events, giving rise to three clades: CYP76ADa, CYP76ADb and CYP76ADg\u003csup\u003e9\u003c/sup\u003e. The CYP76ADa clade includes the\u003cem\u003e CYP76AD1\u003c/em\u003e and \u003cem\u003eCYP76AD3\u003c/em\u003e genes, whose products possess both the tyrosine hydroxylase and L-DOPA 4,5-dioxygenase activities required for L-DOPA and \u003cem\u003ecyclo\u003c/em\u003e-DOPA formation, respectively\u003csup\u003e16\u003c/sup\u003e. The CYP76ADb clade includes the \u003cem\u003eCYP76AD5\u003c/em\u003e, \u003cem\u003eCYP76AD6\u003c/em\u003e, and \u003cem\u003eCYP76AD15\u003c/em\u003e genes, which possess only the tyrosine hydroxylase activity required for L-DOPA formation\u003csup\u003e16\u003c/sup\u003e. However, the functions of the genes in the CYP76ADg clade in betalain biosynthesis have not been determined. Duplication events in the \u003cem\u003eDODA\u003c/em\u003e gene lineage also gave rise to two major clades: DODAa and DODAb. The function of DODAb is unknown, but the evolution of L-DOPA 4,5-dioxygenase activity in betalain-producing plants was proposed to be led by DODAa\u003csup\u003e9\u003c/sup\u003e. Nevertheless, only one paralogous gene from the DODAa clade shows high L-DOPA 4,5-dioxygenase activity in each species, and others exhibit barely detectable activity\u003csup\u003e12,21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAn increased understanding of the betalain biosynthesis pathway has facilitated the metabolic engineering of betalains, providing new sources for basic research studies and commercial applications\u003csup\u003e22,23\u003c/sup\u003e. For example, the fluorescent betaxanthins produced by the expression of \u003cem\u003eMjDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e in yeast were used as chemical biosensors to reveal the tyrosine hydroxylase activity of BvCYP76AD1\u003csup\u003e15\u003c/sup\u003e. The production of semisynthetic betaxanthins by spontaneous condensation between fluorescent betalamic acid and the amino groups of proteins provides an alternative method for labeling proteins for multiple biological applications\u003csup\u003e24\u003c/sup\u003e. Fine-turning the content ratio of betacyanins and betaxanthins via the differential expression of \u003cem\u003eBvCYP76AD1\u003c/em\u003e and \u003cem\u003eBvCYP76AD6\u003c/em\u003e in non-Caryophyllales makes it possible to create a range of color patterns in the background of anthocyanin-producing plants\u003csup\u003e6\u003c/sup\u003e. The heterologous production of betalains via the coexpression of \u003cem\u003eBvCYP76AD1\u003c/em\u003e, \u003cem\u003eBvDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eMjcDOPA5GT\u003c/em\u003e in tobacco enhances the resistance of transgenic plants to \u003cem\u003eBotrytis cinereal\u003c/em\u003e infection\u003csup\u003e6\u003c/sup\u003e. In addition, betanin rice generated through the coexpression of \u003cem\u003emeloS\u003c/em\u003e, \u003cem\u003eBvCYP76AD1\u003c/em\u003e, and \u003cem\u003eBvDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e in rice endosperm shows higher antioxidant activity and provides a functional food\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAmaranth species are economically important crops containing large amounts of betalains. They can be classified into three categories: vegetable \u003cem\u003eAmaranthus\u003c/em\u003e, grain \u003cem\u003eAmaranthus\u003c/em\u003e, and weed \u003cem\u003eAmaranthus\u003c/em\u003e species\u003csup\u003e26\u003c/sup\u003e. \u003cem\u003eAmaranthus tricolor\u003c/em\u003e L., a vegetable \u003cem\u003eAmaranthus\u003c/em\u003e species, is widely distributed in warm and tropical regions and is cultivated as a leafy vegetable. \u003cem\u003eA. tricolor\u003c/em\u003e is rich in natural antioxidants and is able to tolerate abiotic stresses; it has been used as a traditional Chinese medicinal herb for the treatment of eruptive fever, pain, sore throat, dysentery, anemia, bronchitis, colic, etc.\u003csup\u003e27\u003c/sup\u003e. \u003cem\u003eA. tricolor\u003c/em\u003e cultivars with various contents of betacyanins and betaxanthins, resulting in striking colors ranging from red to green, have been commercially produced\u003csup\u003e27,28\u003c/sup\u003e. However, the molecular basis underlying betalain biosynthesis in \u003cem\u003eA. tricolor\u003c/em\u003e remains largely unknown. Recently, virus-induced gene silencing was applied to elucidate the function of \u003cem\u003eCYP76AD1\u003c/em\u003e in producing betalain pigments in \u003cem\u003eA. tricolor\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e. In addition, a comparative analysis of a transcriptome database constructed from different leaf samples of \u003cem\u003eA. tricolor\u003c/em\u003e cv. Dahong was performed to construct a putative metabolic pathway of betalains in \u003cem\u003eA. tricolor\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e. Candidate genes encoding enzymes catalyzing the formation of L-DOPA, \u003cem\u003ecyclo\u003c/em\u003e-DOPA, betalamic acid, \u003cem\u003ecyclo\u003c/em\u003e-DOPA-glucoside, and betanin were obtained from a transcriptome database and showed higher expression levels in red areas of \u003cem\u003eA. tricolor\u003c/em\u003e leaves than in in green areas\u003csup\u003e30\u003c/sup\u003e. However, functional characterization and enzyme activity analyses are still needed to elucidate the roles of these candidate genes in the betalain biosynthesis pathway of \u003cem\u003eA. tricolor\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, \u003cem\u003eA. tricolor\u003c/em\u003e cultivars with different colors were chosen for comparative transcriptome analysis. The key gene showing elevated expression in a red-leaf cultivar of \u003cem\u003eA. tricolor\u003c/em\u003e was identified, and the results indicated that the dual activities of tyrosine hydroxylase and L-DOPA oxidase are important for producing red betalain pigments in \u003cem\u003eA. tricolor\u003c/em\u003e. The core betalain biosynthesis pathway of \u003cem\u003eA. tricolor\u003c/em\u003e was further constructed based on the functional characterization of betalain biosynthesis genes through the heterologous engineering of betalain pigments in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e and\u003cem\u003e in vitro\u003c/em\u003e enzymatic assays of L-DOPA 4,5-dioxygenase activities. These results provide novel insights into betalain biosynthesis and evolution in \u003cem\u003eA. tricolor\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAmCYP76AD1 is highly expressed in a red-leaf cultivar of A. tricolor\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA. tricolor\u003c/em\u003e cultivars are important leafy vegetables that display leaf colors ranging from red to green depending on the betalain content\u003csup\u003e28\u003c/sup\u003e. To elucidate the genetic factors that influence betalain pigment accumulation in red- and green-leaf cultivars of \u003cem\u003eA. tricolor\u003c/em\u003e (hereafter referred to as AMR and AMG, respectively) (Fig. 1a, b, Supplementary Fig. S1a, b), specific primer pairs were designed based on available sequence information from the NCBI database or previously published studies to selectively examine the transcript levels of genes related to the betalain biosynthesis pathway by qRT-PCR (Supplementary Table S1). In 3-week-old \u003cem\u003eA. tricolor\u003c/em\u003e, \u003cem\u003eAmCYP76AD1\u003c/em\u003e and \u003cem\u003eAmPPO\u003c/em\u003e showed higher expression levels in AMR than in AMG (Fig. 1c). Notably, only \u003cem\u003eAmCYP76AD1\u003c/em\u003e exhibited a highly differential expression pattern, showing an ~200-fold difference between AMR and AMG. In contrast, \u003cem\u003eAmDODA\u003c/em\u003e, \u003cem\u003eAmcDOPA5GT\u003c/em\u003e, \u003cem\u003eAmB5GT\u003c/em\u003e,\u003cem\u003e AmUGT79B30-like 4\u003c/em\u003e, \u003cem\u003eAmMYB1\u003c/em\u003e, \u003cem\u003eAmADH\u003c/em\u003e, \u003cem\u003eAmCATPO\u003c/em\u003e, and \u003cem\u003eAmTyDC\u003c/em\u003e did not show a significant differential expression pattern between AMR and AMG (Fig. 1c). The highly differential expression pattern of \u003cem\u003eAmCYP76AD1\u003c/em\u003e between AMR and AMG was also observed in 4-week-old \u003cem\u003eA. tricolor\u003c/em\u003e (Supplementary Fig. S1c). Moreover, as a key element in the initiation of the betalain biosynthesis pathway, \u003cem\u003eAmCYP76AD1\u003c/em\u003e transcript levels displayed a high correlation with betalain pigment contents (Fig. 2a, b). Further phylogenetic reconstruction and LOGO analysis revealed that AmCYP76AD1 belongs to the CYP76ADa clade (Fig. 2c, d), whose members possesses both the tyrosine hydroxylase and L-DOPA oxidase activities required for L-DOPA and \u003cem\u003ecyclo\u003c/em\u003e-DOPA formation, respectively (Fig. 1d). These results suggest that the elevated expression of \u003cem\u003eAmCYP76AD1\u003c/em\u003e is necessary for betalain pigment accumulation, which leads to an obvious red-violet color in the leaves and stems of AMR, but not in those of AMG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAmDODA\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e exhibits a marginal level of L-DOPA 4,5-dioxygenase activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough candidate transcripts related to betalain biosynthesis were identified previously in \u003cem\u003eA. tricolor\u003c/em\u003e\u003csup\u003e30,31\u003c/sup\u003e, their functional and enzymatic activities have not yet been characterized. To functionally characterize the enzyme activities of AmCYP76AD1, AmDODA, and AmcDOPA5GT in the core pathway of betalain biosynthesis (Fig. 1d), \u003cem\u003e35S\u003c/em\u003e promoter-driven cDNAs encoding C-terminal YFP- or FLAG (SFP)-tagged AmCYP76AD1, AmDODA, and AmcDOPA5GT were transiently coexpressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves by agroinfiltration. Upon expression, only a small amount of betalain pigment was produced in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves, which was barely detectable (Fig. 3a). In contrast, as a positive control, high production of betalain pigments with red-violet color was observed when the \u003cem\u003eBeta vulgaris\u003c/em\u003e tyrosinase gene (\u003cem\u003eBvCYP76AD1\u003c/em\u003e), the \u003cem\u003eB. vulgaris\u003c/em\u003e L-DOPA 4,5-dioxygenase gene (\u003cem\u003eBvDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e), and the \u003cem\u003eMirabilis jalapa\u003c/em\u003e\u003cem\u003ecyclo\u003c/em\u003e-DOPA 5-\u003cem\u003eO\u003c/em\u003e-glucosyltransferase gene (\u003cem\u003eMjcDOPA5GT\u003c/em\u003e), were coexpressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves (Fig. 3a). To elucidate the \u003cem\u003eA. tricolor\u003c/em\u003e genes responsible for the negligible activity of betalain synthesis in transient analysis, a series of coinfiltration assays were carried out by replacing the positive control genes individually with \u003cem\u003eAmCYP76AD1\u003c/em\u003e, \u003cem\u003eAmDODA\u003c/em\u003e, and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e. The replacements of \u003cem\u003eBvCYP76AD1\u003c/em\u003e and \u003cem\u003eMjcDOPA5GT \u003c/em\u003eby \u003cem\u003eAmCYP76AD1\u003c/em\u003e and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e, respectively, resulted in high amounts of betalain pigment production in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves (Fig. 3b). However, \u003cem\u003eAmDODA\u003c/em\u003e failed to replace the function of \u003cem\u003eBvDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e. The coexpression of \u003cem\u003eBvCYP76AD1\u003c/em\u003e, \u003cem\u003eAmDODA\u003c/em\u003e, and \u003cem\u003eMjcDOPA5GT\u003c/em\u003e only produced marginal levels of betalain pigments, which were barely detectable (Fig. 3b). Together with the comparable levels of proteins detected by western blotting (Fig. 3c), these results suggest that the L-DOPA 4,5-dioxygenase activity of AmDODA is very low compared to that of BvDODAa1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTwo DODA\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e homologues are present in \u003cem\u003eA. tricolor\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecently, a phylogenetic study of Caryophyllales suggested that at least two \u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e genes are present in betalain-pigmented species, including \u003cem\u003eAmaranthus\u003c/em\u003e\u003cem\u003ehypochondriacus\u003c/em\u003e\u003csup\u003e12\u003c/sup\u003e. To identify the DODAa homologue exhibiting a high level of L-DOPA 4,5-dioxygenase activity in \u003cem\u003eA. tricolor\u003c/em\u003e, the RNA sequencing of aerial tissues derived from AMR and AMG plants was performed on the Illumina HiSeq 4000 platform. Two transcript libraries of AMR and AMG were built from the high-quality reads through \u003cem\u003ede novo\u003c/em\u003e assembly and functional annotation (Supplementary Table S2, S3). The relative abundance of transcripts between AMR and AMG was illustrated in an MA plot (Fig. 4a). In addition, the relevant genes involved in the synthesis of betalain pigments were identified through \u003cem\u003ein silico\u003c/em\u003e analysis and further highlighted in the MA plot (Fig. 4a, Supplementary Table S4). As expected, only \u003cem\u003eAmCYP76AD1\u003c/em\u003e was expressed at a significantly higher level in AMR than in AMG (Fig. 4a). These results suggest that AmCYP76AD1 is the key enzyme responsible for betalain pigment accumulation in AMR and that the loss of \u003cem\u003eAmCYP76AD1\u003c/em\u003e expression in AMG results in the green color phenotype.\u003c/p\u003e\n\u003cp\u003eAdditionally, two \u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e homologues, \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e and \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e (referred to as \u003cem\u003eAmDODA\u003c/em\u003e), were recovered through \u003cem\u003ein silico\u003c/em\u003e analysis (Supplementary Fig. S2, Table S4). This indicated that gene duplication has occurred at least once in the \u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e lineage of \u003cem\u003eA. tricolor\u003c/em\u003e. A reduced phylogenetic tree of DODAa was further generated using AmDODAa1, AmDODAa2, and previously characterized DODAa homologues from \u003cem\u003eB. vulgaris\u003c/em\u003e, \u003cem\u003eCarnegiea gigantea\u003c/em\u003e, \u003cem\u003eChenopodium quinoa, Mesembryanthemum crystallinum\u003c/em\u003e,\u003cem\u003e M. jalap\u003c/em\u003ea, \u003cem\u003eParakeelya mirabilis\u003c/em\u003e, and \u003cem\u003eStegnosperma halimifolium\u003c/em\u003e (Fig. 4b). Two clades, DODAa1 and DODAa2, were obtained, and each of them presented seven previously identified conserved residues that are functionally important for high and marginal activities of L-DOPA 4,5-dioxygenase, respectively (Fig. 4c). Among these sequences, AmDODAa1 belongs to the DODAa1 clade and contains seven residues (DDYNDEI) associated with high L-DOPA 4,5-dioxygenase activity; AmDODAa2 (AmDODA) belongs to the DODAa2 clade and contains seven residues (YGFKNNT) associated with marginal L-DOPA 4,5-dioxygenase activity. These results suggest that AmDODAa1 may exhibit the high level of L-DOPA 4,5-dioxygenase activity required for betalain pigment production in \u003cem\u003eA. tricolor\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmDODA\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e1, but not AmDODA\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e2, exhibits a high level of L-DOPA 4,5-dioxygenase activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a key step in betalain biosynthesis, L-DOPA 4,5-dioxygenase can convert L-DOPA into betalamic acid, the basic structural unit of all betalains\u003csup\u003e1,32\u003c/sup\u003e. To functionally characterize the L-DOPA 4,5-dioxygenase activity of AmDODAa1, \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e was coexpressed with \u003cem\u003eBvCYP76AD1\u003c/em\u003e and \u003cem\u003eMjcDOPA5GT\u003c/em\u003e by agroinfiltration. As a result, high production of betalain pigments was observed when comparable amounts of proteins were expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. (Fig. 3b, c). These results indicate that AmDODAa1, but not AmDODAa2, exhibits a high level of L-DOPA 4,5-dioxygenase activity, similar to that of BvDODAa1.\u003c/p\u003e\n\u003cp\u003eTo verify enzyme activity \u003cem\u003ein vitro\u003c/em\u003e, AmDODAa1 and AmDODAa2 were expressed as SUMO-fused recombinant proteins in an \u003cem\u003eEscherichia coli\u003c/em\u003e expression system (Fig. 5a). Enzymatic reactions were conducted following the method described by Sasaki et al. (2009)\u003csup\u003e32\u003c/sup\u003e, in which crude extracts prepared from \u003cem\u003eE. coli\u003c/em\u003e were used. After incubation for 5 min at 30\u0026deg;C, a bright yellow color derived from betalamic acid was observed in the reaction mixture containing L-DOPA, ascorbic acid, and a crude extract prepared from \u003cem\u003eE. coli\u003c/em\u003e harboring \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e or \u003cem\u003eBvDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, but not \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e (Fig. 5b). However, only a very weak yellow color was observed when the reaction mixture contained twofold crude extract prepared from \u003cem\u003eE. coli\u003c/em\u003e harboring \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e (Fig. 5b). As a control, a reaction mixture containing the crude extract was prepared from \u003cem\u003eE. coli\u003c/em\u003e harboring only the vector, and no color was observed (Fig. 5b). The reaction products were then subjected to LC-MS/MS analysis and revealed that the clear peak at a retention time of 7.5 min was betalamic acid (Fig. 5c). These results confirm that AmDODAa2 exhibits marginal levels of L-DOPA 4,5-dioxygenase activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReconstruction of the core betalain biosynthesis pathway of \u003cem\u003eA. tricolor\u003c/em\u003e in \u003cem\u003eN. benthamiana\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we also attempted to use TRV-based virus-induced gene silencing (VIGS) to examine the functional activities of genes involved in betalain biosynthesis in \u003cem\u003eA. tricolor\u003c/em\u003e. However, the transient silencing of \u003cem\u003eAmCYP76AD1\u003c/em\u003e in \u003cem\u003eA. tricolor\u003c/em\u003e was particularly challenging and failed in our hands. In addition, the attempted overexpression of \u003cem\u003eAmCYP76AD1\u003c/em\u003e to complement the betalain pigments in the leaves of AMG was unsuccessful using an agroinfiltration system. These differences might have resulted from the different varieties and low transformation efficiency of \u003cem\u003eA. tricolor\u003c/em\u003e\u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo reconstruct the core betalain biosynthesis pathway of \u003cem\u003eA. tricolor\u003c/em\u003e, \u003cem\u003eAmCYP76AD1\u003c/em\u003e, \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e were transiently overexpressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves by agroinfiltration for the heterologous engineering of betalain pigments. Similar to the vector-only control, the heterologous expression of single \u003cem\u003eAmCYP76AD1\u003c/em\u003e, \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, or \u003cem\u003eAmcDOPA5GT\u003c/em\u003e was not sufficient to produce any betalain pigment in \u003cem\u003eN. benthamiana \u003c/em\u003e(Fig. 6a). However, low production of betalain pigments was observed when \u003cem\u003eAmCYP76AD1\u003c/em\u003e and \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e were coexpressed in \u003cem\u003eN. benthamiana\u003c/em\u003e (Fig. 6a). In contrast, no betalain pigment was observed when \u003cem\u003eAmCYP76AD1\u003c/em\u003e and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e or \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e were coexpressed in \u003cem\u003eN. benthamiana\u003c/em\u003e (Fig. 6a). Only the coexpression of \u003cem\u003eAmCYP76AD1\u003c/em\u003e, \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e together was sufficient to produce high amounts of betalain pigments in \u003cem\u003eN. benthamiana\u003c/em\u003e, which resulted in a strong red-violet color (Fig. 6a). The strong red-violet color was similar to that in the positive control in which \u003cem\u003eBvCYP76AD1\u003c/em\u003e, \u003cem\u003eBvDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eMjcDOPA5GT\u003c/em\u003e were coexpressed in \u003cem\u003eN. benthamiana \u003c/em\u003e(Fig. 6a). As expected, the coexpression of \u003cem\u003eAmCYP76AD1\u003c/em\u003e, \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e, and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e only produced marginal levels of betalain pigments, which were barely detectable (Fig. 6a). Together with the comparable amount of proteins detected by western blotting (Fig. 6b), our results suggest that the enzyme activities of AmCYP76AD1, AmDODAa1, and AmDOPA5GT are sufficient to construct the core betalain biosynthesis pathway of \u003cem\u003eA. tricolor\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMolecular genetics have shed light on the betalain biosynthesis pathway and its evolutionary significance in Caryophyllales. Based on phylogenetic analysis, CYP76AD homologues can be classified into \u0026alpha;, \u0026beta;, and \u0026gamma; clades\u003csup\u003e9\u003c/sup\u003e. To date, only the functions of CYP76AD\u0026alpha; and CYP76AD\u0026beta; clade homologues, such as \u003cem\u003eCYP76AD1\u003c/em\u003e and \u003cem\u003eCYP76AD6\u003c/em\u003e, have been reported\u003csup\u003e10\u003c/sup\u003e. For example, the cosilencing of \u003cem\u003eCYP76AD1\u003c/em\u003e and \u003cem\u003eCYP76AD6\u003c/em\u003e represses the production of betacyanins and betaxanthins in \u003cem\u003eB. vulgaris\u003c/em\u003e, causing a green leaf phenotype\u003csup\u003e16\u003c/sup\u003e. In this study, a \u003cem\u003eCYP76AD6\u003c/em\u003e-like (\u003cem\u003eAmCYP76AD6\u003c/em\u003e) gene, belonging to the CYP76AD\u0026beta; clade according to phylogenetic construction and LOGO analysis (Fig. 2c, d), was also identified in \u003cem\u003eA. tricolor\u003c/em\u003e through transcriptome analysis (Supplementary Fig. S3). However, the expression of \u003cem\u003eAmCYP76AD6\u003c/em\u003e was extremely low and was difficult to detect in AMR and AMG. As a result, it is difficult to functionally connect \u003cem\u003eAmCYP76AD6\u003c/em\u003e with the production of betalains in \u003cem\u003eA. tricolor\u003c/em\u003e. In addition, although \u003cem\u003ePPO\u003c/em\u003e, a polyphenol oxidase gene, and \u003cem\u003eCATPO\u003c/em\u003e, a catalase-phenol oxidase gene, were previously proposed to be involved in betalain biosynthesis via monophenolase activity\u003csup\u003e34,35\u003c/sup\u003e, their transcripts did not show highly differential expression patterns between AMR and AMG (Fig. 1c, Supplementary Fig. S1c). As a result, we propose that the elevated expression of \u003cem\u003eAmCYP76AD1\u003c/em\u003e is necessary for the occurrence of a red-violet color phenotype in \u003cem\u003eA. tricolor\u003c/em\u003e; in contrast, the loss of \u003cem\u003eAmCYP76AD1\u003c/em\u003e expression results in a green color phenotype in \u003cem\u003eA. tricolor\u003c/em\u003e (Fig. 1a-c). The existence of the \u003cem\u003eAmCYP76AD1\u003c/em\u003e gene in AMG examined by PCR using genomic DNA as a template confirmed the loss of \u003cem\u003eAmCYP76AD1\u003c/em\u003e expression in AMG (Supplementary Fig. S4). Together with the functional characterization of the enzymatic activity of AmCYP76AD1 through the heterologous engineering of betalain pigments in \u003cem\u003eN. benthamiana\u003c/em\u003e (Fig. 3b, 6a), we conclude that AmCYP76AD1, a CYP76AD\u0026alpha; homologue required for the initiation of the betalain biosynthesis pathway, plays a key role in betalain pigment accumulation in \u003cem\u003eA. tricolor\u003c/em\u003e. Accordingly, the transcript levels of \u003cem\u003eAmCYP76AD1\u003c/em\u003e displayed a high correlation with betalain pigment contents (Fig. 2a, b).\u003c/p\u003e\n\u003cp\u003eIn recent years, with the elucidation of the central committed steps of the betalain biosynthesis pathway, comparative transcriptome analyses have been intensively applied to identify genes involved in regulating betalain biosynthesis in Caryophyllales\u003csup\u003e16,20,30,36,37\u003c/sup\u003e. However, numerous duplication events have led to difficulty in elucidating the functional activities of key enzymes in betalain-pigmented species through annotation\u003csup\u003e10\u003c/sup\u003e. For example, duplication events gave rise to two major clades of DODA homologues, DODAa and DODAb, but only one gene paralog in the DODAa clade of each species exhibits high levels of L-DOPA 4,5-dioxygenase activity\u003csup\u003e12,21\u003c/sup\u003e. Thus, it is necessary to examine the possible involvement of annotated genes in betalain biosynthesis on the basis of experimental evidence. In this study, the \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e and \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e genes, which belong to the DODAa clade according to phylogenetic construction and LOGO analysis (Fig. 4b, c), were identified in \u003cem\u003eA. tricolor\u003c/em\u003e through transcriptome analysis (Supplementary Fig. S2, Table S4). Based on the heterologous engineering of betalain pigments in \u003cem\u003eN. benthamiana\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e biochemical studies (Fig. 3b, 5b), we report that AmDODAa1 displayed a high level of L-DOPA 4,5-dioxygenase activity to produce betalamic acid, but such activity was barely detectable for AmDODAa2. These results indicate that at least one duplication event has occurred in the DODAa lineage of \u003cem\u003eA. tricolor\u003c/em\u003e, and the primary function of AmDODAa2 remains to be further studied.\u003c/p\u003e\n\u003cp\u003eBetalains are composed of betacyanins and betaxanthins. In contrast to betaxanthins, which are derived from betalamic acid via spontaneous condensation with amino acids or other amines, a large number of betacyanins are composed of betanidin conjugated with glycosyl moieties\u003csup\u003e9,10\u003c/sup\u003e. We characterized the function of \u003cem\u003eAmcDOPA5GT\u003c/em\u003e, a \u003cem\u003ecyclo\u003c/em\u003e-DOPA 5-\u003cem\u003eO\u003c/em\u003e-glucosyltransferase gene, through the heterologous engineering of betalain pigments in \u003cem\u003eN. benthamiana\u003c/em\u003e. The coexpression of \u003cem\u003eAmCYP76AD1\u003c/em\u003e, \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e enabled the production of high levels of betalain pigments with a dark red color (Fig. 6a). In contrast, low production of betalain pigments was observed when \u003cem\u003eAmCYP76AD1\u003c/em\u003e and \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e were coexpressed (Fig. 6a). Our results suggest the importance of \u003cem\u003eAmcDOPA5GT\u003c/em\u003e in the glycosylation reaction during betalain biosynthesis in \u003cem\u003eA. tricolor\u003c/em\u003e. In fact, the metabolic pathway of betalain biosynthesis is very complex due to multiple glycosylation steps, and different betacyanins have been identified\u003csup\u003e10,38\u003c/sup\u003e. For example, betanin, the most common betacyanin, is not only produced by\u003cem\u003e cyclo\u003c/em\u003e-DOPA 5-\u003cem\u003eO\u003c/em\u003e-glucosyltransferase but is also produced by betanidin 5-\u003cem\u003eO\u003c/em\u003e-glucosyl-transferase through the glycosylation of betanidin\u003csup\u003e39,40\u003c/sup\u003e. In this study, \u003cem\u003eAmB5GT\u003c/em\u003e, a betanidin 5-\u003cem\u003eO\u003c/em\u003e-glucosyl-transferase gene, was also identified through comparative transcriptome analyses (Supplementary Table S4). Although \u003cem\u003eAmcDOPA5GT\u003c/em\u003e showed higher expression levels than \u003cem\u003eAmB5GT\u003c/em\u003e in both AMR and AMG (Supplementary Table S4), it remains to be determined which of the two glycosylation routes is more important for the formation of betanin in \u003cem\u003eA. tricolor\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eRecently, betalain biosynthesis in different pitaya species, such as \u003cem\u003eHylocereus polyrhizus\u003c/em\u003e, \u003cem\u003eHylocereus costaricensis\u003c/em\u003e, \u003cem\u003eHylocereus undatus\u003c/em\u003e, and \u003cem\u003eHylocereus megalanthus\u003c/em\u003e, has been intensively studied through comparative transcriptome analysis\u003csup\u003e36,37,41,42\u003c/sup\u003e. However, further studies remain to be conducted to provide experimental evidence and strengthen the understanding of the roles of candidate genes in betalain biosynthesis. Here, complementation assays conducted through the heterologous engineering of betalain pigments in nonbetalain-producing plants provided a solution for the easy and rapid comparison of the functional activities of genes involved in the core betalain biosynthesis pathway between betalain-pigmented species of Caryophyllales. Using the coexpression of \u003cem\u003eBvCYP76AD1\u003c/em\u003e, \u003cem\u003eBvDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eMjcDOPA5GT\u003c/em\u003e in \u003cem\u003eN. benthamiana\u003c/em\u003e as a positive control, the functional activities of\u003cem\u003e A. tricolor\u003c/em\u003e genes responsible for betalain synthesis could be compared through a series of complementation assays (Fig. 3b, c). We showed that comparable amounts of betalain pigments were observed when the functional activities of positive genes were individually replaced with \u003cem\u003eAmCYP76AD1\u003c/em\u003e, \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e in transient coexpression assays (Fig. 3b, c). Our results indicate that \u003cem\u003eAmCYP76AD1\u003c/em\u003e, \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e exhibit high tyrosinase, L-DOPA 4,5-dioxygenase, and\u003cem\u003e cyclo\u003c/em\u003e-DOPA 5-\u003cem\u003eO\u003c/em\u003e-glucosyltransferase activities, respectively, which are similar to those in \u003cem\u003eB. vulgaris\u003c/em\u003e and\u003cem\u003e M. jalap\u003c/em\u003ea. Accordingly, \u003cem\u003ein vitro\u003c/em\u003e biochemical studies demonstrated that AmDODAa1 displayed comparable L-DOPA 4,5-dioxygenase activity to BvDODAa1 in producing betalamic acid (Fig. 5b). These results provide novel insights into betalain biosynthesis and evolution in \u003cem\u003eA. tricolor\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn conclusion, a comparative transcriptome analysis combined with functional and enzymatic studies were performed to reveal the core betalain biosynthesis pathway of \u003cem\u003eA. tricolor.\u003c/em\u003e The heterologous engineering of betalain pigments through the coexpression of \u003cem\u003eAmCYP76AD1\u003c/em\u003e, \u003cem\u003eAmDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eAmcDOPA5GT\u003c/em\u003e in \u003cem\u003eN. benthamiana\u003c/em\u003e enabled the production of high amounts of betalain pigments with a red-violet color similar to those in the red-leaf cultivar of \u003cem\u003eA. tricolor\u003c/em\u003e. Although the metabolic pathway of betalain biosynthesis is very complex, the core betalain biosynthesis pathway of \u003cem\u003eA. tricolor\u003c/em\u003e constructed here not only provides a basal framework for examining genes related to betalain biosynthesis within the species of \u003cem\u003eAmaranthaceae\u003c/em\u003e but also sheds light on the evolution of the betalain biosynthesis pathway in Caryophyllales.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growth \u003c/strong\u003e\u003cstrong\u003econditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA. tricolor\u003c/em\u003e, \u003cem\u003eB. vulgaris\u003c/em\u003e, \u003cem\u003eM. jalapa\u003c/em\u003e, and \u003cem\u003eN. benthamiana\u003c/em\u003e plants were grown at 26\u0026deg;C in a semicontrolled walk-in chamber under a 16:8-h light:dark photoperiod. Soil (Jiffy) mixed with vermiculite and pearlstone was used. Seeds of \u003cem\u003eA. tricolor\u003c/em\u003e cv. Hung Hsien (red-leaf cultivar) and \u003cem\u003eA. tricolor\u003c/em\u003e cv. Pai Hsien (green-leaf cultivar) were purchased from KNOWN-YOU SEED CO., LTD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBetalain \u003c/strong\u003e\u003cstrong\u003epigment\u003c/strong\u003e\u003cstrong\u003e extraction and measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor betalain pigment measurement, betalain contents were determined as described previously with some modification\u003csup\u003e43\u003c/sup\u003e. Briefly, leaves of seedlings were collected and ground into powder in liquid nitrogen. Betalain pigments were extracted with extraction buffer (methanol:chloroform:H\u003csub\u003e2\u003c/sub\u003eO [1:2:1]). After centrifugation, the upper (hydrophilic) layer was collected to measure the absorbance at 538 nm and 476 nm for betacyanins and betaxanthins, respectively. The relative betalain content was calculated with the following equation: (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e538\u003c/sub\u003e\u0026nbsp;+\u0026nbsp;\u003cem\u003eA\u003c/em\u003e\u003csub\u003e476\u003c/sub\u003e)/gram).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll plasmid constructs were generated using standard restriction site reconstruction methods and confirmed by DNA sequencing. \u003cem\u003eAm\u003c/em\u003e\u003cem\u003eCYP76AD1\u003c/em\u003e, \u003cem\u003eAm\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, \u003cem\u003eAm\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e, \u003cem\u003eAm\u003c/em\u003e\u003cem\u003ecDOPA5GT\u003c/em\u003e, \u003cem\u003eBv\u003c/em\u003e\u003cem\u003eCYP76AD1\u003c/em\u003e, \u003cem\u003eBv\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eMj\u003c/em\u003e\u003cem\u003ecDOPA5GT\u003c/em\u003e were amplified from \u003cem\u003eA. tricolor\u003c/em\u003e, \u003cem\u003eB. vulgaris\u003c/em\u003e, or \u003cem\u003eM. jalapa\u003c/em\u003e cDNA libraries using AccuPrime pfx DNA polymerase (Invitrogen). For the transient expression of C-terminal YFP- or FLAG (SFP)-tagged proteins in \u003cem\u003eN. benthamiana\u003c/em\u003e,\u0026nbsp;PCR products encoding\u003cem\u003e Am\u003c/em\u003e\u003cem\u003eCYP76AD1\u003c/em\u003e, \u003cem\u003eAm\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, \u003cem\u003eAm\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e, \u003cem\u003eAm\u003c/em\u003e\u003cem\u003ecDOPA5GT\u003c/em\u003e, \u003cem\u003eBv\u003c/em\u003e\u003cem\u003eCYP76AD1\u003c/em\u003e, \u003cem\u003eBv\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eMj\u003c/em\u003e\u003cem\u003ecDOPA5GT\u003c/em\u003e were subcloned into pBA-C-SFP or pBA-C-YFP vectors under the control of a \u003cem\u003eCauliflower mosaic virus\u003c/em\u003e\u0026nbsp;(\u003cem\u003eCaMV\u003c/em\u003e) \u003cem\u003e35S\u003c/em\u003e promoter\u003csup\u003e44\u003c/sup\u003e. To produce N-terminal SUMO-tagged recombinant proteins, PCR products encoding \u003cem\u003eAm\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, \u003cem\u003eAm\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e, and \u003cem\u003eBv\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e were subcloned into the pET-SUMO (Invitrogen) vector\u003csup\u003e45\u003c/sup\u003e. For the VIGS assay, a cDNA fragment of \u003cem\u003eAm\u003c/em\u003e\u003cem\u003eCYP76AD1\u003c/em\u003e was amplified and subcloned into the pTRV2 vector\u003csup\u003e46\u003c/sup\u003e. The primer sequences used for plasmid construction are listed in Supplementary Table S5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time polymerase chain reaction (qRT-PCR) and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTRIzol\u003csup\u003eTM\u003c/sup\u003e (Invitrogen)-extracted total RNA was reverse transcribed using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) according to the manufacturer\u0026rsquo;s instructions. Briefly, each sample was prepared from the leaves of three biologically distinct 3-week-old\u0026nbsp;or 4-week-old \u003cem\u003eA. tricolor\u003c/em\u003e\u0026nbsp;plants. Then, cDNA was synthesized from 1 \u0026mu;g of total RNA using a mixture of random hexamers and oligo(dT)\u003csub\u003e20\u003c/sub\u003e under the following conditions: 25\u0026deg;C for 10 min, followed by 50\u0026deg;C for 40 min. The cDNA was employed as a template for qRT-PCR using the KAPA SYBR Fast qPCR Kit (Kapa Biosystems). Three technical replicates were performed on a CFX96\u003csup\u003eTM\u003c/sup\u003e Real-time System (Bio-Rad) under the following conditions: 95\u0026deg;C for 3 min, followed by 40 cycles of 95\u0026deg;C for 10 s and 55\u0026deg;C for 30 s. The expression levels of selected genes were determined by normalization to the reference gene\u003cem\u003e Actin\u003c/em\u003e. Statistically significant differences were determined using Student\u0026rsquo;s\u0026nbsp;\u003cem\u003et\u003c/em\u003e-test in SPSS version 20.0. The primer sequences employed for qRT-PCR analyses are listed in Supplementary Table S1. PCR analyses using genomic DNA extracted from AMR and AMG as a template were performed to confirm the specificity of the primers (Supplementary Fig. S4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransient \u003c/strong\u003e\u003cstrong\u003ecoexpression\u003c/strong\u003e\u003cstrong\u003e assay and western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmids for the transient expression of AmCYP76AD1-YFP, AmDODAa1-SFP, AmDODAa2-SFP, AmcDOPA5GT-SFP, BvCYP76AD1-YFP, BvDODAa1-SFP, or MjcDOPA5GT-SFP were transformed into the \u003cem\u003eAgrobacterium tumefaciens \u003c/em\u003estrain ABI. C-terminal tagged proteins were coexpressed using a mixture of \u003cem\u003eA. tumefaciens \u003c/em\u003ecarrying the desired constructs in \u003cem\u003eN. benthamiana \u003c/em\u003eleaves by agroinfiltration following the method described previously\u003csup\u003e47\u003c/sup\u003e. After three days, the infiltrated leaves were photographed and ground into a powder in liquid nitrogen for total cell extract preparation. Briefly, 0.1 g of sample powder was added to 0.2 ml of 2.5\u0026times; SDS sample buffer (5 mM EDTA, 5% SDS, 0.3 M Tris\u0026ndash;HCl, pH 6.8, 20% glycerol, 1% \u0026beta;-mercaptoethanol, and bromophenol blue), which was then heated at 95\u0026deg;C in a dry bath for 10 min. After centrifugation at 13,000\u0026times; \u003cem\u003eg \u003c/em\u003efor 10 min, the supernatant was obtained, and total proteins were separated by SDS-PAGE. Western blotting assays were performed to monitor protein levels using specific polyclonal and monoclonal antibodies against YFP- and FLAG-tag, respectively. Chemiluminescence signals generated by ECL reagents (PerkinElmer) were captured with an ImageQuant LAS 4000 mini imager (GE Healthcare). All experiments were repeated at least three times using biologically distinct samples prepared from two infiltrated leaves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eL-DOPA 4,5-dioxygenase activity assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn\u003cem\u003e in\u003c/em\u003e\u003cem\u003e vitro\u003c/em\u003e L-DOPA 4,5-dioxygenase activity assay was performed according to the method described previously with some modifications\u003csup\u003e32\u003c/sup\u003e. Briefly, plasmids for the expression of N-terminal SUMO-tagged \u003cem\u003eAm\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e, \u003cem\u003eAm\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e and \u003cem\u003eBv\u003c/em\u003e\u003cem\u003eDODA\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e were transformed into \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003estrain BL21 (DE3). The transformants were grown in 50 ml LB medium, and the recombinant proteins were induced with 0.2 mM IPTG at 22\u0026deg;C for 16 hours. Harvested cells were washed, resuspended, and disrupted by sonication in 50 mM sodium phosphate buffer (pH 7.0). The crude extract (supernatant) was used for the enzyme activity assay after centrifugation at 14,000x \u003cem\u003eg\u003c/em\u003e for 15 min. The amount of recombinant protein was quantified with Protein Assay Reagent (Bio-Rad) and via Coomassie blue staining SDS-PAGE with BSA as the standard. Basically, the reaction (100 \u0026mu;l) was performed with the crude extract containing 8 \u0026mu;g DODA protein, 27 mM ascorbic acid, and 6.75 mM L-DOPA at 30\u0026deg;C for 5 min.\u003c/p\u003e\n\u003cp\u003eLC-MS/MS was performed using a Dionex UltiMate 3000 system (Thermo Fisher Scientific) linked with an amaZon speed-ion trap mass spectrometer (Bruker). Betalamic acid was detected on a Waters BEH shield RP18 column with two eluting solvent systems: (A) H\u003csub\u003e2\u003c/sub\u003eO with 0.1% formic acid, (B) 100% acetonitrile. The gradient elution program was set as follows: 0-3 min (100% A), 9 min (55% A and 45% B), 12-13 min (100% B). The flow rate was 0.3 ml min\u003csup\u003e-1\u003c/sup\u003e, and the detector wavelength was 424 nm. The electrospray ionization mass parameters were set as follows: 4.5 kV capillary, 500 V end plate offset voltage, 40.0 psi nebulizer pressure, 8.0 l min\u003csup\u003e-1\u003c/sup\u003e dry gas, and 230\u0026deg;C dry temperature. The measurement was operated in multiple reaction-monitoring (MRM) with the positive ion mode.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNext-generation sequencing and MA plot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo perform next-generation sequencing, aerial tissues derived from three biologically distinct 3-week-old\u0026nbsp;\u003cem\u003eA. tricolor\u003c/em\u003e plants were collected. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer\u0026rsquo;s instructions. RNA quality was examined via 1.2% (wt/vol) formaldehyde gel electrophoresis and with an Experion RNA analysis kit (Bio-Rad, Munich). Only high-quality RNA was used for next-generation sequencing performed on the Illumina HiSeq 4000 platform with 150 paired-end reads. For each dataset (AMR and AMG), 100 million reads were generated, and de novo assembly was performed with the Trinity tool. The assembled transcripts were annotated with BlastX in UniProt. Gene expression levels were normalized as FPKM values, and differentially expressed genes were identified according to an\u0026nbsp;FDR\u0026nbsp;\u0026lt; 0.05 and logFC \u0026gt; 2 or \u0026lt; -2 (Supplementary Table S2, S3). An MA plot was generated based on the average concentration (logCPM) and fold-change (logFC) values to show the relative abundances of transcripts between AMR and AMG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic tree reconstruction and LOGO analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetic trees were reconstructed using MEGA-X software based on the protein sequence comparisons of CYP76AD and DODA homologues from different betalain-producing species. Multiple sequence alignments were performed using the MUSCLE program and were processed to generate a maximum likelihood phylogenetic tree via the Jones-Taylor-Thornton (JTT) model with bootstrapping to perform molecular evolutionary analysis. The numbers at the branch points are bootstrap values representing the percentages of replicate trees based on 1000 repeats. LOGO analyses were performed via WebLogo (http://weblogo.berkeley.edu/logo.cgi) based on selected conserved amino acids of CYP76AD and DODA homologues reported previously\u003csup\u003e9,12,21,48\u003c/sup\u003e. The species, families, and accession numbers of CYP76AD and DODAa homologues are available in Supplementary Table S6.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported (in part) by grants-in-aid from the Ministry of Science and Technology (MOST-109-2628-B-005-006) and the Advanced Plant Biotechnology Center from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.-C.C. carried out most of the experiments; Y.-C.C. and Y.-L.C. designed and carried out the enzymatic assays. N.-W.T. performed the LC-MS analysis. C.-M.T., Y.-H.C., and P.-C.L. performed RNA-seq and plasmid constructions. Y.-C.L., L.-C.H. and S.-Y.W. contributed to the interpretation of data. J.-Y.Y. conceived and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePolturak, G. \u0026amp; Aharoni, A. \u0026ldquo;La Vie En Rose\u0026rdquo;: biosynthesis, sources, and applications of betalain pigments. \u003cem\u003e Plant\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, 7-22 (2018).\u003c/li\u003e\n\u003cli\u003eJain, G. \u0026amp; Gould, K.S. Are betalain pigments the functional homologues of anthocyanins in plants? \u003cem\u003e Exp. Bot. \u003c/em\u003e\u003cstrong\u003e119\u003c/strong\u003e, 48-53 (2015).\u003c/li\u003e\n\u003cli\u003eLi, G., Meng, X., Zhu, M. \u0026amp; Li, Z. 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Tyrosine hydroxylation in betalain pigment biosynthesis is performed by cytochrome P450 enzymes in beets (\u003cem\u003eBeta vulgaris\u003c/em\u003e). \u003cem\u003ePLoS ONE\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, e0149417; 1371/journal.pone.0149417 (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Amaranthus tricolor, betalain, CYP76AD1, DODA, cDOPA5GT","lastPublishedDoi":"10.21203/rs.3.rs-110761/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-110761/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmaranthus tricolor L., a vegetable Amaranthus species, is an economically important crop containing large amounts of betalains. Betalains are natural antioxidants and can be classified into betacyanins and betaxanthins, with red and yellow colors, respectively. A. tricolor cultivars with varying betalain contents, leading to striking red to green coloration, have been commercially produced. However, the molecular differences underlying betalain biosynthesis in various cultivars of A. tricolor remain largely unknown. In this study, A. tricolor cultivars with different colors were chosen for comparative transcriptome analysis. The elevated expression of AmCYP76AD1 in a red-leaf cultivar of A. tricolor was proposed to play a key role in producing red betalain pigments. The functions of AmCYP76AD1,\u0026nbsp;AmDODAa1, AmDODAa2, and AmcDOPA5GT were also characterized through the heterologous engineering of betalain pigments in Nicotiana benthamiana. Moreover, high and low L-DOPA 4,5-dioxygenase activities of AmDODAa1 and AmDODAa2, respectively, were confirmed through in vitro enzymatic assays. Thus, comparative transcriptome analysis combined with functional and enzymatic studies allowed the construction of a core betalain biosynthesis pathway of A. tricolor. These results not only provide novel insights into betalain biosynthesis and evolution in A. tricolor but also provide a basal framework for examining genes related to betalain biosynthesis among different species of Amaranthaceae.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAccession numbers: \u003c/strong\u003eThe nucleotide sequences reported in this article have been submitted to [GenBank] under accession numbers [MT740230, MT741954, MT741955, MT741956].\u003c/p\u003e","manuscriptTitle":"Elucidation of the Core Betalain Biosynthesis Pathway in Amaranthus tricolor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-25 19:38:35","doi":"10.21203/rs.3.rs-110761/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-12-30T11:28:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-12-04T16:34:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6b1ae7a2-8511-492f-8071-4386ed7ee511","date":"2020-11-26T10:50:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1c4ee8a4-f078-460b-8a55-008fce51a5ba","date":"2020-11-23T04:58:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-11-23T04:16:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-11-21T05:21:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-11-20T13:10:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-11-20T09:52:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2020-11-18T06:50:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d964e0e5-1be8-4481-8423-e5710258610a","owner":[],"postedDate":"November 25th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1203356,"name":"General Biochemistry"},{"id":1203357,"name":"Epigenetics \u0026 Genomics"},{"id":1203358,"name":"Bioinformatics"}],"tags":[],"updatedAt":"2021-08-18T19:33:15+00:00","versionOfRecord":{"articleIdentity":"rs-110761","link":"https://doi.org/10.1038/s41598-021-85486-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2021-03-17 19:02:42","publishedOnDateReadable":"March 17th, 2021"},"versionCreatedAt":"2020-11-25 19:38:35","video":"","vorDoi":"10.1038/s41598-021-85486-x","vorDoiUrl":"https://doi.org/10.1038/s41598-021-85486-x","workflowStages":[]},"version":"v1","identity":"rs-110761","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-110761","identity":"rs-110761","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}