1. Volume 11, Issue 1, Pages 189-204 (January 2018)
Transcriptome and Metabolic Profiling Provides Insights into Betalain Biosynthesis and Evolution in Mirabilis jalapa 
Guy Polturak, Uwe Heinig, Noam Grossman, Maor Battat, Dena Leshkowitz, Sergey Malitsky, Ilana Rogachev, Asaph Aharoni 
Molecular Plant 
Volume 11, Issue 1, Pages (January 2018)
DOI: /j.molp
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2. Figure 1 The Betalain Biosynthetic Pathway.
Genes taking part in the pathway are shown in magenta. Enzymatic reactions: EI, hydroxylation of tyrosine; EII, DOPA-4,5 dioxygenase; EIII, oxidation of DOPA; EIV, glucosylation of cyclo-DOPA; EV, glucosylation of betanidin; EVI, additional glucosylation of betacyanins; EVII, acylation of betacyanins. Predicted spontaneous reactions: SI, condensation (aldimine formation). Enzymatic reactions EII and EIII are followed by a spontaneous cyclization reaction and are therefore marked with an asterisk. EV can alternatively be catalyzed by a betanidin 6-O-glucosyltransferase, leading to formation of gomphrenin instead of betanin. Dashed lines designate reactions of an alternative pathway, which was shown to occur in Mirabilis jalapa, in which cyclo-DOPA is first glycosylated and then condensates with betalamic acid to form betanin.
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3. Figure 2
Quantitative RT–PCR and Co-expression Analyses of Betalain-Related Candidate Genes in M. jalapa.
(A) qRT–PCR analysis of candidate genes in M. jalapa floral tissues. Relative expression of 17 M. jalapa genes in mature petal, immature petal, and sepal tissues was analyzed by qRT–PCR. Six genes showed high relative expression levels in mature petal versus other tissues. These included three glycosyltransferases (Mj2, Mj11, Mj12), two BAHD acyltransferases (Mj6, Mj10), and an ABC transporter (Mj7). Values indicate means of three biological replicates ±SD. Asterisks denote statistical significance of differential expression in comparison with mature petals. *p < 0.05; **p < 0.01.
(B) In a co-expression analysis of a transcriptome dataset derived from 24 M. jalapa tissues, UDP-glycosyltransferase (Mj2) was found to be highly co-expressed with the betalain-related cyclo-dopa-5-O-glucosyltransferase (cDOPA5GT; Pearson correlation r = 0.93). Two BAHD acyltransferases (Mj6, Mj10) co-expressed with the betalain-related cytochrome P450 CYP76AD3 (r = 0.95 and 0.93, respectively). Data of CYP76AD3 and cDOPA5GT expression in the M. jalapa transcriptome dataset were also previously shown in Polturak et al. (2016).
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4. Figure 3
Recombinant Expression of CYP76AD15 in Nicotiana benthamiana Enables Betaxanthin and L-DOPA Production.
(A) Co-infiltration of agrobacteria harboring plasmids for expression of CYP76AD15 and BvDODA1 in N. benthamiana leaves causes yellow pigmentation in the infiltrated area.
(B) LC–MS analysis of yellow-pigmented tissue shows occurrence of several betaxanthins, including dopamine-betaxanthin [M + H = 347.1] and valine-betaxanthin [M + H = 311.1]. XIC, extracted ion chromatogram.
(C) UV–VIS absorption of peaks representing dopamine-betaxanthin and valine-betaxanthin in analyzed tissue.
(D) Mass spectra of L-DOPA peak identified in leaf tissue of N. benthamiana expressing CYP76AD15 shows L-DOPA molecular ion [M + H = ] and typical transitions [m/z 198 → 181, 152, 139].
Molecular Plant  , DOI: ( /j.molp )
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5. Figure 4
Transient Expression of Mirabilis jalapa Hydroxycinnamate Glucosyltransferase (MjHCGT) in Nicotiana benthamiana.
(A) Expression of MjHCGT in N. benthamiana leaves leads to accumulation of hydroxycinnamoyl-glucoses (HCGs), detected by LC–MS analysis. HCGs were not detected following expression of empty plasmid (3α1). Shown are extracted ion chromatograms (XIC) of masses corresponding to cinnamoyl-glucose (+Na+) (m/z = 333.1), coumaroyl-glucose (+Na+) (m/z = 349.1), caffeoyl-glucose (+Na+) (m/z = 365.1), and feruloyl-glucose (+Na+) (m/z = 379.1). 1, 1-O-caffeoyl-β-D-glucose; 2, 1-O-coumaroyl-β-D-glucose isomer I; 3, 1-O-coumaroyl-β-D-glucose isomer II; 4, 1-O-feruloyl-β-D-glucose; 5, 1-O-cinnamoyl-β-D-glucose.
(B) Expression of MjHCGT together with betalain biosynthetic genes (pX11 vector) in N. benthamiana leads to formation of acylated betalains. Acylated betalains were not detected when pX11 was co-expressed with empty plasmid (3α1). Shown are extracted ion chromatograms (XIC) of masses corresponding to cinnamoyl-betanin (m/z = 681.2), coumaroyl-betanin (m/z = 697.2), caffeoyl-betanin (m/z = 713.2), feruloyl-betanin (m/z = 727.2), and their respective isomers. 1′, caffeoyl-betanin isomer I; 2′, caffeoyl-betanin isomer II; 3′, coumaroyl-betanin isomer I; 4′, coumaroyl-betanin isomer II; 5′, feruloyl-betanin isomer I; 6′, feruloyl-betanin isomer II; 7′, cinnamoyl-betanin isomer I; 8′, cinnamoyl-betanin isomer II.
(C) MS/MS fragmentation spectrum of 1-O-caffeoyl-glucose including structure of the putatively identified molecule. The main ion M + Na+, the molecular ion M + H+, and three major fragments are indicated.
(D) Reaction scheme for acyl-glucose-dependent acylation of betanin in M. jalapa. MjHCGT catalyzes formation of hydroxycinnamoyl-glucoses from UDP-glucose and free hydroxycinnamic acids (e.g., caffeic acid, shown in figure). HCGs serve as acyl donors for formation of acylated betacyanins by a currently unidentified acyl-glucose-dependent acyltransferase, most likely from the serine-carboxy-peptidase-like (SCPL) family.
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6. Figure 5
An ANTHOCYANIDIN SYNTHASE Homolog (MjANS) Is Expressed in Mirabilis jalapa
(A) Quantitative RT–PCR analysis of MjANS expression in three M. jalapa floral tissues. Values indicate means of three biological replicates ±SD. Asterisks denote statistical significance of differential expression in comparison with mature petals. ***p <
(B) ANS typically converts leucoanthocyanidins to anthocyanidins (e.g., leucopelargonidin to pelargonidin) in a 2-oxoglutarate-dependent oxygenase reaction.
(C) Sequence truncation is found in both MjANS transcript and gene sequences, indicative of a gene deletion mutation rather than a splicing event. Blue text shows last four codons before deletion, red text shows first four codons after deletion.
(D) Protein sequence alignment of M. jalapa anthocyanidin synthase (MjANS). MjANS is aligned with ANS orthologs from pokeweed (Phytolacca americana, accession BAE ), spinach (Spinacia oleracea, accession BAE ), buckwheat (Fagopyrum esculentum, accession ADT ), and carnation (Dianthus caryophyllus, accession AAB ). All plant species belong to the Caryophyllales order. Pokeweed and spinach are betalain producers, while buckwheat and carnation produce anthocyanins. Sequence of the 2OG-Fe(II) oxygenase superfamily domain (pfam03171) is indicated by a red box. The MjANS deletion area is highlighted.
(E) Expression of MjANS and MjDFR in Arabidopsis mutants. ANTHOCYANIDIN SYNTHASE genes from M. jalapa (MjANS) or Gerbera hybrida (GhANS), driven by a CaMV 35S promoter were expressed in the background of the Arabidopsis tds4-4 mutant (ans). Seed and seedling pigmentation showed that expression of 35S::GhANS in the Arabidopsis ans mutant restores the wild-type phenotype, while expression of 35S::MjANS in the same background does not. DIHYDROFLAVONOL-4-REDUCTASE gene from M. jalapa (MjDFR), expressed in the background of the Arabidopsis tt3 mutant (dfr), restored the wild-type phenotype. Top rows, 3- to 4-day-old seedlings, grown in anthocyanin-inducing conditions (constant light, 5% sucrose); bottom rows, wild-type (WT) or transformant seeds, observed under a binocular microscope.
Molecular Plant  , DOI: ( /j.molp )
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7. Figure 6
Expression Profile of Anthocyanin/Flavonoid Pathway Genes in M. jalapa Flowers
(A) Anthocyanin biosynthesis from phenylalanine. Flavonols can be further modified by glycosylation to produce flavonol glycosides.
(B) Analysis of M. jalapa petals RNA-seq data shows expression of all genes of the anthocyanin/flavonoid pathway from PAL to 3-GT. Five developmental stages of petals are ordered from left to right (shown in top left diagram). Normalized count values for each library (each developmental stage) are calculated as the number of reads per contig divided by the total number of reads. Values represent one biological replicate. PAL, PHENYLALANINE AMMONIA LYASE; C4H, CINNAMATE 4-HYDROXYLASE; 4CL, 4-COUMARATE-CoA LIGASE; CHS, CHALCONE SYNTHASE; CHI, CHALCONE ISOMERASE; F3H, FLAVANONE 3-HYDROXYLASE; FLS, FLAVONOL SYNTHASE; DFR, DIHYDROFLAVONOL REDUCTASE; ANS, ANTHOCYANIDIN SYNTHASE; 3-GT, ANTHOCYANIDIN 3-O-GLUCOSYLTRANSFERASE; Qu, quercetin; Ka, kaempherol; Is, isorhamnetin; Pet, petal.
Molecular Plant  , DOI: ( /j.molp )
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8. Figure 7
Identification of Flavonol Glycosides in M. jalapa Mature Petals with LC–MS
Total ion chromatogram (TIC) in positive ionization mode and extracted ion chromatograms (XIC) of masses corresponding to kaempferol (m/z = 287.1), isorhamnetin (m/z = 317.1), and quercetin (m/z = 303.1). 1, Kaempferol-Hexose-di-Deoxyhexose isomer I; 2, Kaempferol-Hexose-di-Deoxyhexose isomer II; 3, Kaempferol-Hexose-Deoxyhexose; 4, Isorhamnetin-Hexose-di-Deoxyhexose; 5, Isorhamnetin-Hexose-Deoxyhexose isomer I; 6, Isorhamnetin-Hexose-Deoxyhexose isomer II; 7, Quercetin-Hexose-di-Deoxyhexose; 8, Quercetin-Deoxyhexose-Hexose; 9, Quercetin-3-O-rutinoside (rutin). AU, absorbance units. UV–VIS absorption spectra (PDA) and MS spectra of rutin standard and rutin in M. jalapa are shown.
Molecular Plant  , DOI: ( /j.molp )
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