Phytochemistry 117 (2015) 267–295Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Review Plant betalains: Chemistry and biochemistry Mohammad Imtiyaj Khan ⇑, P. Giridhar Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore 570020, India a r t i c l e i n f o a b s t r a c t Article history: Betalains are vacuolar pigments composed of a nitrogenous core structure, betalamic acid [4-(2-ox- Received 6 January 2015 oethylidene)-1,2,3,4-tetrahydropyridine-2,6-dicarboxylic acid]. Betalamic acid condenses with imino Received in revised form 29 May 2015 compounds (cyclo-L-3,4-dihydroxy-phenylalanine/its glucosyl derivatives), or amino acids/derivatives Accepted 2 June 2015 to form variety of betacyanins (violet) and betaxanthins (yellow), respectively. About 75 betalains have been structurally unambiguously identified from plants of about 17 families (known till date) out of 34 families under the order Caryophyllales, wherein they serve as chemosystematic markers. In this review, Keywords: all the identified betalain structures are presented with relevant discussion. Also, an estimated annual Betacyanins Betaxanthins production potential of betalains has been computed for the first time. In addition, mutual exclusiveness Biosynthesis of anthocyanins and betalains has been discussed in the wake of new evidences. An inclusive list of beta- Regulation lain-accumulating plants reported so far has been presented here to highlight pigment occurrence and Ecophysiological factors accumulation pattern. Betalain synthesis starts with hydroxylation of tyrosine to DOPA, and subsequent cleavage of aromatic ring of DOPA resulting to betalamic acid formation. This pathway consists of two key enzymes namely, bifunctional tyrosinase (hydroxylation and oxidation) and DOPA dioxygenase (O2-de- pendent aromatic ring cleavage). Various spontaneous cyclisation, condensation and glucosylation steps complement the extended pathway, which has been presented here comprehensively. The biosynthesis is affected by various ecophysiological factors including biotic and abiotic elicitors that can be manipulated to increase pigment production for commercial scale extraction. Betalains are completely safe to con- sume, and contribute to health. Ó 2015 Elsevier Ltd. All rights reserved. Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 2. Betalain structure, occurrence, and commercial production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 2.1. Structure elucidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 2.1.1. Betaxanthins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 2.1.2. Betacyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 2.2. Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 2.2.1. Mutual exclusiveness of anthocyanins and betalains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 2.2.2. Accumulation in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 2.3. Commercial production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 3. Biosynthesis of betalains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 3.1. Biosynthesis pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 3.2. Tyrosinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 3.3. DOPA dioxygenase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 3.4. Regulation of betalain biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 4. Ecophysiological factors influencing betalain accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 ⇑ Corresponding author present address: Department of Biotechnology, Gauhati University, Guwahati 781014, Assam, India. E-mail addresses:
[email protected],
[email protected] (M.I. Khan). http://dx.doi.org/10.1016/j.phytochem.2015.06.008 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved. 268 M.I. Khan, P. Giridhar / Phytochemistry 117 (2015) 267–295 4.1. Influence of light and other physical factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 4.2. Abiotic and biotic elicitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 5. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 1. Introduction a number of studies on antioxidant and biological activities vis-a- vis bioavailaibility of betalains. Several researchers have presented Betalains are vacuolar nitrogen containing pigments having a periodic updates in the past on betalain structure identification, core structure (protonated 1,2,4,7,7-pentasubstituted 1,7-diaza- occurrence (Strack et al., 1993, 2003; Cai et al., 2005), stability, heptamethin system) known as betalamic acid [4-(2-oxoethyli- chromatic and functional importance (Delgado-Vargas et al., dene)-1,2,3,4-tetrahydropyridine-2,6-dicarboxylic acid] (1). 2000; Herbach et al., 2006; Stintzing and Carle, 2008; Moreno Condensation of betalamic acid with cyclo-DOPA (L-3,4-dihy- et al., 2008), chemosystematics (Brockington et al., 2011), produc- droxy-phenylalanine)/its glucosyl derivatives, and amino acids/its tion in vitro (Georgiev et al., 2008), biosynthesis (Strack et al., 2003; derivatives leads to formation of two categories of betalains viz. Gandía-Herrero and García-Carmona, 2013), and biological activity violet betacyanins and yellow betaxanthins, respectively (Esatbeyoglu et al., 2014; Gandía-Herrero et al., 2014). However, in (Fig. 1a and b). Until 1957, betalains were considered as antho- past two decades, there has been no update on the chemistry vis-a- cyanins (Fig. 1c) or more precisely nitrogenous anthocyanins vis biosynthesis and ecophysiological factors regulating betalain (Fig. 1d) (Peterson and Joslyn, 1958), however crystallisation of accumulation, and elicitation in situ/ex situ for improving betalain betanin and its hydrolysis to betanidin (Wyler and Dreiding, production. An estimate of the annual production potential of beta- 1957), and subsequent report on isolation of indicaxanthin lains has been computed first time and relevant future directions of (Piattelli et al., 1964a) provided evidence that they were a different studies on occurrence/production, ecophysiological factors affect- set of pigments containing a 1,7-diazaheptamethin system (Fig. 1e) ing biosynthesis of betalains have been discussed. responsible for their chroma (Mabry et al., 1967). This concept was further strengthened by the unique biosynthetic pathway of beta- 2. Betalain structure, occurrence, and commercial production nin (Miller et al., 1968) and indicaxanthin (Impellizzeri and Piattelli, 1972) through DOPA incorporation. 2.1. Structure elucidation Over the past five and half decades, flurry of research activities has generated enormous literature on betalains, including some Owing to their hydrophilic nature, betalain extraction from misunderstandings about their chemical nature. Because of pres- plant sources could be in pre-cooled water, aq. methanol, or ence of ‘1,7-diaza’, some researchers claimed it as chromoalkaloids methanol at pH 5 supplemented with ascorbic acid (Strack et al., (Reznik, 1981), but the stability in slightly acidic pH seem to 2003), preferably, at 0.25% (w/v) for enhanced stability (Khan negate the claim (Delgado-Vargas et al., 2000). Concern about bee- and Giridhar, 2014a). The extracts could be processed through pre- turia as a result of betalain consumption was raised before it was cipitation of the pectic substances, and ion-exchange chromatogra- dispelled by a systematic review that attributed beeturia to varia- phy for removal of free sugars and organic acids (Stintzing et al., tion in the ability to metabolise food products (Mitchell, 2001). The 2002a). Further, purification of betalains may involve either con- fact that beeturia was not a phenomenon to worry about prompted ventional anion-exchange column chromatography on Dowex 4 R 1O 5 9 3 a H b H R c R1 R 2O 6 8 N+ 2 COOH N + 7 10 OH 11 2 + B 12 3 O HO 13 R2 14 18 4 A C H H OGlc 16 HOOC 15 N 17 COOH HOOC 5 N COOH 19 H 20 H OH Betacyanins R= amino acid, amine or R1= OH, R2= H; Cyanidin-3-glc (Anthocyanin) derivatives: Betaxanthins R1= H, R2= H; Betanidin HO HO O d OH e R1= HO (glucosyl) or derivatives, R2= H; + OH Betanin group RO O OH HO Bougainvillein-r group HO O + N N N R1= HO (2-glucuronyl glucosyl) or derivatives, H HOOC R2= H; Amaranthin group HO O O OH HO R= Glc; Nitrogenous anthocyanin 1,7-Diazaheptamethin OH R1= H, R2= Glucosyl or derivatives; Gomphrenin group Bougainvillein-v group Fig. 1. Representative structures. During the early years of structure elucidation of betalains. pres. owing to the requirement of acidic chemical and enzymatic hydrolysis. 2006) or size-exclusion chromatography on sepha. (HMBC) afforded 2D carbon to hydrogen connectivity data TLC.2. Wybraniec et al. 3a and 3b). 2003.. 2001... Gandía-Herrero et al. Nemzer et al. presence of the 1. The chiral centre from tyrosine is emulsin or b-glucosidase) hydrolysis of betalains could suggest retained at C-6 of 1. 1971). positive mode acid and the conjugated dienes of 1. the isoform Wybraniec et al. In addition. Other structural features including the nat. betalains was result of incorporation of primary/secondary amines zomethane methylation and subsequent degradation with alkali at aldehydic group of 1. L-tyrosine is hydroxylated. alkaline 1 showed [M+H]+ at m/z 212 and typical signal of decarboxylation hydrolysis releases the acyl group for further characterisation. the structure was confirmed as the chromophore of all beta- methods of determining the linkage are also used (Piattelli. Compound 1 was identified as a naturally the configuration of the sugar linkage and subsequent carbohy.4-tetrahydropyridine-2.1. Absorbance spectra in UV and visible range sition and methylation at COOH of aliphatic acids in acylated together give valuable information about substitutions. Giridhar / Phytochemistry 117 (2015) 267–295 269 1X8 employing acidified aqueous eluents (Piattelli. However. ful in NMR data interpretation. Position of sugar substitution was often analysed by dia. Further studies revealed that its kmax in giving 5-hydroxy-6-methoxyindole-2-carboxylic acid. derivatives (kmax 340–360 nm) that exhibited bathochromic shifts 6. the references for structure elucidation.3. such as HPLC. decompo- (Piattelli. chemical or enzymatic methods have been routinely used (1) (for biosynthetic enzymes see Section 3. Mass spectrum of C-5. Heuer et al. 2012). deduce coupled spin systems (Strack et al. 1D 1H NMR experiments involves 2010) only recently on the structural implications of fluorescence mainly total correlated spectroscopy (TOCSY) that provides chem- associated with betaxanthins. information on the main compound and the possible fragments 2010). M. water shifts from 424 nm to around 480 nm or 540 nm when oxy-6-hydroxyindole-2-carboxylic acid indicating substitution at incorporated with amino acids or cyclo-DOPA. Condensation of 1 with cyclo-DOPA/its derivatives 1D and 2D 1H NMR spectroscopy provided the confirmation of forming betacyanins result in shift of absorption maximum in the chemical properties and proposed structures (Strack et al. the most striking difference between the subcategories of cose in case of C-20 . All these processes are recommended Stintzing et al. 2007.and 13C NMR signals have not been recorded owing determined by permethylation and subsequent acid hydrolysis giv.7-di- ments was based on methylation with diazomethane yielding azaheptamethin system could be found in the range 8. Gandía. 1976. the value was little are artefacts generated during extraction and not naturally occur. betacyanins and betaxanthins is their absorbance glucuronic acid and configuration could be inferred from b-glu. 2006). This infers that betalain isoforms 6. Kobayashi et al.:UVAbs. After prolonged exposure to 5% citric acid in H2O. the fluorescence was lost. and 5-meth.14 Hz.5–5. whereas dilute alkali treatment in specific J14a–15/J4a–5 and J14b–15/J4b–5 for betacyanins (Fig. respectively. 1993). as nuclear overhauser enhancement spectroscopy (NOESY).7-diazahep. while Dreiding (Wyler and Dreiding.7-diazaheptamethin system in all betalain pig. to poor stability in acidic conditions. 1964a). 1993).4. 1993. All betalains exhibit absorption maxima in both nique for separation and purification of fractions for mass spec- UV and visible regions owing to the phenolic nature of betalamic troscopy or NMR analysis... It was established that the aldimine bond present in 1976).6–7.. 1993). infrared spectra. Presence of betalains viz. an array of advanced analytical instruments Herrero et al. 2004a. 2004a. To this end.35 or above.6-dicarboxylic acid] or betalamic acid tral data. lains. which is recommended just in case the pigments are 2D 13C NMR spectroscopy experiments are increasingly employed poorly stable in anion-exchange chromatography or the eluates for structure elucidation of betalains (Strack et al. For betalains. and C-6. (Strack et al.2 ppm with respect to methylsilane of 80–100 nm in acidic solution (Mabry et al. give an average value (Piattelli and Minale. LC-NMR. 1D and dex LH-20. respectively.6-tri-O-methyl-glucose. 2011). tum coherence (HSQC). 1b). Heteronuclear single quan- ring genuine pigments.0–5. ical shifts and coupling constants. and structures.8 ppm and 6.. et al. (Gandía-Herrero et al. 1a) and the absence of oxygen reverses the isomerisation process betaxanthins (Fig.. (Stintzing et al.70 ppm) corresponding to 1. higher (Stintzing et al. In addition. betalamic acid (15R) tends to change to (15S). 2010). 1993).. 1994. There have been few reports on fluorescence of betalains.. the In plants. but on extension of the resonance to the indole ring. of 1.) of 1:0. Also. Synthesis. in darkened cold ambience to avoid pigment degradation.. in correlation spectroscopy (COSY) produce spectra difference to case of betacyanins.36 ± 0.. acid or enzyme (almond therein and Figs. The fluo. In general. Strack betacyanins).5 ppm. 1987. Based on chemical anal- nature of the sugar moiety. whereas in acidified D2O. 1964a.. Wybraniec and Nowak-Wydra. Position of substitutions including acylation could be chemically however 1H. Mass spectra provide ture. To complement the spec. The structure of 1 is well accepted. occurring yellow pigment in Beta vulgaris.. Celosia cristata and drate analysis or treatment with glucose oxidase could reveal the Portulaca grandiflora (Kimler et al. H2O from 424 nm to 541 ± 9 nm owing to extension of the . 1b) 12 Hz. d(H). P. or C-60 substitution. 1a) or J2–3 (betaxanthins. thins. 1. the Apart from providing valuable information on absorbance major determinant of chemical nature was absorbance spectra in spectra in UV–Vis regions. 2006). and functional group tests conditions that may lead to undesirable isomerisation. 13C NMR data acquisition has been limited (Strack hydroxycinnamoyl group produces higher bathochromic shift in et al. 2007. 2007. respectively (Piattelli et al. Inspite of common presence ing rise to 3. Wyler and ence of carboxyl group increased the fluorescence intensity. the absorption spectrum than aliphatic acid attachment. Khan. and mass spectral characteristics owing to their distinct chemical curonidase treatment and analysis of the product. 1962). electrospray ionisation MS works well. the intensity (Gandía-Herrero et al.. The confirmation for Fig. LC-high resolution MS (LC-HRMS)...4-tri-O-methyl-glu. The chemical shift.. Other more cumbersome chemical ysis. but not with betacyanins.3.I... betacyanins. respectively.. 1984) observed that betalains electron donating groups like hydroxyl and aromatic ring reduced essentially exhibit a coupling constant J11–12 (betacyanins. 2001) is that in acidified CD3OD. Fig. In case of acyl substituents. Another com. values in acidified DOH (4.7-diazaheptamethin substruc. C-6 (in 1) and their corresponding carbon in betaxan. followed by ring cleavage former substitution absorbs high at around 320 nm and results in and spontaneous cyclisation to form [4-(2-oxoethylidene)- a ratio (VisAbs. chromatography (paper. or 2. and tamethin. HPLC has emerged as a major tech- UV–Vis region. heteronuclear multiple quantum coher- ure of substituting side chains were characterised through ence (HMQC) and heteronuclear multiple bond coherence characteristic electrophoretic migration.. 1976). or chemical residues calculated through mass difference are help- but a systematic study was conducted (Gandía-Herrero et al. 1992. are to be processed for NMR. whereas 2D experiments such rescence might be due to electronic resonance in 1. Another important attribute exhibited mon structural feature among betalains is diastereomeric C-15 (in by several studies (Wybraniec and Nowak-Wydra. gel). 2006. analytical column chromatography. Bright Lights Kugler et al.5 331 Mirabilis jalapa L. (2001) Serine-Bx (21) 468 299 Beta vulgaris L. cv. Stintzing et al. Khan. (2008) Ethanolamine-Bx (31) –b –b Beta vulgaris L. [M+H]+ Betalamic acida (1) 424 212 Beta vulgaris L. b Data not available. (1965c) and Kugler et al. Bright Lights Hempel and Böhm (1997) and Kugler et al.463. (2004) Portulacaxanthin III (5) 470 269 Portulaca grandiflora/Beta vulgaris L.309 Portulaca grandiflora (Hook. (1965c) and Stintzing et al. cv.] Alef. (1965b) Miraxanthin III (12) 473.483 325c Rivina humilis L. ssp. Stintzing et al. Stintzing et al. ssp. Piattelli et al.] Alef. (2007b) Phenethylamine-Bx (33) 475 315. Piattelli et al. cv.] Alef. (1971) and Gandía-Herrero et al.299 Opuntia sp. (2004) M. it has been included. cicla [L. (2012) Indicaxanthin (2) 260. in this table of yellow colour pigments.264. 485 309 Opuntia ficus-indica L.296 Gomphrena globosa Svenson et al. ssp. Bright Lights Kugler et al. lutea/Beta vulgaris L. the reader should refer the discussion in the text and cited reference. (2007b) Lysine-Bx (30) 458 340. 305. (1965b) and Kobayashi et al.I.] Alef. Piattelli et al. Piattelli et al. Trezzini and Zrÿd (1991) and Kugler et al. (1965b) Miraxanthin II (11) 477 –b Mirabilis jalapa L. ssp. * kmax value differs based on the solvent and instrument. (2002a) Vulgaxanthin II (7) 469 341 Beta vulgaris L. For better understanding of the spectral characteristics. (2001) Histamine-Bx (15) 468 305 Mirabilis jalapa L. (1965b) and Kugler et al. Strack et al. ssp. (2004) Vulgaxanthin III (8) 470 326 Beta vulgaris var./Beta vulgaris L. (1965a) and Castellanos-Santiago and Yahia (2008) Portulacaxanthin II (4) 468 375 Portulaca grandiflora/Beta vulgaris L. (2007a) Putrescine-Bx (32) 461 282 Bougainvillea sp. lutea/Beta vulgaris L. cicla [L. (2005a) Humilixanthin (17) 258. cicla [L. a Betalamic acid is not strictly a betaxanthin. Stintzing et al. Giridhar / Phytochemistry 117 (2015) 267–295 Vulgaxanthin IV (9) 470 325 Beta vylgaris var. cicla [L.] Alef. (2002a) Portulacaxanthin I (3) 483 325. Piattelli et al. (2004) Methionine-Bx (27) 477 343.] Alef. cristata Piattelli et al. Bright Lights Kugler et al. (2002a) Phenylalanine-Bx (23) 472 359 Beta vulgaris L. (1973) and Gandía-Herrero et al. Piattelli et al. However. although the pigment may accumulate in other sources as well. 270 Table 1a List of betaxanthin pigments structurally identified till date. cicla [L. (2001) Miraxanthin V (14) 475. (2012) Tryptophan-Bx (20) 218. (2004) 3-Methoxy tyramine-Bx (13) 461 331 Mirabilis jalapa L. (2002a) Isoleucine-Bx (24) 470 325 Beta vulgaris L. cristata Schliemann et al. (2004) Dopaxanthin (16) 472 391 Glottiphyllum longum Impellizzeri et al. Castellanos-Santiago and Yahia (2008) Bx – betaxanthins. (2007a) Arginine-Bx (29) 469 368 Gomphrena globosa Kugler et al. P. (1965b) and Kugler et al./Celosia argentea var. Kimler et al. (2004) Miraxanthin I (10) 475 –b Mirabilis jalapa L. Bright Lights Kugler et al. # MS peaks are acquired in positive mode following electrospray ionisation. (1965b) and Schliemann et al. cicla [L. c Data in negative mode. (2002a) Valine-Bx (22) 470 311 Beta vulgaris L. Piattelli et al. $ Sources from which the pigment was reported first time./Beta vulgaris L. Plant betaxanthin Spectral characteristics Source$ References * # kmax (nm) MS peak . Biswas et al. . (2002a) Alanine-Bx (25) 468 283 Beta vulgaris L. ssp. (1987) c-Aminobutyric acid-Bx (18) 459 297 Beta vulgaris L. Kugler et al. Hence. Stintzing et al. (2004) Vulgaxanthin I (6) 470 340 Beta vulgaris L.) Mill./Beta vulgaris L. (2004) Muscaaurin VII (26) 472 349 Beta vulgaris L.471 398 Celosia argentea var. it is yellow in colour. (1964a) and Stintzing et al.270 Opuntia spp. cv. Trezzini and Zrÿd (1991) and Kugler et al. (2002a) Methylated arginine-Bx (19) 478 383 Amaranthus tricolor L. cv. Bright Lights Hempel and Böhm (1997) and Kugler et al. (2005) Threonine-Bx (28) –b –b Beta vulgaris L. Piattelli et al.)/Opuntia ficus-indica (L. cv.5 347 Mirabilis jalapa L. Stintzing et al. Piattelli et al. . However. This structure was reconfirmed after reacquiring miraxanthin I (10).7-diazaheptamethin system. Integrated proton spectrum of the compound showed flowers. 1965b). In MS profile. and between C-8 and H-10 confirmed linkage position of the reader may refer Stintzing et al. 1999) (Table 1a). Miraxanthin II (11) was identified as tetracar- 1 H NMR chemical shift data many years later (Wyler and boxylic acid compound which on acid hydrolysis released aspartic Dreiding. six more betaxanthins were identi- proline signals and portions similar to that of betanidin. These compounds gave [M+H]+ at m/z tion with H2O2 established (S)-configuration at C-2 and C-11. Further attempts resulted in tion experiments with semi-synthetic reference compounds and identification of portulacaxanthin III (5) from the same flowers mass spectra obtained (Schliemann et al.. betalain specific a change from (E). tyramine and dopamine betaxanthins were identi- detected at m/z 309 (Stintzing et al.. 1965c). 1991). Compound 2 has been dopamine to betalamic acid as N-9. fied as miraxanthin III (12) and V (13). This compound (kmax 260... Based on the Zrÿd.. Acid hydrolysis and oxida. In Mirabilis jalapa respectively. 1965a) and accumulation plausibly does not ensure presence of 2-descar- later confirmed by MS (Castellanos-Santiago and Yahia. In addition to the pro. Comparing the spectral and chemical shift indicating the presence of (9E). line and betalamic acid specific chemical shift pattern. 2008).1. amino acid feeding trials involving on alkali fusion in the absence of oxygen yielded proline and 4. M. Similarly. 2002a). which Among the condensation products of 1 and primary/secondary were later confirmed by respective MS profiles (Stintzing et al. respectively (Kugler et al. 1965b). Betaxanthins tified as vulgaxanthin I (6) and II (7) (Piattelli et al. histamine-betaxanthin (15) were confirmed. grandiflora flowers and its structure was 2-descarboxy-betacaynins (discussed in Section 3). boxy-betacaynins. . Identities of vulgaxanthin III (8) and was first isolated and identified from Opuntia ficus-indica IV (9) were proposed to be asparagine and leucine betaxanthins (Piattelli et al. 2006). 1965c). 2001. these pigments were iden- 4 HO COOH 3 5 + O N 2 COOH + N N COOH 6 HO 2 7 3 8 4 9 5 9 10 14 H H H H HOOC 6 N 8 COOH HOOC 11 N 13 COOH HOOC N COOH HOOC N COOH 10 H 11 15 H 16 H H 1 2 3 4 2. though miraxanthin has been detected in mem- From the same flowers.7-diazaheptamethin system to the diphe. Kugler et al. portulaxanthin II (4) was isolated and iden. a betaxanthin containing 4-hydroxy-pro. Wybraniec and Nowak-Wydra. hairy roots of B. Miraxanthin could serve as precursor to line was identified from P. structures of 3-methoxy-tyramine-betaxanthin (14) and recorded later in Swiss chard extracts (Kugler et al.. The structure was confirmed by NMR data showing chemical data presented by Piattelli et al. its elucidated as portulacaxanthin I (3) (Piattelli et al..6-dicarboxylic acid. the identity of the amino acid residue in each corresponding to Sections 2. 2004).. amines (Schliemann et al.1. similar to double-bond detected in plants from almost all the betalain-accumulating fam. isomerism of 2... tified as tyrosine conjugate of betalamic acid based on HPLC data. indicaxanthin (2) 2002a. respectively (Piattelli et al. 1964a).9 Hz and long range correlation between C-10 and H- shift of 4–6 ppm (for complete NMR annotation of rotamers of 2.. P.. Upon mild acid absorbance maximum at 471. Giridhar / Phytochemistry 117 (2015) 267–295 271 electronic resonance of 1. 2004). Khan. This observation reaffirmed that proline moiety was lain specific J10–11 = 13 Hz (Piattelli et al.5 ± 13.. Castellanos-Santiago and Yahia.2). 2001.1. A more detailed linked directly to 1.. lutea and confirmed by semi-synthesis methylpyridine-2. vulgaris var. 2002a. the main ion [M+H]+ of 2 was acid. 2008). 2008.6-dicarboxylic acid and an amino acid mary/secondary amines with 1 forming betaxanthins result in on alkaline hydrolysis (Piattelli et al. (Hempel and Böhm.I. 1984).. 485 nm) based on absorbance spectra. 2004.. A tricarboxylic acid compound that lib- these observations. 2006). Kugler et al. between H-7 and C-2 or C-5a/b from gradient enhanced (g) HSQC 2004). two yellow pigments were isolated that nolic aromatic ring of cyclo-DOPA. 1964a). Svenson and amino acid analysis of the hydrolysis product (Trezzini and et al.to (Z)-configuration of 2 produced a highfield J10–11 = 11. Among other characteristic signals. Based on fied (Piattelli et al. pigment was established as glutamine and glutamic acid. Mass spectra of miraxanthins III (12) and V (13) confirmed data (Stintzing et al.. (Stintzing et al. whereas condensation of pri. HPLC co-injec- tyrosine and betalamic acid signals. In addition. 1991).5 nm (data presented in tables hydrolysis and TLC. yielded 4-methylpyridine-2. compound 13 exhibited a characteristic highfield ilies under Caryophyllales... (Trezzini and Zrÿd. the structure of 2 was proposed (Piattelli erated methionine sulphoxide on acid hydrolysis was identified as et al. From B. Also. Kugler et al. 13C measurement of 1H and 13C shift was done recently (Stintzing NMR data suggested that double-bond isomerism at N-1 causing et al. 2007. 8a/b. 305. in addition to 2 and 6. vulgaris roots.1.. Corresponding mass profiles were 2004). 1997). 326 and 325. 2004).. (1965b)..and (9Z)-configurations properties with that of 2. Combining spectral and chemical data.. 2006) established long range correlations their respective structures (Kobayashi et al. bers of Cactaceae and Chenopodiodeae (Stintzing et al. The proton shift spectra of miraxanthin V (13) showed beta- experiments.1 and 2. Kugler et al.. 13C NMR 1965b). 2006). mass spectra. Though dopaxanthin sively elucidated the structure of the betaxanthin as occurs in many sources. Further confirmation was done through thesised 5-hydroxynorvaleric acid. and methylated argi- was 6. 1973).3– absorbance profile. MS peak at m/z 325 conclu- HPLC and MS (Gandía-Herrero et al. in Glottiphylum genus and certain yellow humilixanthin (17) (Strack et al. and HPLC co-injection experi- 1. 1987). 2005a).. 2002a). mass and 1H NMR spectra (Strack et al.1 Hz and average of J10a–11 and J10b–11 c-aminobutyric acid (Stintzing et al.2% dry weight) and identified as a genuine betaxanthin based on ments with semi-synthetic compounds and acid hydrolysis prod- absorbance.. 2005a). in addition to the 1H shift signals corresponding to nine (Biswas et al. been identified from various plant sources (Table 1a).. amino acids . 2012) were proposed. P. Identity of the amino acid residue was confirmed by acid hydrolysis products and semi-synthetic compounds co-chromatography of the hydrolysed product and chemically syn- (Impellizzeri et al. The authors detected 17 flowering members of the genus Lampranthus (specially in members of Aizoaceae.3 Hz. structures of betaxanthins (18–19) containing spectrum showed J7–8 = 12. Based on From the berries of Rivina humilis.. a pigment was isolated (0. 1. Lampranthus productus) of Aizoaceae family.7-diazaheptamethin substructure and 2-amino-5-hydroxy-va- identified as dopaxanthin (16) based on co-chromatography of the leric acid..272 M.. it is the sole pigment Several amino acid derivative and betalamic acid conjugates have present (Gandía-Herrero et al. Giridhar / Phytochemistry 117 (2015) 267–295 O O COOH COOH COOH H2N COOH H2N HO N N N O N H H H H HOOC N COOH HOOC N COOH HOOC N COOH HOOC N COOH H H H H 5 6 7 8 O COOH HO COOH COOH S N O N N N HO H H H H HOOC N COOH HOOC N COOH HOOC N COOH HOOC N COOH H H H H 9 10 11 12 HO 1 O 7 HO 6 8 HO N N NH N HO N HO COOH N 10 11 13 17 H H H H HOOC N COOH HOOC 14 N COOH HOOC N COOH HOOC N COOH H H H H 13 14 15 16 NH 5 3 4 2 COOH HOOC COOH HO N N H H N N N 7 8 10 14 H H H HOOC 11 N COOH HOOC N COOH HOOC N COOH H H H 17 18 19 DOPA derived betaxanthin was isolated from Glottiphylum longum. Similarly.. Portulacaceae and Chenopodiodeae. Khan.I. 1987). 1H NMR ucts of betalains. it is not easy to formulate a yielded 4-methylpyridine-2. methionine. (Table 1a). the available NMR data reported to conjugate with betalamic acid to form betaxanthins point to a particular possibility. the a-amino (20–30) (Table 1a). 1987). Histidine-betaxanthin or muscaaurin VII (26) was first COOH COOH COOH HO N N COOH N N N H H H H H HOOC N COOH HOOC N COOH HOOC N COOH HOOC N COOH H H H H 20 21 22 23 N NH COOH COOH S COOH N N N COOH N H H H H HOOC N COOH HOOC N COOH HOOC N COOH HOOC N COOH H H H H 24 25 26 27 NH OH COOH H2N COOH COOH N H2N H N N N H H H HOOC N COOH HOOC N COOH HOOC N COOH H H H 28 29 30 HO H2N N N N H H H HOOC N COOH HOOC N COOH HOOC N COOH H H H 31 32 33 identified in extracts from cap skin of Amanita muscaria (Musso. 1a). phenylalanine. arginine. Empirical formula of aglu- nucleophilic addition of the amino/imino group (Schliemann cone betanidin (34) was described based on degradation experi- et al. During betaxanthin biosynthesis. Due to din (34) were accounted for on alkaline and acid hydrolysis that paucity of NMR data of betaxanthins. Betacyanins 1979). For amino acids.. This is a typical Schiff base condensation reaction ments that yielded indole and pyridine moieties (Wyler and (acid catalysed. in this case. in slightly acidic pH) wherein loss of Dreiding. Khan. his. Giridhar / Phytochemistry 117 (2015) 267–295 273 tryptophan. M. amines or imino acids Core structure of all betalains is 1.1. Amines such as ethanolamine. 1959).2. P. However. 1999). putrescine and group is most likely to conjugate with betalamic acid to form phenethylamine have been detected in betaxanthins (31–33) aldimine bond as observed in 17 (Strack et al..I. rule of thumb as to which amino/imino group of a compound con- tidine. valine. alanine. 2. whereas betanidin (34) is the conjugate with betalamic acid to form aldimine bond through backbone of all betacyanins (Fig. threonine. and lysine have been jugates with betalamic acid. 3- . serine. All the carbons in the chemical formula of betani- a water molecule stabilises the unstable carbinolamine. 6-dicarboxylic acid and 2. isoleucine. H-7. while the C- mined the correlation between these carbons and protons assigned 17/C-18 diene migrated to C-14/C-15 (Wilcox et al. linkage. H. H-14a/b and H-15. H-3a/b. so far. P. In addition to the signature of aglucone 5.08 ppm). was identified in extracts J 12. 1970).06 ppm between H-4 and H-7. 38 In violet flowers of Carpobrotus acinaciformis. In addi. 5. H-3a/b and H-2 with respective carbons as well betacyanins. which was later supported by 1H NMR spectrum (Heuer et al.. the decar- ing data acquisition (Stintzing et al. instead a triplet indicating two protons was observed. characteristic of COOH at C- pared to 15S-configuration (Heuer et al. Its glucosyl 4. 2001). H-2 and H-12 indicated the s-trans conforma. 13C chemical shifts of betanin (35) were published after extensive search for the suitable medium that could ensure stability of the compound dur. an acyl derivative of 37 was identified as 60 -O-malonyl- (d = 0. HO 6' 4 4' HO 5 9 3 HO 5' O RO O HO H 3' 2' 1' HO O O OH H HO + HO 6 8 N 2 COOH N + OH 7 HO COOH 10 + 11 HO N 12 13 14 18 H H HOOC 15 N 17 COOH N HOOC COOH H 19 H 20 H HOOC N COOH H 34 35 R= H.3-dihydroindole (Piattelli and Impellizzeri. The proton spectra did form tends to show slightly higher J14a–b and lower J14b–15 com. also. Based on its chemical properties and synthesis of loss of pentose and glucose moiety subsequently) and NMR spectra authentic standard through exchange of proline moiety with (Wybraniec et al. It cidation. structure elucidation of 36 (Kobayashi et al. 1957) and on subsequent chemical and physical H studies. 1992). The structure of betanidin (34) was partially elu- cidated by 1H NMR as part of the attempt to generate first NMR + HO N data of betalains (Mabry et al. 1984). 1962). Through ESI-MS.. The 2-descarboxy-betacyanins have been shown to origi- is known that 34 and 35 are the most common betacyanins among nate from 13 through the oxidation and subsequent the betalain-accumulating families under Caryophyllales.. Complete 1H NMR assignment of betanidin (34) was achieved later (Wyler and Dreiding. b-Glucosidase treat- and H-11 and. The major pig- H ment in red beet was crystallised by Wyler and Dreiding (Wyler HOOC N COOH and Dreiding. not show presence of doublet at H-2.. 1964b). A coupling constant of derivative. [M+H]+ at m/z 345 was detected concluding the cone or 34 were the 1H-spin systems comprising of H-2. inferred from the chemical shift difference between H-4 and H-7 Similarly. completing the structure elu- become important reference for betalain structure elucidation. a minor compound In Hylocereus polyrhizus. intramolecular cyclisation (Kobayashi et al. In addition to tion to them. 2001). characteristics of aglu.80 ppm (1H) . In order to exclude the possibility that 36 was an artefact. 1H NMR spectra indicated presence of signals at the identity of 2-descarboxy-betanidin (36) was apparent. structures of 16 pigments having 34 or 35 as C. 1965). 1965). vulgaris.5 Hz between H-11 and H-12 combined with the rotating.. by 1H NMR analysis of betanin (35). 2-descarboxy-betanin (37).15S) and (2S. carboxylated betacyanins were reported along with 17-descarboxy- tions of H-4. Similar cou. 2006). identified in Mammillaria spp. 6-dihydroxyindole-2-carboxylic acid (Wyler and HO Dreiding. in addition to characteristic signals long-range correlation indicated by a difference of d 144 ppm of 37 that showed a downfield chemical shift at H-60 a and H-60 b between H-10 anomeric proton and phenolic carbon C-5/6.42 ppm of an anomeric proton H-100 along with 3. 1-D (TOCSY) and 2-D (COSY) 1H NMR spectra confirmed C-5 pling constant J11–12 values indicate the possibility of the as the position of sugar linkage owing to the chemical shift differ- compounds being (2S.6-dihydroxy-2. ment and analysis of the products confirmed presence of b-glucose tion of the dienyl-moiety (N-1/C-11 and C-12/C-13). from fodder beet seedlings through LC-ESI-MS (kmax 532 nm and frame overhauser enhancement (ROE) correlation between H-7 m/z 507.... 2007). a pentose sugar derivative of 35 was charac- showing similar spectral characteristics to 34 was observed to have terised through UV–Vis. and its glucoside. These data have betacyanins (Wybraniec et al. MS (kmax 539 nm. The indicating acylation at C-60 . The b-configuration of the glucose moiety was 2-descarboxy-betanin (38) based on LC-MS (kmax 535 nm. The 13C NMR spectra for all these 2-de- dihydroindolic system was established through gHMQC correla. 37 R= Malonyl. but not at C-2. 1962). In addition to 2 as in 35. 2-descarboxy-betacyanins have been common backbone have been elucidated (Table 1b). H-7.274 M. typical signal pattern of glucose. ence of 0. H-12.. m/z at 683 as main proto- decarboxylated C-2 instead of the characteristic COOH group of nated ion and daughter ion fragments at 551 and 389 representing amino acids. Giridhar / Phytochemistry 117 (2015) 267–295 dihydro-5.I. acinaciformis and B.. non-naturally occurring degradation products of as ROE correlation between H-4 and H-3a/b. its substitution at C-5 was This confirmed the structure of 2-descarboxy-betanin (37). Khan. H-11. while further studies revealed the presence of stereo configuration at C-2 and C-15 (Wilcox et al. m/z 593 established by a medium-range coupling constant J 7 Hz and a and 345) and 1H NMR spectra. The data obtained boxylation pattern of 34 was studied.. 2007). as well (Wybraniec and Nowak- Wydra. the structure of the compound was confirmed to be betani- 36 din-5-O-b-D-glucose (35) (Piattelli et al. decarboxylation takes place at C-15. 2004a). 1992). Briefly. It was earlier observed that from gHSQC enabled assignment of carbons and gHMQC deter. 345 indicating loss of glucosyl group). 15R-iso.15R)-stereoisomers. 322.#.L. P. Kobayashi et al. Alard et al. a Data not available.Br.551 Phyllocactus hybridus Piattelli and Minale (1964b) 20 -O-Apiosyl-phyllocactin (43) 538 683. Piattelli and Impellizzeri (1969) Lampranthin II (48) 288.345 Beta vulgaris L.633 Hylocereus polyrhizus Wybraniec et al.389 Hylocereus ocamponis Wybraniec et al. (2007) Phyllocactin (42) 539 637.551. (1996) 20 -O[500 -O-(E)-sinapoyl]-Apiosyl-betanin (41) 330. Imperato (1975a) Neobetaninb (51) 267.389 Hylocereus ocamponis Wybraniec et al. (1996) Rivinianin (50) 253. 1957 and Kobayashi et al. (2007) 20 -O[500 -O-(E)-feruloyl]-Apiosyl-betanin (40) 331. (Mes. 275 .389.149 Beta vulgaris L.$. Kobayashi et al.345 Beta vulgaris L.E.756 Schlumbergera buckleyi Kobayashi et al.593. (1988) Prebetanin (49) 538 –a Beta vulgaris/Phytolacca americana L.306.470 549. (2001) Betanin (35) 537 551. It does not have C-15 chiral centre unlike all other betacyanins. (2001) 2-Descarboxy-betanidin (36) 533 345 Carpobrotus acinaciformis L. (2001) Lampranthin I (47) 290.538 –a Lampranthus sp. (1967) and Schliemann et al. [M+H] M.389 Hylocereus ocamponis Wybraniec et al.541 –a Rivina humilis L. (1985) See Table 1a footnotes for ⁄. Khan. Piattelli and Impellizzeri (1970) and Kobayashi et al.320. Wyler and Dreiding.683./Lampranthus sociorum (L.651.593.) Piattelli and Impellizzeri (1969) and Strack et al. Giridhar / Phytochemistry 117 (2015) 267–295 Betanidin (34) 541 389. Wybraniec et al. (2011) and Kobayashi et al.) N.Table 1b List of betalain pigments (betanin group) structurally identified till date.677.Bol. Wyler et al.387 Beta vulgaris L. 538 727 Lampranthus sp.549 945.C. Pigment Spectral characteristics Source$ References ⁄ # + kmax (nm) MS peaks . (2000) 40 -O-Malonyl-betanin (45) 538 619. Schliemann et al. (2007) Hylocerenin (46) 541 695.I.Bol.551. (2001) 20 -O-Apiosyl-betanin (39) 539 683.619.550 889.548 859 Phytolacca americana L. (2001) 60 -O-Malonyl-2-descarboxy-betanin (38) 535 593. (2001) 2-Descarboxy-betanin (37) 532 507 Beta vulgaris L.551. Wyler and Dreiding (1959). b Neobetanin is orange betacyanin pigment having spectral characteristics similar to betaxanthins. (2000) 20 -O[500 -O-(E)-feruloyl]-Apiosyl-phyllocactin (44) 328.551.389 Schlumbergera buckleyi Kobayashi et al. 43 R= 1'' (Malonyl). R2= (Feruloyl). H-500 (2H). tion with hydroxycinnamic acid (Heuer et al. 42 HO 2'' R1= Malonyl. and the methoxyl group attached to C-3000 . A similar acid linked to 20 -O-apiose was identified from Phytolacca americana compound with sinapoyl residue attached to C-500 of apiose moiety cultures as 20 -O(5-O-feruloyl)-b-apiosyl-betanin (40) (kmax 331. R1O HO O O HO R2O O H O + 5'' HO N COOH 1'' HO OH H HOOC N COOH H R1= H. The absolute was proposed based on absorbance (kmax 328 nm).6 and The identity of the acyl group was revealed by the vicinal coupling 63. lished by comparing the coupling constant with that of apiose moiety nals were indicative of a pentose sugar linkage to C-20 . structure of 40 was confirmed. Structure of. in other compounds. and 3.5 ppm. the first betalain pigment having aliphatic acid as well as addition.. These sig. R2= Feruloyl. 1996). two methylenes at C-400 and C-500 as indicated by signals at 73. mass (m/z 945.6–0. 44 showed characteristic signals of betanin and the apiose moiety. The bathochromic shift of (Wybraniec et al. structure of the betacyanin was con. 548 was identified as 20 -O[500 -O-sinapoyl]-apiosyl-betanin (41) and m/z 859) (Schliemann et al.. Further con.I.. R2= H.9 Hz) and long-range coupling between H-2000 firmed as 20 -O-b-apiosyl-betanin (39) (Wybraniec et al.02 ppm (1H) implying H-400 a and H-400 b. 39 O 9''' 1''' R1= H. H-2 showed a slight downfield shift owing to copigmenta. complete structure eluci- et al. In addition. Based on these data. 2007). polyrhizus fruit reported later agreed with the previous 1H and the linkage between the sugars was unambiguously assigned to NMR data for 20 -O-apiosyl-phyllocactin (43) (Wybraniec et al. 13C chemical shifts revealed the presence of at H-500 a and H-500 b of apiose moiety indicated acylation at C-500 . a complex betanin derivative having a hydroxycinnamic spectral characteristics. the downfield shift of 0. These pigments are yet to be detected in 9 nm compared to 39 indicated intramolecular association or copig. 40 HO O OCH3 9''' H3CO 1''' R1= H. 20 -O-apiosyl-phyllocactin (43) with moiety. of an AB configuration of the anomeric proton of the pentose sugar was estab- quartet methylene. The confirmed by the mass spectrum of its methyl ester... 1992).. this compound exhibited absorbance ratio (VisAbs. 1992). R2= H. other sources. 20 -O[500 -O-feruloyl]-apiosyl-phyllocactin (44). R2= (Sinapoyl). of H-7000 and H-8000 (15. Carbohydrate downfield shift of C-60 in 13C NMR spectra of the pigment from analysis confirmed the presence of glucose as well as pentose sugars H. Based on these data and Similarly. P.7 ppm firming apiose linkage. NMR spectra of the compound 2007).37 and on alkaline hydrolysis yielded ferulic acid. 41 HO OCH3 RO R1O 6' 4' HO O HO 5' O O O HO HO 3' 2' 1' R2O O H OH H O + + HO N COOH HO N COOH HO OH H H HOOC N COOH O O HOOC N COOH H H 3'' R1= Malonyl. In probably.55 ppm of a singlet.:UVAbs.. mentation with acyl groups such as hydroxycinnamic acid (Heuer From flowers and fruits of Cactaceae. Giridhar / Phytochemistry 117 (2015) 267–295 and 4.) malonyl group substitution at C-60 based on 1D and 2D 1H NMR of 1:0.276 M. Further suggesting presence of a hydroxycinnamoyl dation of phyllocactin (42). 2003). . C-20 following methylation analysis. 2007). respectively. which was spectra has been summarised elsewhere (Strack et al. aromatic acid linkage. Khan. .. on methylation of the acyl group methyl group (singlets of H-300 ) were seen. this was the first betanin were determined and the complex compounds were instance of acyl identified as lampranthin I (47) and II (48). 1992.. 1964b. 2007). previously for characterisation of 42 and its derivatives. A pigment purified from H. 1969). this compound isomerised was readily identified following the chemical methods employed partially to 42 during prolonged exposure to room temperature. polyrhizus fruit extract showed migration product of 42 was misidentified earlier from H. 2002b). which was confirmed by further studies the absence of NMR spectra... Wybraniec methylation in basic condition (to exclude any acyl residue). 2004) and Phytolaccaceae (Schliemann et al. With respect to betacyanins. p-coumaroyl and feruloyl substitutions in nyl-betanin (45). 2008). 46 and their spectra of alditol acetate obtained following a mild methylation derivatives have been identified from Cactaceae members only process. Svenson Further. et al. Kugler et al. Khan. Whereas fur- O O ther characterisation of 47 was limited to absorbance and MS HO spectra. M. 389). 619. The malonyl group linkage NMR data. 1996).. Strack et al. R2= H. Giridhar / Phytochemistry 117 (2015) 267–295 277 756) and the data obtained for 42 and 43 (Kobayashi et al... 1988). Based on Hence. 2000).I. this compound’s structure was unambiguously eluci- to sugar moiety was analysed and confirmed as C-40 by the GC–MS dated as hylocerenin (46).. 389) and chemical properties were recorded (2H). However. respectively. 48 R2O HO O O R1O OH H + HO N COOH H HOOC N COOH H R1= H. (Piattelli and Impellizzeri. whereas presence of glucose moiety was confirmed by (Piattelli and Minale. (singlets of H-400 and AB quartet of H-200 a/H-200 b) and 3H of nate ester. OH H + Lampranthins accumulate in Cactaceae (Wybraniec and Nowak- HO N COOH Wydra. A possible acyl 2007). 551. 48 was conclusively characterised based on [M+H]+ at HO O O O m/z 727 and 1H NMR spectra (1D and 2D).. Furthermore. while new signals corresponding to two methylene groups (Wybraniec et al. 46 R= (Feruloyl). Betacyanins such as 42. 593. Based on MS and confirming presence of a malonyl group. Chenopodiodeae (Bokern et al.. different sets of spectra (kmax 538 nm and m/z to that of 42 except for the absence of malonate protons H-200 637. 2000. In migration phenomenon. 2007. polyrhizus linkage of aliphatic carboxylic acid 3-hydroxy-3-methyl-glutaric fruit extract as betanin-60 -O-3-hydroxy-3-butyric acid based on acid at C-60 of betanin resulting in a characteristic MS fragmen- absorbance and mass spectra (Stintzing et al. feruloyl group linkage to C-500 of apiose as in 40. 2001.. chemical properties. as identified by GC–MS. Aromatic acyl group substitution in betanin (35) firmed the release of 34. from Lampranthus sp. which bore signatures HO of a feruloyl substitution at C-60 and 35 (Strack et al. 47 OH HO (3-Methyl-3-hydroxy methyl glutaryl). Wybraniec and Nowak-Wydra. Kobayashi et al. et al. 1988. P. 50 . 49 R1= HSO3 . the authors relied on earlier report of on malonyl migration at different pH values (Wybraniec et al. 551. tation pattern (m/z 695. Svenson et al. R2= HSO3. This compound formed a dimethyl malo. 2007. H HOOC N COOH H 45 RO HO O O RO HO HO O OH H O + HO N COOH HO OH H + HO N COOH O H HOOC N COOH H 9''' H HOOC N COOH H 1''' R= HOOC CH2 C H2COC R= (p-Coumaroyl). alkaline deacylation and subsequent acidification con. the compound was unambiguously confirmed as 40 -O-malo. 1H NMR spectrum was similar on reinvestigation. 2008).. O. Enzymatic hydrolysis confirmed presence of glucose. 1966. the simplest of them was unambiguously identified HOOC N COOH as amaranthin (52) based on complementary spectral data (kmax H 270. 13C NMR spectra of neobetanin (51) was the and protons from glucose. 1988). 1967. Sinapoyl-amaranthin (56) 1 H NMR spectrum was identical to that of 52 except for the Table 1c List of betacyanin pigments (amaranthin group) structurally identified till date. Sinapoyl-amaranthin (56) has been tentatively identi- fied based on absorbance and mass spectra of extracts from Gomphrena globosa petals (Kugler et al.. 294. The NOE difference confirmed that b-glucose was attached of prior 13C chemical shift data. The spectral data (kmax J10 –20 of 6. and. the structure of celosianin O HO O II (55) was conclusively identified as betanidin-5-O[20 -O(200 -O-fer- HO OH H uloyl)glucouronyl]-glucoside or 600 -feruloyl-amaranthin (Strack + HO N COOH et al. In the absence dues. OH. et al.. similar to of 52 was characterised conclusively from Chenopodium rubrum 34 signals. Amaranthin (52) of hydrolysed products with authentic standards. The proposed identities of the compounds overnight in citric acid solution indicating absence of a chiral cen- were found to be different when sophisticated techniques investi- tre at C-15. could be alkaline R1= H R2= feruloyl. R2= 3-hydroxy-3-methyl glutaryl. b-configuration was revealed by a coupling constant suspension cultures (Strack et al. 1988). R2O 6' HO O HOOC N COOH O H HO 1' H HOOC 6'' 51 O HO O + HO N COOH HO A group of betalain pigments having 20 -O-glucuronic acid substitu. (1988) and Cai et al. 1962). Thus.. Celosianin I (54) This compound had absorbance at kmax 537 nm. P.389 Celosia cristata L. Minale et al. Although neobetanidin was identified as degradation product of (2001) also confirmed the presence of 3-hydroxy-3-methyl-glu- 34 and 35 (Mabry et al. Minale et al. This products and strong absorbance at UV region (kmax 319 nm) orange pigment failed to produce C-15 diastereoisomer on holding (Minale et al.8 Hz... the assignment was done by com- to C-5 of 34 and C-20 had a b-glucuronic acid substitution. Cai et al. . (2001) Celosianin II (55) 312. So far. 1967).546 903 Celosia cristata var. An aliphatic car. 1985). the structure elucidation of ire- neobetanin (51). was detected in several plants including B.. 2001. Its R1= H. R1= H. Zygocactus truncatus. revealed the presence of two sharp singlets at 3 and 1. 1966). a naturally occurring glucone. CH 2CO dated from R. fied as celosianin I (54) based on absorbance and mass spectra. R2= H. 1966). 2001). grandiflora.58 and 1. whereas paring with literature on appropriate compounds. The linkage alkaline hydrolysis and subsequent analysis of the products (Cai of the glucose to C-5 was confirmed based on the difference of et al. 1985).7 ppm.28 ppm corresponding to OCH3 and.65. Giridhar / Phytochemistry 117 (2015) 267–295 A minor red beet pigment. 324. Minale et al. taric acid from MS spectrum. Kugler et al. 0. two sugar molecules and a feruloyl moiety. ex Lindl. (2001) Sinapoyl-amaranthina (56) 540 933. Methanolysis of the pigment indicated the linkage of H3C CH sulphuric acid to C-60 of 35. 2001) (Table 1c)..3 ppm between the signals of H-4 and H-7. which was in between that of doublets such as J2– 1 298. 2. respectively. vul- sinin I (53) was completed. humilis as betanin-30 -O-sulphate or rivinianin (50) (Imperato. Phytolacca substituted 52 derivatives were reported based on their hydrolysis bogotensis and Rhipsalis warmingiana (Alard et al. 389) and co-chromatography R1= H.I. the spectra showed a downfield shift at H-200 characteristic of an HO acylation.62 ppm corresponding presence of sulphur. OR1 1'' tion in betanin was reported in members of Amaranthaceae (Schliemann et al. [M+H] Amaranthin (52) 536 727. Based on these observations.. cristata Cai et al. ficus-indica. (1966) and Cai et al. Comparing the 1H NMR spectra with the acid revealed presence of a hydroxyl group and 1H NMR spec- that of 35.. while partial hydrolysis released 35 and sul- to CH2CO and. 541 nm and m/z 727.. (2001) Celosianin I (54) 306. respectively. Owing to better stability in acidic medium required Irradiation at H-10 produced negative NOE spectrum over H-4 for data acquisition. R1= H R2= p-coumaroyl.546 872 Celosia cristata var. of 3-hydroxy-3-methyl-glutaric acid was confirmed.278 M. p-Coumaric acid derivative of 52 was identi- revealed m/z 549 and 387 indicating the loss of hexose moiety. Cai et al. a Tentatively identified. f. glucuronic acid and aromatic acid resi- first for a betalain compound (Alard et al. d 33. Iresinin I (53) boxylic acid derivative of 52 was detected in Iresine herbstii leaves. 1975a). Pigment Spectral characteristics Source$ References ⁄ # + kmax (nm) MS peaks . 538 nm and m/z 903) indicated presence of one aromatic 3a and J2–3b or J60 a–60 b in H NMR spectrum.551.. (2001) Iresinin I (53) 298. The IR spectrum of phuric acid.540 871 Iresine herbstii Hook. 551. P.389 Gomphrena globosa L. Similarly. Ferulic acid derivative ca. Celosianin II (55) hydrolysed to yield amaranthin and an optically inactive acid. the pigment was identified as prebetanin (49) (Wyler trum (CCl4) of the acid indicated presence of three singlets at 3. From IR and 1H NMR spectra presence reported from other plants. cristata Strack et al. Following a preliminary 1H NMR assignment (Minale H et al.. (1966) and Cai et al. Positive ion fast atom bombardment mass spectra gated the structure. Khan.$ See Table 1a footnotes for . compounds 49 and 50 have not been and H3C C . Structures of two aromatic acid garis. in the absence of NMR spectra. R2= sinapoyl.#.551. This was further con- 13 acid and 1D (including NOE) and 2D 1H NMR spectra was charac- firmed by long-range C chemical shift of C-10 from C-5/6 by teristic of 34. structure of a derivative of 35 was eluci. on elemental analysis. 2007b).. (2007b) ⁄. 551.b or H-500 /H- tion was suggested by fragmentation pattern itself. 551 + 162). are recognised degradation products. (kmax 540 nm). 2010). R2= Malonyl.8 ppm between H-4 and H-7 indicating C-6 time in the fruits of M. This compound exhibited a characteristic signal of 34 and chemical 2007). 13C (Wybraniec et al. alkaline hydrolysis that yielded p. 2-descarboxy betacyanins 2007). P. A p-coumaric ments (Kobayashi et al. Feruloyl-bougainvillein-r I (59) identified in bracts of Bougainvillea glabra var. RO 6' O R2O HO O R1O O HO O 1' H HO HO 6'' O HO H HO O + O HO N COOH O + HO HO HO N COOH 1'' OH HO OH H COOH H HOOC N H HOOC N COOH H R= H. arising out of processing of the sample. 59 R1= Malonyl. (Wybraniec and Nowak-Wydra. 57 R1= H. Khan. 389 nals confirmed the malonyl moiety presence. 551 (betanin). which too released sively elucidated as betanidin-6-O-sophoroside. 2004a) in that it on absorbance and mass spectra. 1:0. R2= H. while two decarboxylated mammillarinins were and C-20 provided information about the b-linkage of the second identified as 2-descarboxy-mammillarinin (62) and 17-descar- glucose molecule to the first glucose. Characteristic downfield shift at H-60 a/b indi- product of permethylation. coronata. 551. Compound 57 is present in 1970b). The betanin characteristic signals. 62 713 (bougainvillein-r I. sanderiana based on was identified in Ullucus spp. coumaric acid + bougainvillein-r I and acid hydrolysis that released Similar to 57. the identity of the compound was established clearly as cose and betanidin. 600 a. betanidin + glucose and betanidin. The integrated proton (Wybraniec and Nowak-Wydra. firmed after detection of dimethyl malonate ester on GC analysis Based on these data. 58 R= Feruloyl. 2007). 1970a). 1H NMR and GC–MS were used (Heuer et al. For further structure elucidation of the simplest 6-O-diglu- Mammillaria spp. and mass (m/z 713. Recently.. was identified based on respectively). respectively. ocam. betanidin + glu. H. a putative artefact din + 2 glucose moieties. coside compound. a new compound was identified for the first shift difference of d = 0. and multiplets at H-50 /H-60 a. apart from spectra obtained from absorbance ponis (Wybraniec et al. respectively).. Acylation of 63 was . Butt’’ was investigated (Piattelli and Imperato. sensitivity to b-glucosidase and mammillarinin (60) or betanidin-5-O(60 -O-malonyl)-sophoroside 1 H NMR spectrum (Wybraniec et al. absorbance and mass spectra (Wybraniec and Nowak-Wydra. and Basella sp. Table 1d enlists all the identified betani- spectrum (kmax 312.. spectra (Svenson et al. bougainvillein-r III (58). malonic acid. The mass difference of 86 (799–713) indicated constant (J 8 Hz) of doublet signal corresponding to anomeric presence of an acyl compound. 2001)... 2007). M.b. The intersugar linkage was confirmed at C-20 by GC analysis light when pigment composition of purple bracts of Bougainvillea of processed permethylated product. a possible acyl migration product had readily recognisable patterns of 34 and glucose moiety. 2008). these data confirmed the structure of are considered as naturally occurring dopamine based genuine pig- betanidin-5-O-sophoroside (bougainvillein-r I) (57). betacyanins with 6-O-sophoroside linkages were 34 (Piattelli and Imperato. In addition. a betacyanin has been charac. cated the malonyl substitution at C-60 and it was confirmed by the terised elaborately from Hylocereus ocamponis based on absor. 61 RO HO O O HO HO O HO O + HO N HO OH H HOOC N COOH H R= Malonyl. Whereas the 17-decarboxylated betacyanins from methylation product analysis by GC–MS (Wybraniec et al. tubers based on absorbance and mass absorbance and chemical properties (Piattelli and Imperato. and position of acyla. 2007) of 60 was identified as 4-O-malonyl-bou- chemical shifts measured by gHMBC correlation between H-100 gainvillein-r (61). 1970a). Taken together. 1H NMR spectra showed ‘‘Mrs. It was con. 539 nm (VisAbs. 17-descarboxy-bougainvillein-r I. 541 nm). din-5-O-sophorosides. proton H-10 or H-100 .. 62 awaits elaborate struc- acid derivative of 57 was partially characterised and considered a ture elucidation and biosynthetic studies to be considered a genuine pigment.:UVAbs. (Lin et al.... The b-configuration of the sugar residues were identified by vicinal 389 (betanidin). The intersugar linkage was analysed as in 57. H-2000 a and H-2000 b proton sig- bance and mass spectra (kmax 538 nm and m/z 713. results.. Based on these corresponding to betanidin + 2 glucose moieties. In the same study. structure of bougainvillein-v (63) was conclu- of methylated product of alkaline hydrolysis.I. This as well was inferred boxy-mammillarinin. 1994). 389 suggesting betani- Additionally. This compound showed kmax sugar susbstition. based on absorbance genuine betalain pigment. 2007). 60 R= p-Coumaroyl. 637 (799–162). and intersugar linkage was indicated structure (57) was deduced mainly based on chemical properties by 13C chemical shift data acquired through gHMBC showing corre- pertaining to 34 and diglucoside revealed by analysis of hydrolysed lation of H-100 to C-20 . based spectrum resembled that of 35 (Stintzing et al. For now.16) and MS peaks [M+H]+ at m/z 799. 2007). Giridhar / Phytochemistry 117 (2015) 267–295 279 Presence of 5-O-sophoroside substitution in betacyanins came to 57. gHMBC correlation of H-60 a/b to C-1000 . 713.593. Bougainvillea-v – violet-red pigments from B.1021. R2= H. HO R1O H 6' O + HO O N COOH HO 200 -O[60 . Mammillaria spp. 545 nm indicating bathochro- Wybraniec and Nowak-Wydra (2007) Wybraniec and Nowak-Wydra (2007) Wybraniec and Nowak-Wydra (2007) mic shift of 5 nm owing to copigmentation with hydroxycinnamic acid.541 316. 65 ⁄ kmax (nm) R1= H. Table 1d presents data on betani- din-6-O-sophorosides. R2= Glucosyl. Bougainvillein-r IIIa (58) Bougainvillein-r Ia (57) Betacyanins with 6-O-glucoside were extracted from inflorescence Bougainvillein-v (63) Data not available. R2= p-Coumaroyl.551. and 200 -O{[60 -O-caffeoyl]-[600 -O-p-coumaroyl]}- Ullucus tuberosus Caldas Bougainvillein-r – red-violet pigments from Bougainvillea glabra var. structures of 60 -O-p-coumaroyl-bou- References gainvillein-v (65). and Piattelli and Imperato (1970b) mass spectra (m/z 875. 1H NMR exhibiting lowfield of 4. 68 HO 200 -O{[60 -O-(E)-caffeoyl]-[600 -O-(E)-p-coumaroyl]}-Sophorosyl-bougainvillein-v (71) Tentatively identified compounds based on absorbance and chemical properties. (1994) Heuer et al. (1994) Heuer et al.#. and substitution at Table 1d C-6 confirming the structure of gomphrenin I (72) as betanidin-6- O-glucoside (Heuer et al. R2= Glucosyl.713 1183. [M+H] HOOC N COOH H 713. (1994) Heuer et al. Piattelli and Imperato (1970a) Piattelli and Imperato (1970a) and Wybraniec et al. 71 40 -O-Malonyl-bougainvillein-r I (61) 2-Descarboxy-mammillarinin (62) Feruloyl-bougainvillein-r Ic (59) See Table 1a footnotes for ⁄. 637. 1021.551. In the absence of mass and NMR spectra. 64 889 1''' –b –b HO R1= p-Coumaroyl. the structure of 600 -O-rhamnosyl-bougainvillein-v (68) was proposed based on absorbance and chemical properties (Imperato.280 M. Khan.1 ppm at H- 60 a/b characteristic of C-60 acylation and signals in between 6. R2= H. 69 600 -O-(E)-p-Coumaroyl-bougainvillein-v (66) 60 -O-(E)-p-Coumaroyl-bougainvillein-v (65) R1= p-Coumaroyl. 70 60 -O-(E)-Caffeoyl bougainvillein-v (64) 600 -O-Rhamnosyl-bougainvillein-v (68) R1= Caffeoyl..859. R3= p-Coumaroyl. R2= H3O OH (Rhamnose). Mammillarinin (60) of G. 713.600 -di-O-(E)-p-coumaroyl]-Glucosyl-bougainvillein-v (70) 1' R3O 6'' O HO O HO 1'' OR 2 H HOOC N COOH 60 . 713.697. 389 + MS peak . 60 .548 306.551 755. Butt’’.1183. sugar moiety.$.547 312. glabra Choisy such as 200 -O{[60 -O-caffeoyl]-[600 -O-p-coumaroyl]}-glucosyl- bougainvillein-v (69). (1994) Heuer et al. Mass and 1H NMR spectra showed character- Pigment istic signals indicating betanidin.600 -di-O-p-coumaroyl]-glucosyl bou- Bougainvillea glabra Choisy Bougainvillea glabra Choisy Bougainvillea glabra Choisy Bougainvillea glabra Choisy Bougainvillea glabra Choisy Bougainvillea glabra Choisy Bougainvillea glabra Choisy Bougainvillea ‘‘Mrs.549 307.30 Svenson et al.389 1167. 637. sophorosyl-bougainvillein-v (71).389 859. 1992).545 312. (1994) Heuer et al.389 H 1005. 1:0. (1994) Heuer et al. (2007) bougainvillein-v). (2008) and 7. Butt’’ gainvillein-v (70). acid hydrolysis and analysis of the alkali fusion product of methylated compound (Minale et al.713.669.755.I.1005.345 1345. 507. 1975b). Butt’’ Bougainvillea ‘‘Mrs. Similarly.755. R3= p-Coumaroyl.389 713.389 875. R1= Caffeoyl. 67 538 534 539 538 533 541 540 6''' H C O 1''' R1= H. P.49 ppm confirmed the structure of the compound as 60 -O- Heuer et al.600 - di-O-p-coumaroyl-bougainvillein-v (67) were elucidated (Heuer et al. HO Source$ R1O H 6' O + HO O N COOH HO 1' R2O 6'' O O HO 799. glabra varieties except ‘‘Mrs. sanderiana glucosyl derivatives of 63 have been as well identified in B. 1967).540 R1= p-Coumaroyl. 1994).389 HO 1'' OH 799. Giridhar / Phytochemistry 117 (2015) 267–295 evident from absorbance (kmax 316. 63 # 9''' Spectral characteristics R1= HO (Caffeoyl). based on absorbance.389 859. 200 -O{[60 -O-(E)-caffeoyl]-[600 -O-(E)-p-coumaroyl]}-Glucosyl-bougainvillein-v (69) List of betacyanin pigments (bougainvillein group) structurally identified till date.548 307.713. 697. sanderiana Bougainvillea glabra var. (1994) Imperato (1975b) caffeoyl-bougainvillein-v (64). Butt’’.540 306. 66 312. R3= p-Coumaroyl. Mammillaria spp. 200 -O[60 .697.551 O R1= H. Mammillaria spp. mass and 1H NMR spectra.600 -di-O-(E)-p-Coumaroyl-bougainvillein-v (67) H Tentatively identified based on spectral data. R2= Sophorosyl.551.:UVAbs.. R2= H.. R2= p-Coumaroyl. Complex Bougainvillea glabra var. globosa and indetified through absorbance.45 inferring hydroxycinnamoylation). Similarly. ‘‘Mrs. bougainvillein-v + acyl compound. gomphrenin II (73) and a c b gomphrenin III (74) structures were elucidated as betanidin-6- .713. 600 -O-p-coumaroyl-bougainvillein-v (66). VisAbs. 40 or above and a bathochromic shift ranging Caryophyllales. Kugler et al. In the absence of 1H unique type (P3) of plastid in sieve element (APG III. 75 that although anthocyanin biosynthetic genes dihydroflavonol 4- reductase (DFR) and ANS were naturally expressed in betalain pro- ducing taxa (Shimada et al. 2005. Caryophyllales. except Caryophyllaceae and Molluginaceae.1. b Data not available. Till date.389 Gomphrena globosa L.. 73 and 74 (Kugler tein crystal (Cronquist and Thorne. for plants under Caryophyllales order that accumu- members of some families under the order Caryophyllales. Pigment Spectral characteristics Source$ References ⁄ # + kmax (nm) MS peak . all the pigments in this group showed tematic markers have shown that all families that comprise core VisAbs. probably. 2009). RO H 6' + 2002). In a recent report. 2013). 2004. of 1:0. by selective binding to the recent report based on HR-ESI-MS. had specific amino acid sequence at its catalytic site. O(60 -O-p-coumaroyl)-glucoside and betanidin-6-O(60 -O-feruloyl).2. 1H NMR. firmed that the compound was not a betacyanin rather a beta- 2015. Studies on chemosys- et al. perisperm.5. pro- from 2 nm to 9 nm indicating copigmentation with hydroxycin. and (3) betalain-containing plants HO 1' OH accumulate leucoanthocyanidins too. One of the ways to understand mutually exclusive- they are chemosystematic markers and carry out anthocyanin.389 Gomphrena globosa L. and curved embryos (APG III. 1992).4). which was different from that of non-betalain producing plants (Christinet 2... In addition to this. (1967) and Heuer et al. a transcription factor that regulates betalain biosynthesis. More recently.389 Gomphrena globosa L. studies on comparative genetics R= p-Coumaroyl. apart Betalains are exclusively present replacing anthocyanins in from pigment. citryl-celosianin. the enzyme responsible for aromatic ring cleavage leading 1. but anthocyanin biosynthesis. Heuer et al. Minale et al. ness of anthocyanins and betalains is to conduct reciprocal analogous functions. Plants under Caryophyllales have anatomical characteristic of a glucoside. that betalain producing plants dioxolo]-2H-pyrrolo[2. paper chromatography. (1992) Gomphrenin II (73) 550 697. is a natural assemblage of 34 families having mor. controlled at tran- suaedin was reported as a betacyanin.6.20 S)- (DOD). during evo- H lution. Giridhar / Phytochemistry 117 (2015) 267–295 281 Table 1e List of betacyanin pigments (gomphrenin group) structurally identified till date. Harris et al. (2007b) See Table 1a footnotes for ⁄. It has been observed R= Sinapoyl. anthocyanidin synthase (ANS) underwent selective expres- HOOC N COOH H sion resulting in loss of the enzyme in plants of some families R= H .4). if there is any advantage of betalain biosynthesis over antho- second para). P. [M+H] Gomphrenin I (72) 543 551. 2007).551. properties of gomphrenin-type betacyanins are listed in Table 1e. placentation. but not hydrolysis. ubiquitous hydrophilic plant pigments (Brockington et al. For example.I.1-a]isoquinoline-3-one. earlier known as experiments involving introduction of genes of betalain biosynthe- Centrospermae. 2011). The authors isolated this compound was evident from homology analysis of L-DOPA dioxygenase as lilac powder and the structure was elucidated as (10bS. it is not established is apparently catalysed by different enzymes (see Section 3. Khan. 72 under the Caryophyllales order (Clement and Mabry. these two hydrophilic pigment groups (anthocyanins Biosynthesis of 5-O-glucosides and 6-O-glucosides of betacyanins and betalains) are mutually exclusive.551.. Spectral wise. M. 1967. (1967) and Heuer et al. cyanins. (2) betalains/anthocyanins are accumulated in reproductive O O N COOH HO as well as vegetative plant parts. or there is any specific reason known for the absence of betalains in anthocyanin producing plants. 13C NMR spectra con- promoter region of betalain biosynthetic genes (Hatlestad et al.1. 2013.. 1996. It has been proven recently with the discovery of data obtained from paper electrophoresis. Mutual exclusiveness of anthocyanins and betalains be possible to include DOD as another biochemical marker. Minale et al.$. the most convincing theory that explains this evolutionary paradox seems to be that both the pigments coexisted in pre-historic plants from which. other- namic acid (Minale et al. probably. 74 for the lack of anthocyanins in Caryophyllales. (1992) Gomphrenin III (74) 550 727.. see Section 3.. Except 72. 2009). sis pathway enzymes to examine their expression and activity in phological characteristics such as free-central (sometimes basal) anthocyanin-pigmented taxa. Minale et al. 1992).10b-tetrahydro-8. P3 type NMR data. duce betalain pigments instead of anthocyanins. structure of gomphrenin IV (75) was identified tenta. Gandía-Herrero and García-Carmona. wherein late betalains.. 1994). (1992) Gomphrenin IVa (75) –b 757 Gomphrena globosa L. a Tentatively identified compound. the promoter regions of these genes were different from that of anthocyanin-producing The ambiguity in structure elucidation in some cases has been species (Shimada et al. 2006). of plastids have an arrangement consisting of a peripheral ring of tively as betanidin-6-O(60 -O-sinapoyl)-glucoside by comparing the proteinaceous filaments that surrounds a globular or angular pro- mass fragmentation pattern with that of 72. for more information see Section 3. it could 2. Occurrence et al. 2007). respectively (Heuer et al.9[20 -(200 -hydroxy-00 -carboxylalkenyl)-[10 . Thus. Grotewold. 2007b). 1971)..30 ]- to formation of betalamic acid. (2012) could induce betalain production in anthocyanin producing . tion in the betalain-pigmented taxa is.#.:UVAbs. it cyanin-like (Men et al. (1967) and Heuer et al. In the light of this evolutionary convergence.. and silica-gel TLC (Piattelli and Imperato.2. As (1) there is no evi- HO dence that betalain-accumulating and betalain-devoid families within Caryophyllales have monophyletic lineage (Cuénoud et al. 73 of betalain producing taxa have revealed a possible explanation R= Feruloyl. which are. This infers that anthocyanin produc- resolved with the use of sophisticated techniques. based on scriptional levels.. Core members can be further divided into anthocyanin and betalain-accumulating families. 1999. 1994). jalapa DOD (Nakatsuka Complex pigment evolution in Caryophyllales has attracted a et al. systematic and phylogenetic analyses (Waterman. 2012). and spontaneous production or complete loss of pigmentation trait (Brockington cyclisation to cyclo-DOPA. none of the time as a group of pigments different from anthocyanins. Family names (APG III. 2005a. 2014). expression of Lentinula edodes TYR and M. 1999a) and UDP-glucose:cyclo- was carried out by a cytochrome P450 encoded by CYP76AD1 DOPA-5-O-glucosyltransferase (cD5GT) (Sasaki et al.. 2007) and phylogenetic data (Brockington et al.. 2013). or CYP76AD1 in L-DOPA oxidation leading to cyclo-DOPA formation Nicotiana tabacum and A. Dilleniaceae and Santalales (some plants under the family Santalaceae accumulate anthocyanins such as berries of Santalum 2. 2007. uolar transport. although earlier grandiflora DOD and A. Although metabolic Brockington et al. few Pigment profile of about 20 genera out of about 472 genera studies have suggested that certain genera (e. muscaria L-DOPA dioxygenase gene (DODA) attempt to identify betalain pigment in them was inconclusive constructs and feeding of L-DOPA. 2009). 1999. 2011). 2013. 2014b)) may Betalains have been known to accumulate in leaf.. formation of betanin. the authors analytically et al.. which could be achieved only anthocyanin accumulating plants and reversals to anthocyanin in the presence of diphenol oxidase (DO) activity.. anthocyanins. P. the reason for mutual exclusiveness of these pigments ified with pigment analysis data. Limeum) in (excluding that of Chenopodiaceae) belonging to eight families Fig. may be reflected in vac. thaliana cell cultures by combined (Steiner et al. 2.e. In line with this. inflorescence/flower. In another recent study. petiole. phylogenetic character expressed. root. Giridhar / Phytochemistry 117 (2015) 267–295 cell culture of Solanum tuberosum. Caryophyllales (total 34 families) is divided into core and non-core clades. Going by chemical which is naturally not equipped to do so. However. 2002). The physiological effect of resulted in inclusion of five more families under Caryophyllales biosynthesis of both anthocyanins and betalains in the same plant. 2). Hatlestad et al. that has now 34 families (APG III. reconstruction studies suggested multiple origins of betalains from mation of dopaquinone from DOPA. betalain pigmentation trait seems to be partially supported by These findings indicated that there is DO activity of tyrosinase reports on existence of two enzymes for formation of a betalain (TYR) present in some of the anthocyanin producing plants.. chemosystemtatic (Waterman. A list of 2000) and there is need for inclusion of more families under betalain-containing plants and their parts is provided in Table 2a.. Molluginaceae. 17 out of 25 families forming core engineering has achieved production of betalain in anthocyanin Caryophyllales are most likely to accumulate betalains instead of producing plants indicating phylogenetic single ancestor origin. In line with this.. 1999..b) in (Hatlestad et al..I. pigmentation and agronomic data on some of the promising beta- This particular puzzle is expected to further complicate in the lain sources need to be generated as betalains. storage and their function.. 2009) (Fig... Even if functional DOD gene is (Clement et al. The above reports presented data on production of anthocyanins in advances in molecular phylogeny research on Caryophyllales have the transformed plants/parts or cells. some of these families have not been ver- as of now. petals of Antirrhinum majus and anthocyanin producing taxa. specially betaxan- light of the molecular phylogeny studies which reported that thins. It compound i. and diphenol oxidase (DO) activity of TYR thesis was achieved in non-betalain accumulating plants viz. fruit. Large scale screening of betalain in nature is far from our understanding. Suzuki et al. Khan. are highly desirable water-soluble yellow colours.2.. production of violet coloured betacyanins requires for. This study revealed the possibility of engineering lot of attention since late 1960s when betalains were classified first betalain biosynthesis across species barrier. bract and seed grains. 2011) have been adapted. anthocyanins and betalains (Cuénoud et al. *Pigment analysis data not available.282 M. 2002).g. . However.. To add to this.. 2011). The possibility of multiple phylogenetic origins of confirmed the accumulation of betaxanthins and betacyanins. stem. Caryophyllales order (Cuénoud et al. be related to Caryophyllales (Hoot et al.. may accumulate both seedlings of Arabidopsis thaliana through biolistic introduction of P. involvement of UDP-glucose:betanidin-5-O-gluco- could be also possible that the L-DOPA to dopaquinone oxidation syltransferase (B5GT) (Vogt et al. 2012. Accumulation in plants album (Sri Harsha et al. betalain biosyn. Soltis et al..2. Khan and Giridhar. 14. Gandía-Herrero et al. and elucidation of biological activities of the crude or purified product and betacyanin-like suaedin) have been identified unam. DOD – DOPA dioxygenase. DO – diphenol oxidase. reactions 4. 2008. There are many more pigmented plant investigations on application and possible health benefits of the parts to be explored from the remaining genera. 18. 24. and amaranth have been used in most of the elaborate stud- Caryophyllales have been studied and about 75 betalains (exclud. cD6GT – cyclo-DOPA-6-O-glucosyltransferase. B5GT – betanidin-5-O-glucosyltransferase. 2003 and Gandía-Herrero and García-Carmona. In the last decade. cD5G20 GlcUT – cyclo-DOPA-5-glucose-20 -O-glucuronyl- transferase. extracts (Moreno et al. 23. HOOC N COOH Betalamic acid HOOC N COOH quinone H H H β−Glucosidase Glc HOOC N COOH Betanidin H 2-Descarboxy betanidin 16 UDP-Glc Betaxanthins 15 HOH2C HOH2C HOH2C B5GT O O HO HO O O O HO UDP HO HO O HO OH OGlcU H OH H UDP-Glc UDP-GlcU 17 HO N COOH HO N COOH HO + 19 N COOH H H cD5GT cyclo-DOPA-5-O-glucoside cD5G2'GlcUT cyclo-DOPA-5-O(2'-O- UDP UDP glucouronyl)-glucoside 18 H 20 HOOC N COOH H Betanin Amaranthin Fig. 9. Schematic diagram showing betalain biosynthesis pathway (modified from Strack et al. which is a heating degradation uses. M. 8.I. Such biguously (Tables 1a–1e). dragon fruit. 20. TOH – tyrosine hydroxylase.. 2014). out of a total of 17 betalain-accumulating families under pear. Reactions 3. These studies . cD5GT – cyclo-DOPA-5-O-glucosyltransferase. UDP – uridine-50 -diphosphate. P. Giridhar / Phytochemistry 117 (2015) 267–295 283 COOH 8 H 7 NH2 1 HO COOH 6 2 N Tyrosine 3 5 HO 4 OH 9 N HO Tyrosine H DAA O2 HOOC N COOH AA 1 H TOH COOH H Portulacaxanthin II COOH 25 H H N COOH HOOC O2 6 H 10 NH2 NH2 O2 NH2 5 21 DO/ CYP76AD1 (?) Miraxanthin V DO/ DOPA HO O CYP76AD1 HO decarboxylase CO O2 2 HO O OH DO/ OH 26 CYP76AD1 (?) HO N COOH DOPA-quinone Dopamine DOPA DOPA O2 O O2 7 2 22 DO/ 11 CYP76AD1 (?) O N DOD COOH H COOH H HOOC N COOH NH2 23 AA H N H Dopaxanthin H HO HOOC N COOH 13 HO NH O2 O H 12 OH O HO Miraxanthin V DO/ cyclo-DOPA 4. 27-spontaneous cyclisation. ies concerning pigment extraction and/or purification for food ing 17-descarboxy-mammillarinin.5-seco-DOPA HO CYP76AD1 (?) DAA OH O 27 3 2-Descarboxy 8 cyclo-DOPA O N COOH HO HO O H + + HO N COOH HO N 14 Amino acids/ H R + Amines N H H 24 HOOC N COOH HOOC N COOH 4 H H H H Dopaxanthin. B6GT – betanidin-6-O- glucosyltransferase. Glc – glucose. 3a. Khan. 7. The involvement of question- marked enzyme in the given reaction has not been experimentally verified... cactus ing number of new and potential betalain sources. pigments need to be carried out keeping in perspective the increas- only few betalain sources such as red beet. 11. 25-spontaneous Schiff base condensation. 2013). Schematic diagram showing extended betalain biosynthesis pathway (modified from Strack et al. The non-membranous spherical structures have been tion relationship of various types of betalains reported till date as understood to be formed from vesicles of ER in epidermal cells there has been only a few reports of systematic evaluation of the (Kitamura et al. X – spontaneous Schiff base condensation reactions. Vidal et al. Only a fraction (30%) of all pig- inclusions formed through H-bonding to a protein matrix (Zhang mented plants has been investigated fully and 0. betacyanins transferase gene family in anthocyanin transport to vacuole from and betaxanthins have been observed to accumulate in different cytosol (Markham et al. betalains. Studies on microstructure of antho. refer Fig. On the other hand. 3b. This hypothesis is in agreement with the detec- to accumulate betalains (Ibdah. Khan.3. colour variation based According to Delgado-Vargas et al. the loose ends need to be joined together by elucidating through their antioxidant activity (Shu et al.. betacyanins tion of cytoplasmic DOD (Christinet et al.. lack of simple and efficient methods of extraction and that anthocyanins accumulate in spherical anthocyanic vacuolar purification of these products.. 3a caption. involving certain member of glutathione-S- spp. 2010.5% of it have been . edge about subcellular localisation of betalain synthesis in plants. Bladder cells of epidermal ing suggest synthesis of betalains in cytoplasm and subsequent layer in leaves of Mesembryantheum chrystalinum have been shown import to vacuoles. (Parakeelya) (Chung et al. 2002). et al. In cactus stem. Giridhar / Phytochemistry 117 (2015) 267–295 HO X 5 HO HO O HO O O O HO HO HO H OGlc Betalamic acid O + O HO N COOH HO HO N COOH HO H OH cyclo-DOPA-5-O-sophoroside H 3 ) (? HOOC N COOH 4 Glucosylation (?) i on H lat sy u co Bougainvillein-r 2 Gl β− cyclo-DOPA-5-O-glucoside Betanin G lu X HO co UDP HO si d H as 7 B5GT 8 O e HO + 1 cD5GT cyclo-DOPA UDP-Glc O N COOH 6 Glc HO OGlc Betanidin UDP-Glc cD6GT (?) 11 B6GT 9 H Betalamic acid UDP HOOC N COOH 10 ) H HO (? HO HO HO i on H at Bougainvillein-v yl O HO O + cos HO O N COOH l u O N COOH HO G HO H OH 12 OH cyclo-DOPA-6-O-glucoside X H Glucosylation (?) 13 14 X HOOC N COOH H Gomphrenin HO HO H HO O O N COOH HO HO H O HO O HO cyclo-DOPA-6- OH O-sophoroside Fig. (Iwashina et al.. to protect the underlying photosystems However. (2000). few studies involving tyrosine feed- cell layers (Schliemann et al. 2009). multiple-papillate epidermal DOPA from chloroplasts to cytoplasm and vacuolar import of petal cells in flower of Australian native plant. copigmentation. 2006). nov. duction of natural pigments has been lagging behind due to short- lains is poorly understood. For abbreviations.284 M.. Much akin to intracellular anthocyanin translocation mechanism lainoplast in vacuoles was first reported in tepals cells in Rebutia (Grotewold. 2006).I. and structural complexity of beta. 2010).. the commercial pro- on vacuolar pH. P.. age of significant quantities of highly pigmented fresh plant cyanin-coloured petals using electron microscopy have revealed tissues. which are considered functionally analogous in planta. 2004). Unlike anthocyanins. and cytochrome accumulate in hypodermis and outer layers of chlorenchyma P450 involved in betalain biosynthesis (Hatlestad et al. 2014). 1999).. In red beet cultured cells. may be. 2003)... Subcellular localisation of betalains in vacuoles complements with its hydrophilic nature 2. 1988).. will provide a lot of significant information on structure and func. Analytical confirmation of accumulation of betacyanins in beta. 2012). Commercial production and stability in slightly acidic pH. Betacyanins intracellular biomolecules transport mechanism of tyrosine and/or were observed to express in long. there is lack of knowl- same (Gandía-Herrero et al. 2000). (Mosco. 2004). Calandrinia sp. 2012).. ) Willd. –/fruit 1. Bougainvillea/bract 259. Octopus tree/fruit NA NA Nyctaginaceae (Genera: 28–33. (2015) Spinacia oleraea L. stem 31–117 Cai et al. ex Lindl. stem NA Wang and Yang (2010) Talinaceae (Genera: 2. Cuaresmeno Xoconostle/fruit 10. Pokeberry/fruit 0. (2001) Alternanthera spp. (2005a) and Kugler et al. P. Khan.) N. Giridhar / Phytochemistry 117 (2015) 267–295 Achyranthes spp. (2012) Portulacaceae (Genera: 20. F.100) Delosperma luteum L. Erect spiderling/bark 185. stem 12. Species: 850) Celosia spp. b mmol/100 g dry weight.6 Zampini et al. Red goosefoot/flower NA NA C. (1990) Ruschia spp. Seepweed/leaf 250 Wang et al.Table 2a Betalains content of other plants.7 Kugler et al. a Total number of generas and species have been adapted from APG III (APG III.E. (2013) NA – not available. Purple ice plant/flower 66–417 Gandía-Herrero et al. Djulis/seed grain 94 Tsai et al. leaf <3.) N. quinoa Willd. oleracea L. –/leaf 27–35 Cai et al.5–17.2c Guzmán-Maldonado et al. (2008) Cactaceae (Genera: 110–120. (2004b) Bougainvillea sp. bogotensis H.5 Piattelli et al. Species: 100) Phytolacca americana L.I. Southern pokeweed/fruit NA Strack et al. Common cockscomb/leaf. –/seed 11–28 Cai et al.) Moss rose/petal. Pokeweed/fruit 1400c Forni et al.Br.E. Malabar spinach/fruit 36. –/petal 12b Strack et al. Species: 1. (2010) Ullucus tuberosus Caldas Ulluco/tuber 7 Svenson et al.18 Neamtu et al. decandra L. (1996) Mesembryanthemum crystallinum L. (2001) Iresine herbstii Hook.6–18.6–20. (2005a) Lampranthus productus (Haw.8–31b Strack et al.8c Trezzini and Zrÿd (1991) P. Species: 300–400) Mirabilis jalapa Four o’clock/flower 6. leaf 109. (2001) M. Pitaya/fruit 31–41 Vaillant et al.Br.8c Kugler et al. –/petal. (1983) P.4–551. (2007b) Basellaceae (Genera: 4.Br. d Subfamily of Amaranthaceae (APG III. (2010) Lepismium spp.7–499. (2010) C.3 Swarna et al. K. (1987) Erepsia spp. Family (total number of genera and species)a Species Common name/pigmented part Betalains content References (mg/100 g fw) Aizoaceae (Genera: 126. Christmas cactus/flower 163 Kobayashi et al. (2006) Chenopodium rubrum L. matudae cv.6–55. –/fruit 9. (1990) Dorotheanthus bellidiformis (Burm. Species: 25–30) Talinum triangulare (Jacq. (1979) P. –/petal 0. stem. Pigeonberry/fruit 350 (1700c) Khan et al. Bolus Ice plant/petal NA Strack et al.) Bl. formosanum Koidz. B.) Shwantes –/petal 2b Strack et al.5 Stintzing et al. –/flower 135–138 Gandía-Herrero et al. Common ice plant/flower.) N. (1987) Rivina humilis L. (2000) Hylocereus sp. (2001) Aerva sanguinolenta (L. (2006) Phytolaccaceae (Genera: 15. Species: 2000–3000) Myrtillocactus geometrizans Garambullo tree/fruit 214c Reynoso et al. (1997) Schlumbergera sp. –/leaf 75 Cai et al. Waterleaf/flower. (1990) Oscularia deltoids (L. 285 . inflorescence. (1990) Smicrostigma viride (Haw. 2009). –/inflorescence 15 Cai et al.E. Quinoa/seeds NA Tang et al.1 Wybraniec and Nowak-Wydra (2007) O. Species: 580) Portulaca grandiflora (Hook. Species: 11) Didieria madagascariensis Baill. (2007b) Amaranthaceae (Genera: 74. (2011) Chenopodioideaed (NA) Suaeda salsa L. Spinach/leaf NA NA Didiereacea (Genera: 4. Globe amaranth/inflorescence 7. Species: 25) Basella alba L.2 Vogt et al. (2001) Gomphrena globosa L.2–60.1 Lin et al. –/petal 4–8b Strack et al. f. c Dry weight basis. Livingstone daisy/flower NA Heuer et al. 2009). (1965b) Boerhavia erecta L. (1999b) Glottiphylum spp. (2005) and Shea (2012) Mammillaria spp. 000 acre.45 billion (Manchali et al. are poorly stable resulting in significant loss during processing.8 Gt (Table 2b) in which about components could be expected. (2006) and Shea (2012) Amaranthe (seed) 0. Sources (plant part) Yield (t/ Global production Betalain content (mg/g Total betalain yielda References ha) (kt) fw) (t) Red beetroot (root) 50–70 241985317000 0. reduced our market at the end of 20th century (Downham and Collins. amino derivatives to form betaxanthins through aldimine bonding and (3) the plant parts could also be utilised effectively as animal (reaction 4. 2005a).02 Cai et al. the other hand. The low level of commercial of betalains (Figs. There is no proper The foundation for the currently accepted biosynthesis pathway data for other sources of betalains.000 ton (Manchali et al. 2013). cyclo-DOPA is a common feed. 4000 t only (Hilpert and Dreiding. inflorescence. This led to the tion as loss of betaxanthins during extraction is comparatively conclusion that DOPA was efficient precursor of betalamic acid. d Global production estimate of pitaya is based on the European import. common and feathered cockscomb (Amaranthus sp. Biosynthesis pathway duction of betalains.. For example. In case of betalains. Among the recently reported new sources of betalains. e Global production figure of amaranth is based on annual production in USA..48 Tt annually. R. 2000. humilis (Khan et al. cess. or cyclises to cyclo-DOPA other beneficial phytochemicals reported in them. 2013). (1968) by extraction of betalains could be attributed to the fact that betalains showing that radiolabelled DOPA was incorporated into betanin. c The value represents the average yield of pitaya in Malaysia and USA. Extradiol it may be assumed that ca.) Swiss chardb 35–40 NA 0. Betalain biosynthesis starts with global market for food colours grew by 2. 2006).. 1972). a Total betalain content was calculated based on the lower values of the betalain content in respective sources. Hatlestad et al. biosynthesis was compiled (Strack et al. However. Ullucus tuberosus (Svenson et al. 1991).04–0. In 2009. appear to be promising mainly due to the acid (Gandía-Herrero et al. unlike other amino acids. by the catalytic activity of either DO of TYR or a cytochrome P450 2008). Most of the plant parts including seed. Fig. feeding betalamic acid to non-betalain accumulat- sidered because (1) some plants are easy to maintain and grow ing plants resulted in accumulation of betaxanthins. better quality of pigments devoid of off-flavour. leaves. (2014) suggest the presence of . 2007).5% of the annual production of the betalain pigmented plant parts are uti. efforts on exploring new and eco.001 0. Fig. 2013) have shed light on the advances in betalain improvement in extraction. (2009) Pitaya (fruit) 7–19c 194.. seco-DOPA that non-enzymatically rearranges to betalamic acid. it has not been considered economically viable source (Stintzing and Carle.05 0.286 M. while the quinone gets reduced to its diol (Tsai et al. 3a).08 NA Stintzing and Carle (2008) (petiole) Cactus pear (fruit) 45 23. on reaction with proline produced only betacyanins are considered as economically viable for extrac. a comprehensive update of the different reports on betalain pigment loss. which is US$ 1. and which. 99. More recently. (2005). It is formed by oxididation of DOPA to dopaquinone Suaeda salsa (Wang et al. b Due to about 50% post-harvest loss of Swiss chard petioles. of colour. 1997) (reactions 2–3. (1999) extraction. In the pro- been estimated at 100 million ton annually (Britton et al. biosynthesis in the last decade.5–1.7 Guevara et al. Fig. root. Talinum triangulare form in the presence of biological reducing agent such as ascorbic (Swarna et al. 2012). bougainvillea and cactus pear (O..8 18. 3... few other advantages such as availability of wide spectrum However. In all betacyanins.3 Vaillant et al.. Chenopodium formosanum (encoded by CYP76AD1). It also includes the USA’s 1200 t of pitaya fruit import from Nicaragua (Shea. raw or refined beet extract accounted for ca. P. a lot more technological innovations would be the core structure of betalains (Christinet et al. food vs colourant conflict. 2012). (2) some fruits such as cactus fruits could be used that betalamic acid spontaneously condenses with amino acids and as source of important phytochemicals after pigment extraction. pitaya and cactus pear. 3. possibility of developing betalain-forti- 2000).1 t from other sources such as amaranth. China grows amaranth in 100.. 2010). Khan. In improvement in extraction and subsequent processing to minimise 2003. 2013) and ducing DOPA (Steiner et al. ficus-indica) have the carotenoids production by plants. Fig. stem could be used for pigment extraction. specificity for aldimine bond formation with amino acids and.. Similar results were reported on biogenesis of indicaxanthin after Herbach et al. in EU. If 0..1% during 2009 to reach massive accumulation of tyrosine (Kishima et al.5- betalains (obtained from red beet) supply (Hilpert and Dreiding.. 2004. the global demand hydroxylated by tyrosine hydroxylase activity (TOH) of TYR pro- for food colour was 45. carotenoids constituted only a quarter of the natural col. total pigment production capacity would be 0. In recent times. exhaustively studied (Wissgott and Bortlik. 2008). 1996).. on however mass propagation of plants could be also seriously con. 2010). Malaysia and Vietnam’s production of the fruit in 2005.I. 2003).. Africa and others (103. 2007). 1999) (reaction 1. Plants such as (Hatlestad et al.7d 0.000 acre cultivated with a yield of 1000 pound/acre) in 2005. Le Bellec et al.. Gandía-Herrero and García-Carmona (Gandía-Herrero and García- tion of many new commercial sources and technological Carmona.. substructure. Biosynthesis of betalains lised for pigment extraction. Basella alba (Lin et al. higher than that of betacyanins (Delgado-Vargas et al. 3a and 3b) was laid by Miller et al. 1995). pokeweed. Schliemann et al. 3a). indicaxanthin exhibiting 90% of the radioactivity. Mueller required in coming years to exploit more plant sources for betalain et al. 2006).41 60. Indicating much lower commercial pro.4–20 96794126800 Pavokovic and Krsnik-Rasol (2011) and DAFF (n.99% contribution is from beetroot alone and 79.4 0.. 10% of the demand was fulfilled by aromatic ring cleavage activity of DOD converts DOPA to 4. 2012) (reactions 5–7. This indicated on dry/arid land. Biotechnological production of betalains through noticed that betalamic acid did not show stereoselectivity and in vitro systems could deliver the market demand satisfactorily.1. nomically viable sources of natural colours have resulted in addi.. estimated potential of annual global fied formulations containing certain other bioactive and nutritional production of betalains stands at 96.000–50. 3a).).32–0. This clearly shows the need for technological feeding radiolabelled DOPA (Impellizzeri and Piattelli.99 0. Giridhar / Phytochemistry 117 (2015) 267–295 Table 2b Estimated annual production potential of plant betalains from edible sources. As a result. algae. and dinoflagellates has potential of replacing red beet as source of betalains. processing and application.d. (2012) and Suzuki et al. 3a). (2001) and Myers (2004) NA – not available. pigeon berry. grandiflora by means that yellow beet and parts of various plants belonging to inhibition experiments with Cu2+-chelating agents (Steiner et al. 1999). americana fruit ontogeny was concomitant lamic acid. Gomphrena sp. 2012). 5. Fig. Fig. Lampranthus. 1996).. different stages of P. Owing to the detection of an enzyme cD5GT model plant with broad pigment spectrum to establish a coherent (Bauer. Fig. cyclo-DOPA. 3a). this enzyme was considered a TYR (Km duct may catalyse formation of betanidin through reactions 10 for L-tyrosine.2. 3a). 2008). On the other hand. nordihydrogua- hydroxycinnamoylglucose as acyl donor (Bokern et al. 3b).10. Among the different routes of L-DOPA P. betanin. experimental evidence is yet not available for these proposed reac- This doubt is significant as the discussion in both reports excludes tions leading to bougainvillein-r. globosa and U.. 2005a)... is plant PPO classified under type-3 copper centre proteins having ing plants (Sasaki et al.. 3. The expression of the gene during uptake.. Apart from browning reactions. 2001. 11. DOPA level in some plants) was reported to have no plastid transit peptide before and during betalain synthesis remains constant (Kishima and additional data indicated its localisation in vacuole et al. In some of the betacyanin accumulating plants yse reactions 1. Among the PPOs involved in plant pigment synthesis. In such plants. 2005a. be also formed through condensation of betalamic acid and cD5G gomphrenin. americana (Joy IV et al. It had two anthins do not synthesise cyclo-DOPA at all. two copper-containing nuclei.. while b-glucosidase may revert it considered to be paraphyletic with only 19% amino acid sequence to betanidin (reaction 15–16.1) as well as DO (EC 1. DOPA consumption oxidase. Betanidin is glucosylated by B5GT to pro. 5. It appears that betanidin formation co-accumulate 5-O and 6-O-glucosides of betanidin (Kugler et al. gomphrenin and bougainvillein-v could be condensation 14 (Fig. 1992). B5GT along with B6GT is awaited.. in B.. may be even less. Since then. identity (Vogt. 3a). or both. inseparable catalytic activities indicating dual function: TOH (EC CYP76AD1 expression (Hatlestad et al. 1999) (reaction 8. Through betanidin. and betalains. 1997).. For formation of betanin. or both. sylated to form bougainvillein-v as in B. cyclo-DOPA-5-O-glucoside (cD5G). the maximum is in reaction 2 that forms beta. or 25–27 (Kobayashi et al. Fig. Giridhar / Phytochemistry 117 (2015) 267–295 287 CYP76AD1 or its homologue and regulation of the same by tran... There are other PPOs such as catechol Depending upon a plant’s pigment profile. with unknown function.. to thylakoid membrane. as these plants co-accumulate amaranthin. Based on the substrate affin- cyclo-DOPA formation. a type-3 copper centre protein. However.. glabra (reactions 6–10. thins. globosa or U. differing from TYR in an additional C-terminal region dopamine that gives rise to 2-descarboxy-betanidin through reac. Svenson et al. DO of TYR and CYP76AD1 are present in betalain producing tissues. The model plant could be probably. 3a) may equally contribute to betanidin biosynthesis in product of cyclo-DOPA-6-O-glucoside (cD6G) and cyclo-DOPA-6-O- other plants because of co-presence of DOPA. as a consolidation of the information available acts as precursor to betanin and gomphrenin. 2006) in red In the light of contrasting reports on chemical nature. plant PPOs involve in biosynthesis maroyl or feruloyltransferase using corresponding 1-O. either reaction 8 or 18. tyrosine and oxidis. sophoroside. 2008). bougainvillein-r through formation of either betanin or cD5G-20 - scription factor (Hatlestad et al. ity and catalytic activities.. P.. Svenson et al. a cat- ble that there is equilibrium between DOPA formation (reaction 1) echol oxidase homologue (45% sequence identity) aureusidin and consumption (reactions 2. localised to thylakoid increases as it is involved in reactions 2.. Sasaki et al. and formation of membrane... with betalamic acid (reactions 11–12 ing enzymes.I. 2007b). The polypeptide 19–20. the comprehensive biosynthetic pathway of betalains pre.. Fig. Nevertheless. regulation and requirements in several reactions.b) that catalyses formation of pathway. 1999a). 2005a.. and low acceptance for L-tyrosine as sub- tions 21–24. 1991). Khan. 3b). globosa petals (Kugler idation of the claims made so far about betalain biosynthesis in a et al. Portulaca that accumulate only betax. an enzyme was purified (Steiner et al. cyclo-DOPA TYR/DO. it is believed that betanin could tuberosus. TYR has been proposed to catal- and 12 (Fig. formation of gomphrenin has been sup- to form betanidin (Schliemann et al. 3a). where the enzyme exists in latent form. There is need for val- ulluco tubers (Svenson et al. 1996. 2000). M. 3a) as their prod. may be the right model plant 2000). 3a) as its cyclisation pro. 3b). In amaranthin-accumulat. it has not been established if the betalamic acids (B6GT) in Dorotheantheus bellidiformis (Heuer et al. It is possi. able literatures on TYR do not fully appreciate multiple catalytic cus indicate the possibility of co-occurrence of both reactions 8 and functions. Although the reaction leading to formation of bougainvil- CYP76AD1 (Fig.. This of detection of tyrosine hydroxylation activity in P. Following this and subsequent report duct. Glottiphylum. sented in this review includes both DO activity of TYR and Fig. whereas reactions 5 to 8 and 9 to Logically. Isayenkova et al. cyclo-DOPA may give rise to existence of TOH free of DO in soluble protein fraction was . Involvement of acyltransferase in the formation monomer consists of a transit peptide responsible for localisation of acylated betalains has been demonstrated in case of lampran. logically. G. is also possible via betaxanthins in the absence of cyclo-DOPA 2007b. This assumption can explain the availability of and 13–14. ported by the presence of betanidin-6-O-glucosyltransferase Nevertheless. Till date. synthase responsible for synthesis of aurones (yellow flavonoids ucts do not co-accumulate in the same plant. strate. Fig. which is linked to 1. 2002). TYR 18 in biosynthesis pathway of betalains. glabra. The origin of these enzymes is duce betanin (Vogt et al. Fig.. proposed in red beetroots only. with betalain accumulation. tuberosus that nate from same pool or not. 3b).14. However. the discovery of the presence of (reactions 9–14. 2005. 1984) and BvGT associ. which route is favoured. 1995). 2002). 5 and many other reactions (Fig. otherwise the gene pro. However. Interestingly. Tyrosinase ated with betanin accumulation through betanidin glucosylation (Sepúlveda-Jiménez et al. G. 3a). is required in all betacyanins (Fig.1). 11 and 21. followed by reaction 5 (Fig. 2008) and G. cyclo-DOPA condenses with betalamic acid lein-v needs validation. which are produced from betanin in the presence of p-cou. The only exception seems to be genus Glottiphylum (Nakayama et al. Vogt contributing to formation of betacyanins and betaxanthins origi. 3a)... the deduced amino and Lampranthus (yellow flowering variety) of Aizoaceae family acid sequence of the plant PPO gene involved in biosynthesis of wherein dopaxanthin is the sole betalain pigment (Gandía. of low molecular weight pigments such as aurone. 3a). cD5G acts as substrate for glu. 3a). 2001) (Fig. 3a) (Zakharova and Petrova. of Nyctaginaceae and Cactaceae. 1 mM). it is not clear betaxanthins (Kugler et al. 2015) prompting one to ask if both O-glucoside (reactions 1–3 and 4–5. betanidin formation via deglucosylation has been for investigating if cD5GT/B5GT and cD6GT/B6GT are co-present.3.18.. there is no such report till date betanidin stock for co-accumulation of its various derivatives such because of the absence of cD6GT and cD6G-20 -O-glucosyltrans- as betanidin-5-O-glucosides and betanidin-6-O-glucosides in ferase activities in betalain producing plants. However. the avail- beet. respectively. 2007b. and cloning of cD5GT and B5GT in Amaranthus hypochondria. detection of cD5G (Wyler et al. et al. betalains consisted of a plastid transit peptide as first detected in Herrero et al. Moreover. bougainvillein-r and their derivatives as well as (reactions 17–18.b). Fig. This enzyme has dual function: oxy- curonyltransferase leading to formation of amaranthin (reactions genase and oxidase (Gerdemann et al. iaretic acid. which may be gluco- so far. P. but coenzyme (pterin compounds) dependent (Km Betalain formation was confirmed by HPLC analysis of the extracts for L-tyrosine. the (Casique-Arroyo et al. which too is pro. TYR activity may increase in the presence of purified and characterised from A. If 3. inhibitory effect of IAA on wheat root elongation (Stenlid. and P. gla- and DO (either present in a single or separate enzyme) in order bra. then how their regulation is achieved needs to be answered. the authors purified DOD (monomer. americana. It appears that role of TYR and any other Preliminary observations suggested that DNA methylation or PPO (?) entailed in betalain biosynthesis is subject of intensive rearrangement of genes play a key role in betalain biosynthesis research as this enzyme is obscure with respect to its cloning. Remarkably. salsa (gi: 642023. dopaxanthin.. then how are they regulated to ensure physiological neously cyclises to betalamic acid (reactions 2 and 3. activating both oxygen and the substrate ities have been observed to be commensurate with betalain con- for reaction in which oxygen gets incorporated to the substrate tent (Steiner et al. 2004)... Khan.288 M. ficus-indica. In homeostasis is not clear yet. func. Gandía- oxidation reactions (Hatlestad et al.. regulated independently. 442557134) (Gandía-Herrero and García-Carmona.5-dioxygenase leading to for. 1976). Given this broad distribution. Regulation of betalain biosynthesis this speculation is true. Mammilaria sp. 2001.. subcellu.1). 2002). The plant DOD (present as single copy) was first et al. 2006).I. 2012). 2004). americana to inhibit IAA biosynthetic enzyme IAA oxidase and counteract and S. 1052516. in cultured red beet cells (Girod and Zrÿd.3-dioxygenase responsible for for.9 mM) which sponta- et al. 2014). pendent of DO. the active site. Being key enzymes. Moreover. americana (Takahashi et al. 1999. jalapa (MjDOD) was isolated and shown to involve and DO separately or. 1999). miraxanthin. 1991). 2005b). In general.. As for active site (His-177) (Christinet et al. 2012). It indicated the possibility of existing TOH clone from M. muscaria (Hinz protein (29. DO activity associated with TYR corresponded mation of muscaflavin and L-DOPA-4. 2009. 1995). grandiflora. it is difficult to draw a conclusive picture and bryophytes (Weng et al. The fact that. lycophytes et al. 2013) owing to presence of the char- mutant four o’clock plant with yellow perianth. Fig. The dis.. sequences of PPO involved in betalain biosyn... The deduced amino acid sequences share a conserved region for maintaining constant level of DOPA in cellular environment characterised by Pro-(Ser/Ala)-(Asn/Asp)-x-Thr-Pro close to the during betalains biosynthesis (discussed in Section 3. mulating plants including Iresine sp. it appears that 2.9 kDa) having only L-DOPA-4. It Cu2+ thereby increasing betalain biosynthesis (Morales et al. Spatial and temporal expression of extradiol aromatic ring-cleaving dioxygenases accept an electron TYR or DO vis-a-vis their regulation under biotic and abiotic stress transferred from the catecholic substrate through the reduced need further studies. per- haps. dopamine. P.5-seco-DOPA (Km 6. 1752723. 2005a). Involvement of more than one PPO in betalain synthesis has been 2009). In 3. 3a). 2001. a latent PPO that do no accumulate betalains. If there are more than one PPO associated with 32 kDa) for the first time to homogeneity and it catalysed conver- betalain accumulation (Yamamoto et al. Transcript of the gene was not found in other plant parts earlier suggested (Joy IV et al. 2014)... 2014).. Till date. 3a). of enzymatic oxidation reactions in betalain biosynthesis as TYR it will be worthy to understand their structure function relation- and cytochrome P450 are localised in thylakoid and endoplasmic ship and evolutionary convergence of DOD in betalain producing reticulum membranes (Strack and Schliemann.. a cytochrome P450 (pro.. in the presence of ascor. natural hormone indole-3-acetic acid (IAA) when analysis to deduce features like sequence identity. DOPA dioxygenase addition to IAA.5 mM) and distinct from earlier reported TYR of the transformed cells. and along with them. 3a) sequence of DOD has been reported from few other betalain-accu- reverts to DOPA (Gandía-Herrero et al.5-dioxygenase activity et al. necessitates regulation of TOH Mesembryanthemum crystallinum.4-D) and cytokinin (6-benzylamino purine... addition to the above reports.3. 2012). Fig. This is interesting because betalains have been shown thesis have been reported from Spinacea oleraceae.. a homologue of CYP76AD1. et al. and functionally MYB and basic helix–loop–helix (bHLH) responsive elements in . 1741861.4-D too has a regulatory effect on TYR as its concentration in culture medium was linked to Betalain biosynthetic enzyme L-DOPA dioxygenase (DODA) was enzyme activity.3-dioxygenase activity). salsa and B. 2009) through (without L-DOPA-2. muscaria (Mueller et al.. plants. coli and functionally validated (Gandía-Herrero and García- agents that could induce conformational change without affecting Carmona. The purified enzyme validated successfully by transforming epidermal cells employing from P.4-dichlorophe- vated by the lack of TYR homologous sequences from plants noxy acetic acid (2. dicots. It could be possible that in planta betanin and IAA compete for crete claims on the role of TYR in biosynthesis of betalains need to interaction with a common signal transduction system... Furthermore.4. 3a). Fig. alone was linked proteins catalyse extradiol-type aromatic ring-opening reaction of to red colouration that needs formation of cyclo-DOPA (Suzuki lignin metabolism in monocots. 6- under Caryopyllales.. Casique-Arroyo (Lipscomb. in betalain biosynthesis in red petals of the plant (Sasaki et al. indole-2-carboxylic acid. grandiflora showed TOH (monophenolase activity) inde. This suggests that IAA may mimic beta- lains and cause feedback inhibition of betalain biosynthesis. 2012).. 2013). These changes tional validation. 0. and quinones logues encoded by LigB are present in non-betalain accumulating of dopaxanthin. In general. The situation is further aggra. structure and exogenously applied inhibited betalain biosynthesis (Bhuiyan localisation.. 2004. It was observed that the DOD activity. respectively.. cloned from P. Giridhar / Phytochemistry 117 (2015) 267–295 reported afterwards (Yamamoto et al. S. which be streamlined through heterologous expression of TYR. O. beetroot DOD was isolated. and regulation. Fig. partial or complete coding DNA bic acid. Gandía-Herrero sion of L-DOPA to 4. 1997). has better acceptance for betanin because of the substructure lar localisation and elucidation of crystal structure. having DO activity. hypochondriacus mation of betalamic acid (reaction 2. In support of this. However. a homologous cDNA (Steiner et al. phylogenetically. microprojectile bombardment method (Christinet et al. In another study. characterised in silico as a cytoplasmic DOD is regulated at transcriptional level in A. Sasaki et al. 2008). a transposition acteristic conserved region (Christinet et al. thereby facing a roadblock in bioinformatic BAP). Similarly. to betalain content in different tissues of A. 984206. both TYR and DOD activ- active site metal (Fe2+).. 2004). reaction 6. were effected by synthetic plant hormones like 2. perhaps. gymnosperms. duced by TOH (reaction 1. another PPO with DO activity also exists in soluble fraction. 2005b). plants. served two functions: L-DOPA-2. DOD sequences from betalain accumulat- tein encoded by CYP76AD1) might be also able to catalyse these ing plants are clustered together (Christinet et al. expressed in DO activity of the latent enzyme was activated by protease or other E. the product of DO (dopaquinone. Heterologous expression of the was purified from soluble as well as membrane fraction of red gene in Escherichia coli produced a recombinant protein showing beetroot (Gandía-Herrero et al. LigB event in CYP76AD3. P. 1997). in Herrero and García-Carmona.. Although DOD homo- DO’s role in generating cyclo-DOPA. Tanaka. 2001). In contrast. . Stommel In red beet. Lee et al. tion of secondary metabolites. metabolites. Ecophysiological factors influencing betalain accumulation way of betalain biosynthesis. it was of betalain biosynthesis regulation. 2005). as early as 1966. white light enhances betalain accumulation (Garay and et al.. MYB. cytochrome ological factors affecting the accumulation of these secondary P450. bellidiformis (Heuer et al. As drop in chlorophyll content (Tucker and Grierson. Giridhar / Phytochemistry 117 (2015) 267–295 289 the promoter region. Excision of dTmj1 from the intron led to red colour seedlings involved interaction of phytochrome and a blue light variegation in yellow perianth (mutant). americana (Noguchi et al. Initially it was proposed that element dTmj1 was found in the intron of CYP76AD3. lain biosynthesis through physiological components such as cryp- 1996). Khan. 2012. environmental stress seems to provoke B5GT rently known three distinct gene families encoding transcription too that catalyses betanin biosynthesis (reaction 15. in maintaining hormonal homoestasis.. Although B5GT expression pattern is tissue and genotype-specific. compartmentalisation. oxidative and abiotic stress (discussed in Section 4). salt. and detoxification (Gachon et al. salsa involvement of MYB-type transcription factor (BvMYB1) in regula. ratio. In this regard. phytochrome... 2008. Gene expression studies in S... The functional. 2007a). after watering seedlings with H2O2 (Wang et al. Fig. BvMYB1’s divergent nature points mulation induced by ecophysiological factors is needed as new evi- to the possibility that it regulates betalain biosynthetic genes dence show that in drought and salt stress. In addition. It would be relatively easy thesis. chlorophyllase activity increases have provided evidence for environmental stress regulation of with the onset of climacteric. It appears that BvMYB1 has different metabolite UDP-glucosyltransferases. betacyanin accumulation.. it is yet to establish the interaction of BvMYB1 with genes encoding the increasing number of enzymes involved in the extended path- 4. 2011). jalapa the focus of research shifted to different wavelengths of light and (Suzuki et al. Later on. and CuSO4 tion of betalain biosynthetic genes has been reported in red beet treatment to hydroponic cultures of red beet (Morales et al. was required for the formation of dihydropyridine moiety that The latest physiological component linked to betalain regula- gives rise to betalamic acid (de Nicola et al. salsa calli Looney and Patterson (1967). 2005. 1981). 1996). which are grouped in a residues instead of the ones responsible for interaction with monophyletic clade. research should have to focus shown that phytochrome can participate in signal transduction on sequencing of genes encoding biosynthetic enzymes. The authors presented a logical explanation In addition to oxidative stress. oxidants. Consequent levels of DOD. 2004) and a good requires light.. bHLH and WD40 repeats (WDRs) are cur. reaches maximum activity at the betacyanin accumulation. 2014b).. 2012). transcription factors. Cao et al. in response to certain growth regulator treatment to the changes in enzyme activity. the discovery of BvMYB1 reinforces the notion that anthocyanin and betalain-accumulating plants 4. In agreement with this suggestion.. Vacuolar pigments like antho- ronmental factors such as sunlight. 2005). enzyme activity in relation to (Cockburn et al. 2009).. which correlated with mRNA transcript peak of climacteric. Hatlestad et al. The latter case B5GT are not matching with betacyanin accumulation in A. Further understanding of the role of B5GT in betalain accu- ing plants (Shimada et al. Ca2+ signalling cascade. Isayenkova et al. was also reported in the leaves of S. fol- regulates flavonoid biosynthesis at transcription level (Hichri et al.1. 2005). Khan and Giridhar. That. Apart from DOD. the expression levels of either independently or through MBW complex. 1987)... 2014). chlorophyll pigments mask the colour of these metabolites in fresh 2015) through enhanced expression of DOD (Zhao et al. Sometimes the green Towers. will need a hitherto unknown betalain specific bHLH transcription hypochondriacus (Casique-Arroyo et al. Later. lowed by reports from red beet (Sepúlveda-Jiménez et al.. It factors that interact among each other to form MBW complex that was first characterised from D. 1975).. etc (discussed in Section 4). 2009). tion is a class II DNA transposable element identified in M. bility. 2006) and P.. To gain deeper knowledge responsive cryptochrome (Kochhar et al. The study revealed that low red:far red various ecophysiological factors such as repressors and inducers of light ratio and salt stress acted synergistically to affect indicators the pathway. cyanin biosynthetic genes that are also present in betalain produc. viridis of CYP76AD1. older seedlings requiring more light than younger ones’’ number of functionally validated homologous sequences are avail- (Garay and Towers.. anthocyanins. 2011).. Khan expected. 1996). low red:far red light cyanins and betalains too start accumulation in fruits and leaves. it was established that sunlight able for the study... since 1960s different experiments have However. 2014. 2014). which are green in the initial stages of ontogeny. it was known that ‘‘the system to study the different mechanisms at work for DOD regulation as responsible for amaranthin biosynthesis from tyrosine or DOPA DOD exists as single copy gene (Christinet et al. ulate anthocyanin biosynthesis (Nakatsuka et al. According to (Takahashi et al... 1987. M.. expression pathway leading to accumulation of betacyanin in M. Temporal change in pigment content enables For now. It appears that factor with which it will interact to form MBW complex. 3a). 2011). B5GT was induced by wounding as of how BvMYB1 can selectively activate betalain biosynthetic well as bacterial infection (Sepúlveda-Jiménez et al. UV light. These transcription factors have been already shown to reg. These genes bypassing interaction with anthocyanin bHLH transcription observations are consistent with the roles of plant secondary factors in MBW complex. 2007). 2009).. crystallinum during different stages of ontogeny. 1967.I. of crassulacean acid metabolism (CAM) and pigmentation when . 2014.. This implies that there are many environmental and physi- tochrome 2. regula- bHLH transcription factors of anthocyanins (Hatlestad et al. after which it falls to near zero. and betalains Besides MYB and bHLH responsive elements. P. biochemical and molecular similarities was followed by increase in betanin accumulation (Sepúlveda- among betalains and anthocyanins suggest the likelihood of similar Jiménez et al. BvMYB1 will not bind to promoters of antho. DOD promoter accumulate in plant parts following degradation of chlorophyll region also contains environmental stress responsive element due to action of chlorophyllase (Brady. chloroplast changes to chromo- (Zhao et al. Following this. biochemical studies have revealed plast wherein carotenoids begin to accumulate (Looney and that betalain biosynthesis is complemented by a number of envi- Patterson. Khan et al.. For example. 1966). As a result. it may be said that environmental factors influence beta- plants to adapt to ecophysiological adversities (Lichtenthaler. (Hatlestad et al. a homologue accumulation of amaranthin in Amaranthus caudatus var.. The DNA sequence of the non-autonomous their role in betalain biosynthesis.. Pigments such as carotenoids. enhancing stability and water solu- 2015). with concomitant whereas blue light inhibits the biosynthesis of betalains. B5GT was induced by oxidative stress and the induction et al. Influence of light and other physical factors originated from same ancestors and selective pigment synthesis is controlled at transcription level. the mode of interaction of these physiological agents been conducted to understand the role of light in betalain biosyn- and DOD promoter is not yet known. 1966. 2011). leaves and fruits. reactive oxygen species (ROS) induce biosynthesis regulation. 2015). chitosan and salicylic acid through floral spray to R. it has been demon. MeJA is known to elicit biosynthesis of indol glucosinolates. Triacontanol (upto 20 lM) did not affect 4. 2014). 2007). Khan. ment with MeJA (P10 lM). polyamines.0 mM). 2006). To adjust the cost involved for commercial by cryptochrome 2 in S. unless it is carried out in controlled conditions. flavonoids including anthocyanins and resvera- trum of betalains. In this scenario. non-tubers have been ond stage. 1995. red beet is the most popu- that calcium messenger system is responsive to biotic stress/elici. thujaplicin. use of plant parts appears to be the most dark condition compared to light-grown (14 h light/10 h dark) con. there is no report on large or indirectly this enzyme. applied through soil. lab-to-field plants can ensure sustain- an important role in dark-induced betacyanin accumulation in able production of natural colours.290 M. and innovative farming techniques. et al. 2005).. Currently. straightforward way to fulfil the increasing demand of hydrophilic trol (Wang et al. betalains commensurate with increase in TYR activity (Wang and Application of elicitors to intact plants may be carried out to Wang. 2008). (2010) than blue light as shown by studies on Portulaca cell cultures used crude extract of recombinant DOD to produce large quantity (Kishima et al. scale application of elicitation technique in field or ex situ plants. Giridhar / Phytochemistry 117 (2015) 267–295 widely different red:far red light ratios were used to intervene required for obtaining the ready-to-use product. Wang and Wang. In an attempt thereby maintaining decent level of betalains (Wang et al. calcium toxic) plant parts may have few other associated advantages as is a signalling molecule in plant that changes its intracellular level well such as utilisation of arid land as few betalain pigmented in response to various stimuli (Lichtenthaler. However. 1996). The main reasons for this is the ple. for production of betalains in vitro through the use of immobilised tion of betalains (Vogt et al. It appears food colourants among technologists and consumers. scale production. however less efficient that of plant cell culture. and ethylene are endogenously produced signalling (Georgiev et al. 1999). the role of light may be that it facilitates betalain biosynthesis One problem in this type of experiment is the poor reproducibility and decomposes the accumulated betalains depending upon the of the results because seasonal variations and other environmental ability of the plant to protect the pigments. salicylic acid (SA). isoflavones. maintenance requirements and various processing steps on use of abiotic elicitors on intact plants need to be carried out to . taxol. UV light too enhances accumula. In this direction.2. there may be other mechanisms such as activation of Production cost is expected to be high owing to the use of synthetic other signalling cascades through Ca2+. 1994). Gallardo et al. low temperature treatment reduced pigment degradation. etc (for details see reference Zhao et al. H2O2. SA. etc. This idea may not be easy to execute as TYR is not well reported to accumulate betalains in dark. 2007) indicate that cellular Ca2+ level also regulates directly translate the in vitro findings.. Abiotic and biotic elicitors the biochemical contents of fruit juice including pigments. S. possibly. 2009). and ethylene (>1. humilis plants. However. exploitation of red beet for pigment extrac- and Boland. The response was relatively higher in light on treat- tant with increase in betalain level were convincing. blue light of betalamic acid in vitro from L-DOPA for conjugation with amines was shown to decompose betacyanins and inhibit TYR mediated to obtain betaxanthins. SA. Ibdah. In another study. Taken together all the evidences avail. 2012). 2007. More studies medium. Exogenous application of these compounds at elevated mones. to quench oxidants like H2O2 and stabilise lene were externally applied in light and dark conditions (Cao PSII (Shu et al. selection of vari- events taking place in the dark that results in accumulation of ety. Thus. perhaps.. Ca2+-regulated ion channels. 2005). Interestingly. Jasmonic acid transformed cultures including hairy root have been reviewed (JA). 2002. b- lack of consistent reports on upstream processing including devel. molecules. lar raw material for betalain extraction. and ethy- biosynthesis. opment of high yielding cell lines that accumulate a broad spec. 2007). enzymes and a reaction medium that will be much simpler than 1996).. poses a considerable challenge in downstream processing.. While data on stabilisation of PSII concomi. H2O2. 2007b. a type of UDP-glucose: flavonoid O-glucosyl transferase. Mechanism of of the problem could be attributed to betalains’ poor stability that action is mainly through induction of JA signalling pathway. part trol. Cockburn et al. phtyochrome mediated signal transduction pathway in the pres. 2006). 2006). The observa. and bio-derived colour tag for betalain biosynthesis is in line with the already established facts health-conscious consumers. One possible reason of increase in betacyanin tion is expected to cause food vs colourant conflict in future. which induce or mediate signal enhance betalain accumulation in vitro. H2O2. In contrast. wherein the first stage which are underground and unexposed to sunlight indicates that reaction involves TYR catalysed synthesis of L-DOPA and then. salsa (Wang and Wang. 1999b. but it is also an important tor induced accumulation of secondary metabolites (Vasconsuelo vegetable crop. Apart from growth regulators and hor. the mecha. plants grow well in such soil. The colour spectrum and level of betalains which is reportedly regulated by Ca2+ (Cheng et al. exposure to high temperature enhanced betalain methyl jasmonic acid (MeJA). pigment content significantly (Khan et al. whereas continuous exposure to light decomposed been observed to be superior in terms of uniform pigment content pigments in S. possibility of direct application of tion that calcium and its physiological associates are involved in the juice without purification.. and calmodulin play plants (Khan. that darkness in the germination phase is one of the most impor. benzophenanthridines. triacontanol was was not explained. 2007). unlike DOD. The could be improved through pigment elicitation. Among other physical factors have decisive role in the success or failure of the experi- stress. In line with this hypothesis.. tive sources of betalains. For example. different biotic and abiotic elicitors are also used to concentration exerts abiotic stress. and culture condition optimisation.. 2006). so far. ment. seedlings were observed to accumulate betacyanins in continuous For time being. soyasaponin. In general. to enhance betalain level in Amaranthus mangostanus seedlings.. able. ulluco) to develop a two stage reaction system. Also. P. whereas SA nism involved for the increased accumulation of the pigments and H2O2 were less efficient.. For exam- of betalains at commercial scale. there is no in vitro production tion (Vasconsuelo and Boland. it direct exposure to light may not be absolutely required for betalain gets converted to betalamic acid through DOD activity in the sec- formation. a backward integration step could be introduced Accumulation of betalains in tubers (such as red beet. as a protective mechanism. 2006. Zhao et al. Inspite of many studies transduction pathways leading to secondary metabolite produc- on in vitro production systems. Consumption of edible (non- seedlings of S. Furthermore. Micropropagation technique could be utilised for mass propaga- tant environmental factors for betacyanin accumulation (Wang tion of plants in field as tissue culture derived beetroot plants have et al. salsa seedlings (Wang and Tao. In this accumulation after Ca2+ treatment may be through regulation of context. an alternative strategy could be to develop bio-mimetic system ence and absence of salt stress. Sekiguchi et al. salsa seedlings. as well as growth characteristics.. Hence. it is imperative to develop commercial plants as alterna- B5GT..I.5%) and salicylic acid (100 lM) could enhance Some studies pertaining to elicitation of betalains in cell lines. whereas chitosan (0. compared to seedling grown strated that Ca2+. salsa characterised... if any.. Plant Physiol. 2383–2385. E. Etude des voies métaboliques conduisant à la bétanine dans des against herbivory. 2009.. G. Plant 5. Thorne. iological role of betalains in planta does not seem to hold the clue. or auxin-antago. Carotenoids today and challenges for bance of betacyanins compared to betaxanthins is a result of the future. Bot. Neobetanin: isolation ing as to why betalain biosynthetic genes in acyanic plants are and identification from Beta vulgaris.. respectively.F. J. Rep.-Z. Sun. seedlings. I. I. pigment evolution in the Caryophyllales. 2004. This review presents 75 well characterised betalain structures Pharmacogn... Davies. Britton.. Soltis.). Liaaen-Jensen. M. mutual exclusive. Bauer. S. In: Behnke. pigmentation trait is prone to reversals (Brockington phytochrome in the signal transduction pathway of salt stress responses in et al.. Broad.. Dreiding. Salzresistenz bei pflanzen. De Nicola.C. 49. 2011. Biswas. 1975. W.. Although single ancestor origin of betalains and anthocyanins is Clement.. the Caryophyllales. Zaiko.J. J. South Africa. Strack. J. G. Springer Verlag. atpB.. S. 134. M. Whitelam. M. Few questions pertaining to biosynthesis of betalains are still Cheng.. 1435–1439. Khan is grateful to CSIR. Khan. R. 2009). and matK DNA sequences.. Technol. p... 2002.A....C. cultures de Beta vulgaris L. The data presented in this review high. the reported structures have been extracted pigments from plants of the Amaranthaceae. H. F. Food Chem. M. The participation of et al. T. 1976.d. prospective plants belonging to the families not yet studied should Casique-Arroyo. Clifford.. H. L. Y. J. 247–261. Annu. The bathochromic shift in absor. ‘‘Jewel’’ cell cultures. González de la Vara.. Characterization and to both betacyanins and betaxanthins. Acta 109.S.. on the convergence of TYR and DOD in betalain-accumulating Chung. 265– of betalains takes place.J. Department of Agriculture. Reznik. more information is required 274.. Délano-Frier..J. Smith.. UDP-glucose: flavonoid-O- unanswered such as.. 1992. M.. art facilities. research fellowship. D. cell cultures of Chenopodium rubrum and occurrence in some other members of lamic acid absorbs at 424 nm. Liaaen-Jensen. M. how intracellular trafficking betalain pigment biosynthesis in Portulaca grandiflora.. cyanins absorb at 471. Trends Food Sci. Birkhauser Verlag. Also. 190. Sun. J. H. cell lines (Esatbeyoglu et al. 2015). Acknowledgements Albeit betalains have been considered to deter herbivores from cactus (Lev-Yadun. 1994. C. 1994... 1976. (Eds. Nomenclatural and taxonomic history.. being strengthened by betalain biosynthesis in anthocyanin pro. G. or DOD alone (Harris et al. 1715–1718..8 Gt by plants belonging to about 17 families under Cai. Food Chem. University of Lausanne.. Corke. Apart from transcriptional control.. functional identification of a novel plant 4. Phytochemistry 35. D. ally is about 96. 2011). Berlin. Christinet. Interestingly. Agric. J. whereas betaxanthins and beta. K.W. J. Biol.. D. par incorporation de tyrosine et de dihydroxyphenylalanine. to repress anthocyanin synthesis Casique-Arroyo et al.. P. The chromophore beta. Identification and quantification of could be further studied for commercial scale extraction of the betalains from the fruits of 10 Mexican prickly pear cultivars by high- performance liquid chromatography and electrospray ionization mass pigments.-D. T... Sen. P.. APG III. M. T. P.. M. 3–9. On the other hand. Republic of South Africa. J. H... Fruit ripening... S. spectrometry. Grayer. 2012. Piattelli. Corke. 16. 647–653. Yet. 2001. New Zealand.). Linn. Chemical review and ducing plants after introduction of TYR and DOD (Nakatsuka evolutionary significance of betalains... 376. 1971– 1978. Giridhar / Phytochemistry 117 (2015) 267–295 291 ascertain optimum conditions for reproducible results. 32 betaxanthins biosynthesis of acylated betacyanins: purification and characterization from and 42 betacyanins (including betanidin). etc. Heuer. R..J. An update of the Angiosperm Phylogeny Group classification for the Perhaps. Mabry. Switzerland. Schwinn.. H.. N. J..J. 2013). do the same pool of betalamic acid gives rise glucosyltransferase from strawberry fruit. H. 2010.. 1994. (Eds. Light control of amaranthin late certain gene expression. A liberal estimate of annual betalain production glob. n.5 nm and 541 ± 9 nm.. interact with signalling factors/enzymes.H. 38. 2014) and for many dietary phenolic otic factors such as salinity (Bothe.M... Murakami. 2001). how TYR and CYP76AD1.K. Variation in betalain content and factors affecting the biosynthesis in Portulaca sp. Betalains from Amaranthus tricolor L. It is now obvious that more clue Verlag. 1001–1043. A. Identification and distribution of simple and fluorescence.. V.. It is worth ponder- Alard. Basel. L. G. Glover. Queenstown. New Delhi for awarding senior has not been investigated earlier. these genes have role in local and systemic defence orders and families of flowering plants: APG III. leading to accumulation of betalains in A. Liu. 5. Bot.. 2001.S. Bokern. Conclusions Biotechnol. induced in response to discontinuous and continuous herbivory. in: QMB2010-Plant them.. Harrison. In: Behnke.. 5758–5764. To update. et al. Pfander. Calandrinia sp. A. Jaganath. depending upon the structure. Parts of this review have been updated from mulation had a linear relationship in Amaranthus spp (Niveyro MIK’s doctoral thesis. 360–367. Mabry. as demonstrated for betanin in liver synthesis in isolated amaranthus cotyledons. Characterization and application of betalain Caryophyllales.. Y. Production guidelines for beetroot. Bhuiyan. Williams. H. Plos One 9.. Jiang.. Berlin. 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San Diego. Mizrahi. T... isobetanidin.-S. P. Betacyanins and phenolic compounds from Amaranthus spinosus L. 10 Indian patents and one US and EU patent. betalains. P. Alexandre de Kochko at the Institute Department of Botany of the Kakatiya University in the for Research and Development. steviosides. 40. under the super. Mysore. and his work is currently field of fungal secondary metabolites.I. working in the he became Principal Scientist at CSIR-CFTRI. He is a recipient of spices and dry fruits. caffeine. and regulation of plant secondary metabolites from food value plants that includes Indian Botanical Society (2009). After having switched his research interests towards Sciences. He is bestowed with Fellowship of Academy of Plant Sciences. and has presented several vision of Prof. Prof. The subject of his thesis was invited lectures. Dube Fellowship and worked with plant physiology group under the guidance of Outstanding Young Scientist Award of Indian Society of Mycology and Plant Professor Ewald Komor working in the field of phytoalexins accumulation and Pathology in 2008. Allahabad. 19 and i10-index. Y. France for one year. Khan. along with the development of novel and eco- .M. Warangal. Dr. He Ph. Mysore. He is an elected member of National Academy of Research Institute. in vitro production 2009. India in 2008 and Plant Tissue Culture Association of India in plant biotechnology. India (2008). He obtained his concerned with the metabolic engineering of secondary metabolites from plants. India. Reddy. Murthy to University of Bayreuth. 3 chapters in Kakatiya University. in the area of Mycotoxins in May 1996 at the is currently author of 130 peer-reviewed publications. M. India in 2012. isoflavones etc. Prof. S. (2011). He started his research career in 1992 at the associated with Coffee genomics group of Dr. and Society for Applied Biotechnology.S. 5 reviews.C. Giridhar has h-index. saicin. Subsequently in 1999 he joined as Godhoo young Food technologist Award of Association of Food Scientists and Scientist at Plant Cell Biotechnology Department of CSIR-Central Food Technological Technologists. In 2005 he was awarded Botany in 1991 at the Kakatiya University Warangal. Laljee phloem characteristics in Ricinus communis. books. H. he is working in the area of tissue culture. cap. carotenoids). Best Research Scientist Award of CSIR-CFTRI in 2012. Giridhar / Phytochemistry 117 (2015) 267–295 295 Giridhar Parvatam obtained his Masters degree in friendly methodologies for augmentation of annatto dye. Montpellier. India natural pigments (anthocyanins.D. flavours. DST-BOYSCAST Fellowship of Government of India. M. Reddy. oral and poster communications at national and international the studies on mycotoxigenic fungi associated with Conferences. Germany on DAAD Young Botanist Award of Indian Botanical Society in 2007. During 1996 to 1996 he had been Young scientist award of Academy of Plant Sciences India in 2003. In September 2010 research group of Professor S. As a part of this program he was India.