Betalain production is possible in anthocyanin-producing plant species given the presence of DOPA-dioxygenase and L-DOPA

Abstract

BackgroundCarotenoids and anthocyanins are the predominant non-chlorophyll pigments in plants. However, certain families within the order Caryophyllales produce another class of pigments, the betalains, instead of anthocyanins. The occurrence of betalains and anthocyanins is mutually exclusive. Betalains are divided into two classes, the betaxanthins and betacyanins, which produce yellow to orange or violet colours, respectively. In this article we show betalain production in species that normally produce anthocyanins, through a combination of genetic modification and substrate feeding.ResultsThe biolistic introduction of DNA constructs for transient overexpression of two different dihydroxyphenylalanine (DOPA) dioxygenases (DODs), and feeding of DOD substrate (L-DOPA), was sufficient to induce betalain production in cell cultures of Solanum tuberosum (potato) and petals of Antirrhinum majus. HPLC analysis showed both betaxanthins and betacyanins were produced. Multi-cell foci with yellow, orange and/or red colours occurred, with either a fungal DOD (from Amanita muscaria) or a plant DOD (from Portulaca grandiflora), and the yellow/orange foci showed green autofluorescence characteristic of betaxanthins. Stably transformed Arabidopsis thaliana (arabidopsis) lines containing 35S: AmDOD produced yellow colouration in flowers and orange-red colouration in seedlings when fed L-DOPA. These tissues also showed green autofluorescence. HPLC analysis of the transgenic seedlings fed L-DOPA confirmed betaxanthin production.ConclusionsThe fact that the introduction of DOD along with a supply of its substrate (L-DOPA) was sufficient to induce betacyanin production reveals the presence of a background enzyme, possibly a tyrosinase, that can convert L-DOPA to cyclo-DOPA (or dopaxanthin to betacyanin) in at least some anthocyanin-producing plants. The plants also demonstrate that betalains can accumulate in anthocyanin-producing species. Thus, introduction of a DOD and an enzyme capable of converting tyrosine to L-DOPA should be sufficient to confer both betaxanthin and betacyanin production to anthocyanin-producing species. The requirement for few novel biosynthetic steps may have assisted in the evolution of the betalain biosynthetic pathway in the Caryophyllales, and facilitated multiple origins of the pathway in this order and in fungi. The stably transformed 35S: AmDOD arabidopsis plants provide material to study, for the first time, the physiological effects of having both betalains and anthocyanins in the same plant tissues.

Plant betalains: Chemistry and biochemistry

“…These findings indicated that there is DO activity of tyrosinase (TYR) present in some of the anthocyanin producing plants. It could be also possible that the L-DOPA to dopaquinone oxidation was carried out by a cytochrome P450 encoded by CYP76AD1 (Hatlestad et al, 2012). In another recent study, betalain biosynthesis was achieved in non-betalain accumulating plants viz.…”

“…In line with this, phylogenetic character reconstruction studies suggested multiple origins of betalains from anthocyanin accumulating plants and reversals to anthocyanin production or complete loss of pigmentation trait (Brockington et al, 2011). The possibility of multiple phylogenetic origins of betalain pigmentation trait seems to be partially supported by reports on existence of two enzymes for formation of a betalain compound i.e., involvement of UDP-glucose:betanidin-5-O-glucosyltransferase (B5GT) (Vogt et al, 1999a) and UDP-glucose:cyclo-DOPA-5-O-glucosyltransferase (cD5GT) (Sasaki et al, 2005a,b) in formation of betanin, and diphenol oxidase (DO) activity of TYR or CYP76AD1 in L-DOPA oxidation leading to cyclo-DOPA formation (Steiner et al, 1999;Hatlestad et al, 2012;Suzuki et al, 2014).…”

Are betalain pigments the functional homologues of anthocyanins in plants?

“…This exclusivity remains unexplained, and is, perhaps, surprising given that gene homologues for a key enzyme in betalain biosynthesis are present in anthocyanic plants (Christinet et al, 2004) and that betanidin 6-O-glucosyltransferase, which catalyses the glucosylation of betacyanin, can also efficiently glucosylate the anthocyanidins (Vogt et al, 1997). There is, moreover, no apparent physiological barrier that prevents the two pigments from co-occurring; a recent study transferred part of the betalain biosynthetic pathway into cell cultures of potato, petals of Antirrhinum and shoots of Arabidopsis, and demonstrated that betalain production was possible in these normally anthocyanic plants when they were fed with a betalain intermediate, L-DOPA (Harris et al, 2012). Indeed, Clement and Mabry (1996) posited that the ancestor of the Caryophyllales could have held both types of pigments, but that one or the other was lost as species evolved.…”

Neuroprotective effect of Portulaca oleracea extracts against 6-hydroxydopamine-induced lesion of dopaminergic neurons

“…Catecholamines, such as dopamine, are synthesized from levodopa precursor by tyrosine-hydroxylase and have important roles as neurotransmitters in the central nervous system and in PD, once the dopamine depletion is the responsible for the hallmark symptoms of disease (Missale et al 1998). The presence of cathecolamines such as dopamine has been reported in P. oleracea (Gandía-Herrero et al 2009, Weng et al 2005 as well as the presence of levodopa in P. oleracea and the ability of converting tyrosine to levodopa (Harris et al 2012).…”