Traditional medicinal use is linked with apparency, not specialized metabolite profiles in the order Caryophyllales.

PREMISE: Better understanding of the relationship between plant specialized metabolism and traditional medicine has the potential to aid in bioprospecting and untangling of cross-cultural use patterns. However, given the limited information available for metabolites in most plant species, understanding medicinal use-metabolite relationships can be difficult. The order Caryophyllales has a unique pattern of lineages of tyrosine- or phenylalanine-dominated specialized metabolism, represented by mutually exclusive anthocyanin and betalain pigments, making Caryophyllales a compelling system to explore the relationship between medicine and metabolites by using pigment as a proxy for dominant metabolism. METHODS: We compiled a list of medicinal species in select tyrosine- or phenylalanine-dominant families of Caryophyllales (Nepenthaceae, Polygonaceae, Simmondsiaceae, Microteaceae, Caryophyllaceae, Amaranthaceae, Limeaceae, Molluginaceae, Portulacaceae, Cactaceae, and Nyctaginaceae) by searching scientific literature until no new uses were recovered. We then tested for phylogenetic clustering of uses using a "hot nodes" approach. To test potential non-metabolite drivers of medicinal use, like how often humans encounter a species (apparency), we repeated the analysis using only North American species across the entire order and performed phylogenetic generalized least squares regression (PGLS) with occurrence data from the Global Biodiversity Information Facility (GBIF). RESULTS: We hypothesized families with tyrosine-enriched metabolism would show clustering of different types of medicinal use compared to phenylalanine-enriched metabolism. Instead, wide-ranging, apparent clades in Polygonaceae and Amaranthaceae are overrepresented across nearly all types of medicinal use. CONCLUSIONS: Our results suggest that apparency is a better predictor of medicinal use than metabolism, although metabolism type may still be a contributing factor.

Comparison of betalain compounds in two Beta vulgaris var. cicla and BvCYP76AD27 function identification in betalain biosynthesis.

Beta vulgaris var. cicla is an edible, ornamental and horticultural plant. However, the difference of components and contents of betalain in beets with different leaf color are not well understood. Here, the stress resistance and metabolites of two B. vulgaris var. cicla cultivars were determined. The differences in stress resistance between red leaf-colored chard (RC) and yellow leaf-colored chard (YC) were positively related to betacyanins (BC) and betaxathins (BX) content in the leaves. Furthermore, a total of 3615 distinct metabolites were identified by UPLC-QTOF-MS in two cultivars, including 70 alkaloids and their derivatives, 249 flavonoids, and 264 terpenoids. There were 17 metabolites attributed to betalain biosynthesis pathway, seven of nine BC were up-regulated, and eight BX showed no significant difference in RC compared with YC. The contents of celosianin II and betanin were the highest BC in RC, at approximately 84.38 and 19.97 times that of YC, respectively. The content of portulacaxanthin II was the highest BX in two beets. Additionally, the BvCYP450 genes were identified based on genome, and the members that might be involved in betalain biosynthesis were screened. BvCYP76AD27, a member of the BvCYP76AD subfamily, had a higher expression level in RC than YC under freezing, drought and shading stress. In yeast Saccharomyces cerevisiae, BvCYP76AD5 and BvCYP76AD27 only hydroxylated tyrosine to L-DOPA, which was transformed into portulacaxanthin II by 4,5-DOPA extradiol dioxygenase. The results contribute to illustrating the molecular mechanism of betalain biosynthesis and provide useful information for further investigation of beet chemistry and sufficient utilization of this species.

Metabolic engineering of betacyanin in vegetables for anti-inflammatory therapy.

Betalains, which consist of the subgroups betaxanthins and betacyanins, are hydrophilic pigments that have classically been used for food colorants. Owing to their strong antioxidant property, their usefulness for application for therapeutic use is also expected. In addition, as betalains are mainly naturally available from plants of the order Caryophyllales, including beet (Beta vulgaris), metabolic engineering for betalain production in crops such as vegetables, fruits and cereals may provide new food resources useful for healthcare. Here we conducted metabolic engineering of betacyanins in tomato fruits and potato tubers. The transgenic tomato fruits and potato tubers with coexpression of betacyanin biosynthesis genes, CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase) and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis jalapa, under control of suitable specific promoters, possessed dark red tissues with enriched accumulation of betacyanins (betanin and isobetanin). The anti-inflammatory activity of transgenic tomato fruit extract was superior to that of wild-type fruit extract on macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS), as a result of decreased LPS-stimulated transcript levels of proinflammatory genes. These findings were in accord with the observation that administration of the transgenic tomato fruits ameliorated dextran sulfate sodium (DSS)-induced colitis as well as body weight loss and disease activity index in mice, via suppression of DSS-stimulated transcript levels of pro-inflammatory genes, including Tnf (encoding TNF-alpha), Il6, and Ptgs2 (encoding cyclooxygenae 2). Intriguingly, given the fact that the transgenic potato tuber extract failed to enrich the anti-inflammatory activity of macrophage cells, it is likely that metabolic engineering of betacyanins will be a powerful way of increasing the anti-inflammatory property of ordinary foods such as tomato.

In vitro gastrointestinal stability, bioaccessibility and potential biological activities of betalains and phenolic compounds in cactus berry fruits (Myrtillocactus geometrizans).

Cactus berry (Myrtillocactus geometrizans) is a scarcely studied Mexican wild fruit. These fruits could contribute to reduce the risk of degenerative chronic diseases due to their bioactive profile. The aim of this work was to study the betalains and phenolic profile in cactus berry, their in vitro biological activities and gastrointestinal digestive stability and bioaccessibility. 43 metabolites were identified by HPLC-DAD-ESI-QTOF (8 betaxanthins, 8 betacyanins, 13 flavonoids, 6 phenolic acids). Phyllocactin and Isorhamnetin rhamnosyl-rutinoside (IG2) were the most abundant metabolites (5876 and 396 µg/g dw) which were also bioaccessible (16 and 21%, respectively). Pulps showed higher (p ≤ 0.05) antioxidant activity by the Oxygen Radical Absorbance Capacity (27 mM Trolox equivalents). The anti-hyperglycemic activity was highest (p ≤ 0.05) in peel and pulp tissues (85% α-glucosidase and 8% α-amylase inhibition). An 83% inhibition of hyaluronidase showed high anti-inflammatory activity. Cactus berry fruit should be considered a promising fruit candidate for a sustainable healthy diet.

Effects of betalains on atherogenic risk factors in patients with atherosclerotic cardiovascular disease.

This study evaluated the potential impacts of supplementation with betalain-rich extracts of foods on some atherosclerotic risk factors in coronary artery disease patients. During an acute phase, 48 male patients received about 50 mg betalain/betacyanin, and their blood and urine samples were collected at 3, 8, and 24 hours after supplementations. Also, in a pilot randomized crossover trial, these participants were allocated to two-week interventions (a betacyanin-rich supplement of Opuntia stricta, a betalain-rich supplement of red beetroot and a placebo) with two-week washout periods. Then, their plasma samples were collected at the baseline after a two-week period. The concentrations of betanin in plasma and urine samples were determined using HPLC. Also, homocysteine and glucose levels, lipid profile, and blood pressure were analyzed. Additionally, quality of life and dietary intake were assessed. After these interventions, minimal amounts of betanin were found in plasma and about 0.13-0.93% in urine. Also, both supplements significantly decreased the concentration of homocysteine, glucose, total cholesterol, triglyceride, and LDL. Also, betalain-rich supplements lowered both systolic and diastolic blood pressures. Nevertheless, the clinically meaningful changes were only found in the case of Hcy, LDL, and non-HDL-c concentrations. It seems that food sources of betalains can be considered as functional foods because they improve the lipid profile and levels of homocysteine, glucose, blood pressure, and quality of life to some extent.

Betalains, the nature-inspired pigments, in health and diseases.

Betalains are unique nitrogen-containing pigments found exclusively in families of the Caryophyllales order and some higher order fungi, where they replace anthocyanin pigments. Betalains, consisting of betacyanins and betaxanthins are generally used as color additives in food. This review discusses on the favorable effects of acute and chronic consumption of betalains, whose edible sources consist primarily of red beetroots (Beta vulgaris) and prickly pears (fruit of the Opuntia genus of cacti). Moreover, it encompasses in vivo and in vitro studies about the bioavailability and bioaccessibility of betanin and indicaxanthin. It seems that treatment with betalains and betalain-rich diets is not only nontoxic but could also prove to be a promising alternative to supplement therapies in oxidative stress-, inflammation-, and dyslipidemia-related diseases such as stenosis of the arteries, atherosclerosis, hypertension, and cancer, among others. Due to its toxicological safety, accessibility, low price, biodegradability, and potentially advantageous biological effects on health, the incorporation of betalains in food manufacturing and related industries could pave the way to overcome current concerns over the health risks of artificial colors. Nevertheless, further studies using pure betalains are required to gain a deeper understanding of their precise biological functions.

Preharvest application of ethephon and postharvest UV-B radiation improve quality traits of beetroot (Beta vulgaris L. ssp. vulgaris) as source of colourant.

BACKGROUND: Betanins have become excellent replacers for artificial red-purple food colourants. Red beet (Beta vulgaris L. spp. vulgaris) known as beetroot, is a rich source of betalains, which major forms are betanin (red to purple) and vulgaxanthin (yellow). Betalains and phenolic compounds are secondary metabolites, accumulation of which is often triggered by elicitors during plant stress responses. In the present study, pre-harvest applications of ethephon (an ethylene-releasing compound) and postharvest UV-B radiation were tested as elicitors of betalains and phenolic compounds in two beetroot cultivars. Their effects on quality parameters were investigated, and the expression of biosynthetic betalain genes in response to ethephon was determined. RESULTS: Ethephon was applied as foliar spray during the growth of beetroot, resulting in increased betanin (22.5%) and decreased soluble solids contents (9.4%), without detrimental effects on beetroot yield. The most rapid accumulation rate for betanin and soluble solids was observed between 3 and 6 weeks after sowing in both untreated and ethephon-treated beetroots. Overall, the expression of the betalain biosynthetic genes (CYP76AD1, CYP76AD5, CYP76AD6 and DODA1), determining the formation of both betanin and vulgaxanthin, increased in response to ethephon treatment, as did the expression of the betalain pathway activator BvMYB1. In the postharvest environment, the use of short-term UV-B radiation (1.23 kJ m- 2) followed by storages for 3 and 7 days at 15 °C resulted in increased betanin to vulgaxanthin ratio (51%) and phenolic content (15%). CONCLUSIONS: The results of this study provide novel strategies to improve key profitability traits in betalain production. High betanin concentration and high betanin to vulgaxanthin ratio increase the commercial value of the colourant product. In addition, lowering soluble solids levels facilitates higher concentration of beetroot colour during processing. Moreover, we show that enhanced betanin content in ethephon-treated beetroots is linked to increased expression of betalain biosynthetic genes.

Redirecting Primary Metabolism to Boost Production of Tyrosine-Derived Specialised Metabolites in Planta.

L-Tyrosine-derived specialized metabolites perform many important functions in plants, and have valuable applications in human health and nutrition. A necessary step in the overproduction of specialised tyrosine-derived metabolites in planta is the manipulation of primary metabolism to enhance the availability of tyrosine. Here, we utilise a naturally occurring de-regulated isoform of the key enzyme, arogenate dehydrogenase, to re-engineer the interface of primary and specialised metabolism, to boost the production of tyrosine-derived pigments in a heterologous plant host. Through manipulation of tyrosine availability, we report a 7-fold increase in the production of tyrosine-derived betalain pigments, with an upper range of 855 mg·kg-1·FW, which compare favourably to many in vitro and commercial sources of betalain pigments. Since the most common plant pathway for tyrosine synthesis occurs via arogenate, the de-regulated arogenate dehydrogenase isoform is a promising route for enhanced production of tyrosine-derived pharmaceuticals in diverse plant hosts.

Gain-of-function mutations in beet DODA2 identify key residues for betalain pigment evolution.

The key enzymatic step in betalain biosynthesis involves conversion of l-3,4-dihydroxyphenylalanine (l-DOPA) to betalamic acid. One class of enzymes capable of this is 3,4-dihydroxyphenylalanine 4,5-dioxygenase (DODA). In betalain-producing species, multiple paralogs of this gene are maintained. This study demonstrates which paralogs function in the betalain pathway and determines the residue changes required to evolve a betalain-nonfunctional DODA into a betalain-functional DODA. Functionalities of two pairs of DODAs were tested by expression in beets, Arabidopsis and yeast, and gene silencing was performed by virus-induced gene silencing. Site-directed mutagenesis identified amino acid residues essential for betalamic acid production. Beta vulgaris and Mirabilis jalapa both possess a DODA1 lineage that functions in the betalain pathway and at least one other lineage, DODA2, that does not. Site-directed mutagenesis resulted in betalain biosynthesis by a previously nonfunctional DODA, revealing key residues required for evolution of the betalain pathway. Divergent functionality of DODA paralogs, one clade involved in betalain biosynthesis but others not, is present in various Caryophyllales species. A minimum of seven amino acid residue changes conferred betalain enzymatic activity to a betalain-nonfunctional DODA paralog, providing insight into the evolution of the betalain pigment pathway in plants.

Alternative Strategies in Response to Saline Stress in Two Varieties of Portulaca oleracea (Purslane).

Purslane (Portulaca oleracea) is a globally-distributed plant with a long history of use in folk medicine and cooking. We have developed purslane as a model system for exploring plant responses to stress. We exposed two varieties of purslane to saline stress with the objective of identifying differences between the varieties in the plasticity of morphological and physiological traits. The varieties responded to saline stress with significantly different changes in the measured traits, which included inter alia biomass, flower counts, proline concentrations and betalain pigment concentrations. The alternative responses of the two varieties consisted of complex, simultaneous changes in multiple traits. In particular, we observed that while both varieties increased production of betalain pigments and proline under saline stress, one variety invested more in betalain pigments while the other invested more in proline. Proline and betalain pigments undoubtedly play multiple roles in plant tissues, but in this case their role as antioxidants deployed to ameliorate saline stress appears to be important. Taken holistically, our results suggest that the two varieties employ different strategies in allocating resources to cope with saline stress. This conclusion establishes purslane as a suitable model system for the study of saline stress and the molecular basis for differential responses.

Comparative analysis of two DOPA dioxygenases from Phytolacca Americana.

The comparative analysis of two Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) that may be involved in betalain biosynthesis was carried out. The recombinant protein of PaDOD catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. The role of DOD genes is discussed in the evolutionary context using phylogenetic analysis, suggesting that DOD might have been duplicated early in evolution and that accumulation of base substitutions could have led to the different characteristics of DODs within the betalain-producing Caryophyllales.

Biochemical analysis of Phytolacca DOPA dioxygenase.

The biochemical analysis of Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) was carried out. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. A mutated PaDOD1 protein with a change of 177 Asn to Gly showed reduced specific activity compared with PaDOD1, while DOPA dioxygenase activity was not observed for a mutated PaDOD2 protein which had its conserved motif replaced with that of PaDOD. A three-dimensional (3D) structural model of PaDOD1 and PaDOD2 showed that the conserved motif in DODs was located in the N-terminal side of a loop, which was found close to the putative active site. The difference in stability of the loop may affect the enzymatic activity of PaDOD2.

Trans-epithelial transport of the betalain pigments indicaxanthin and betanin across Caco-2 cell monolayers and influence of food matrix.

PURPOSE: This study investigated the absorption mechanism of the phytochemicals indicaxanthin and betanin and the influence of their food matrix (cactus pear and red beet) on the intestinal transport. METHODS: Trans-epithelial transport of dietary-consistent amounts of indicaxanthin and betanin in Caco-2 cell monolayers seeded on Transwell(R) inserts was measured in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under an inwardly directed pH gradient (pH 6.0/7.4, AP/BL) mimicking luminal and serosal sides of human intestinal epithelium. The effect of inhibitors of membrane transporters on the absorption was also evaluated. Contribution of the paracellular route was investigated after EDTA treatment of the cell monolayer. In vitro digestion of betalainic food was performed to provide a post-intestinal fraction containing bioaccessible pigments. RESULTS: Apparent permeability coefficients (P(app)) in the absorptive direction were (4.4 ± 0.4) × 10⁻6 and (3.2 ± 0.3) × 10⁻6 cm s⁻¹ for indicaxanthin and betanin, respectively. Transport of indicaxanthin was non-polarized, linear as a function of time and concentration, and unaffected by inhibitors of membrane transporters. Betanin exhibited significantly different bidirectional P(app) values and non-linear efflux kinetics. The concentration-dependent betanin efflux was described by a kinetic model including one non-saturable (K(d) = 0.042 µL cm⁻² min⁻¹) and one saturable component identified as the apical multidrug resistance-associated protein 2 (MRP2; K(m) = 275 µM; J(max) = 42 pmol min⁻¹ cm⁻²). Permeation of both betalains increased remarkably after EDTA treatment of the cell monolayer. Neither indicaxanthin nor betanin underwent metabolic transformation. Food matrix did not affect trans-epithelial transfer of indicaxanthin, but reduced the absorption rate of betanin, red beet more than cactus pear. CONCLUSIONS: Dietary indicaxanthin and betanin can substantially be absorbed through paracellular junctions of intestinal epithelial cells. Additional trans-membrane permeation can be considered for betanin, whose absorption is limited by a MRP2-mediated efflux and negatively affected by its food matrix. Present findings are consistent with the quite higher bioavailability of indicaxanthin over betanin established in humans.

The beet R locus encodes a new cytochrome P450 required for red betalain production.

Anthocyanins are red and violet pigments that color flowers, fruits and epidermal tissues in virtually all flowering plants. A single order, Caryophyllales, contains families in which an unrelated family of pigments, the betalains, color tissues normally pigmented by anthocyanins. Here we show that CYP76AD1 encoding a novel cytochrome P450 is required to produce the red betacyanin pigments in beets. Gene silencing of CYP76AD1 results in loss of red pigment and production of only yellow betaxanthin pigment. Yellow betalain mutants are complemented by transgenic expression of CYP76AD1, and an insertion in CYP76AD1 maps to the R locus that is responsible for yellow versus red pigmentation. Finally, expression of CYP76AD1 in yeast verifies its position in the betalain biosynthetic pathway. Thus, this cytochrome P450 performs the biosynthetic step that provides the cyclo-DOPA moiety of all red betacyanins. This discovery will contribute to our ability to engineer this simple, nutritionally valuable pathway into heterologous species.

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

BACKGROUND: Carotenoids 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. RESULTS: The 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. CONCLUSIONS: The 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.

Characterization of recombinant Beta vulgaris 4,5-DOPA-extradiol-dioxygenase active in the biosynthesis of betalains.

Betalains are water-soluble pigments with high antiradical capacity which bestow bright colors to flowers, fruits and other parts of most plants of the order Caryophyllales. The formation of the structural unit of all betalains, betalamic acid from the precursor amino acid 4,5-dihydroxyphenylalanine is catalyzed by the enzyme 4,5-DOPA-extradiol-dioxygenase followed by intramolecular cyclization of the 4,5-secodopa intermediate. This paper describes the purification and the molecular and functional characterization of an active 4,5-DOPA-extradiol-dioxygenase from the best-known source of betalains-Beta vulgaris-after heterologous expression in Escherichia coli. The enzyme is a monomeric protein with a molecular mass of 32 kDa characterized by chromatography, electrophoresis and MALDI-TOF analysis. Enzyme kinetic properties are characterized in the production of betalamic acid, the structural, chromophoric and bioactive unit of plant pigment betalains.

In vitro digestion of betalainic foods. Stability and bioaccessibility of betaxanthins and betacyanins and antioxidative potential of food digesta.

Betalains are considered to be bioactive dietary phytochemicals. The stability of betacyanins and betaxanthins from either fresh foods or manufactured products of cactus pear fruit ( Opuntia ficus indica L. Mill. cv. Gialla and Rossa) and red beet ( Beta vulgaris L. ssp. vulgaris) was assessed in a simulated oral, gastric, and small intestinal digestion and compared with the digestive stability of purified pigments. A minor loss of indicaxanthin, at the gastric-like environment only, and a decrease of vulgaxanthin I through all digestion steps were observed, which was not affected by food matrix. In contrast, food matrix prevented decay of betanin and isobetanin at the gastric-like environment. Loss of betacyanins, either purified or food-derived, was observed during the small intestinal phase of digestion. Betalamic acid accumulated after digestive degradation of purified pigments, but not of food betalains. Betaxanthins were wholly soluble in the aqueous (bioaccessible) fraction after ultracentrifugation of the postintestinal (PI) digesta, whereas release of betacyanins from the matrix was incomplete. PI digesta inhibited dose-dependently the oxidation of methyl linoleate in methanol, an effect not correlated with the betalain content. The data suggest that digestive stability controls bioaccessibility of dietary betaxanthins, whereas additional factors, relevant to the food matrix and style of processing, affect betacyanin bioaccessibility.

Morphometric and biochemical characterization of red beet (Beta vulgaris L.) hairy roots obtained after single and double transformations.

It is known that T-DNA of Agrobacterium rhizogenes affects processes of plant development and activates the synthesis of secondary metabolites in transformed plant cells. In the present investigation, we provide evidence that different strains of A. rhizogenes significantly affect morphometric, morphological and functional characteristics of hairy roots of red beet (Beta vulgaris L.). Infection with four strains of A. rhizogenes (A4, A 2/83, A 20/83 and LMG-150) resulted in ten clones of hairy roots, which were named accordingly as A4(1), A4(2), A4(3), A 2/83(1), A 2/83(2), A 2/83(3), A 20/83(1), A 20/83(2), A 20/83(3) and LMG-150. Their growth characteristics, pigment content, levels of endogenous auxin and T-DNA copy number showed significant differences probably due to the physiological status of the host cell rather than the T-DNA copy number. Although A 2/83 showed highest hairy root induction capacity, the best hairy root clone was obtained with strain LMG-150 that produced highest biomass and pigments. In this root clone, the enzyme peroxidase was found involved in altering the endogenous auxin pool. When root clone LMG-150 was re-transformed to insert additional individual rol genes, two double transformed clones were obtained, one for rolABC and the other for rolC gene where the former produced higher biomass and betalaine than the latter. Despite the established fact that rol genes of T-DNA influence endogenous phytohormones, no direct correlation among the single transformants and the double transformants was found. This is the first report, in our knowledge, where a hairy root clone has been used to obtain double transformants.

Batch and fed-batch production of betalains by red beet (Beta vulgaris) hairy roots in a bubble column reactor.

Hairy root cultures from red beet (Beta vulgaris L.), which could be used for the commercial production of biologically active betalain pigments, were cultivated in a 3 L bubble column bioreactor in batch mode with various rates of air supply. Both the growth of the roots and betalain volumetric yields were highest (12.7 g accumulated dry biomass/L and 330.5 mg/ L, respectively) with a 10 L/h (0.083 vvm) air supply. The air flow rate also influenced the betacyanins/betaxanthins ratios in the cultures. Growth and betalains production were then examined in two fed-batch regimes (with a 10 L/h air supply), in which nutrient medium was fed just once or on five occasions, designated FBI and FBII, respectively. The root mass accumulation was increased in the FBI feeding regime (to 13.3 g accumulated dry biomass/ L), while in FBII the betalains content was ca. 11% higher (15.1 mg betacyanins/g dry weight and 14.0 mg betaxanthins/g dry weight) than in the most productive batch regime. Data on the time course of the utilization of major components in the medium during both operational modes were also collected. The implications of the information acquired are discussed, and the performance of the hairy roots (in terms of both growth and betalains production) in the bubble column reactor and previously investigated cultivation systems is compared.