Engineered gray mold resistance, antioxidant capacity, and pigmentation in betalain-producing crops and ornamentals

Significance

In plants, three major classes of pigments are generally responsible for colors seen in fruits and flowers: anthocyanins, carotenoids, and betalains. Betalains are red-violet and yellow plant pigments that have been reported to possess strong antioxidant and health-promoting properties, including anticancer, antiinflammatory, and antidiabetic activity. Here, heterologous betalain production was achieved for the first time in three major food crops: tomato, potato, and eggplant. Remarkably, betalain production in tobacco resulted in significantly enhanced resistance toward gray mold (Botrytis cinerea), a plant pathogen responsible for major crop losses. Considering the significant characteristics of these molecules, heterologous betalain production now offers exciting opportunities for creating new value for consumers, producers, and suppliers of food crops and ornamental plants.

Abstract

Betalains are tyrosine-derived red-violet and yellow plant pigments known for their antioxidant activity, health-promoting properties, and wide use as food colorants and dietary supplements. By coexpressing three genes of the recently elucidated betalain biosynthetic pathway, we demonstrate the heterologous production of these pigments in a variety of plants, including three major food crops: tomato, potato, and eggplant, and the economically important ornamental petunia. Combinatorial expression of betalain-related genes also allowed the engineering of tobacco plants and cell cultures to produce a palette of unique colors. Furthermore, betalain-producing tobacco plants exhibited significantly increased resistance toward gray mold (Botrytis cinerea), a pathogen responsible for major losses in agricultural produce. Heterologous production of betalains is thus anticipated to enable biofortification of essential foods, development of new ornamental varieties, and innovative sources for commercial betalain production, as well as utilization of these pigments in crop protection.

Betalains are water-soluble, nitrogen-containing plant pigments that are synthesized from tyrosine. The betalain class contains a wide array of compounds, which are generally classified into two groups: the red betacyanins and the yellow betaxanthins (1). Betalains have attracted both scientific and economic interest (2–4). They have long been in use as food colorants, with pokeberry juice being used as early as the 19th century to enhance the color of red wine (5). Their stability in a wide pH range has also made them a pigment of choice for the food industry today, in which they are widely used as natural dyes for dairy, confectionery, and meat products (4). Their noted antioxidant properties (6, 7) have led to commercialization of a variety of betalain-based products in the dietary supplements industry and have prompted extensive studies into their potential health-promoting properties, including anticancer, hypolipidemic, antiinflammatory, hepatoprotective, and antidiabetic activities (8–13).

In the plant kingdom, betalains are limited to a single order, Caryophyllales, and found in very few edible plants, with red beet being the only major source for betalain extraction in commercial use today (14). Their beneficial biological activities, together with their limited availability in nature and particularly in the human diet, have sparked research into characterization of the betalain biosynthetic process, as well as the attempted production of betalains in naturally nonproducing organisms. However, progress in heterologous betalain engineering was hindered primarily due to the lack of knowledge of the gene(s) involved in the first step of the pathway, namely, 3-hydroxylation of tyrosine to form l-DOPA (15). Attempts for biotechnological betalain production were therefore focused on development of Beta vulgaris cell or hairy root culture (16). Likewise, the wide range of colors that can be produced by betalains makes them an excellent target for development of new ornamental varieties. However, as genetic engineering of betalains has been obstructed due to the lack of a fully decoded biosynthetic pathway, genetic engineering of flower color has thus far focused on modification of pathways generating flavonoid/anthocyanin or carotenoid pigments (17).

In a recent study we reported on two cytochrome P450-type enzymes, CYP76AD1 and CYP76AD6, that act redundantly to catalyze the first step of betalain biosynthesis in red beet, namely, the 3-hydroxylation of tyrosine to form l-DOPA (18). Elucidation of the last unknown step in the core pathway demonstrated the ability for stable, heterologous betalain production in tobacco (Nicotiana tabacum). More specifically, expression of three genes from the pathway, namely, the cytochrome P450 CYP76AD1, BvDODA1 dioxygenase, and the cDOPA5GT glycosyltransferase, in a single binary vector (pX11), resulted in entirely red, betalain-accumulating tobacco plants (18).

Here we conducted metabolic engineering for betalain production in three important food crops: tomato, potato, and eggplant, as well as in the ornamental plant species Petunia × hybrida. Additionally, tobacco plants with various flower colors were generated via expression of different combinations of betalain-related genes, resulting in accumulation of betalains in different betacyanin/betaxanthin ratios. Stable betacyanin and betaxanthin production without substrate feeding was also attained in tobacco (BY-2) cell-suspension culture. Finally, we report significantly increased resistance in betalain-producing transgenic tobacco plants against leaf infection by gray mold fungus (Botrytis cinerea), a necrotrophic plant pathogen that infects more than 200 plant species and is responsible for annual crop losses estimated at 10–100 billion US dollars worldwide (19). Thus, betalain engineering is demonstrated in a number of plant species and in cell cultures, opening new possibilities in biotechnological production of betalains, biofortification of food crops, development of new ornamental varieties, and crop protection.

Results

Biofortification of Tomato, Potato, and Eggplant with Betalain Pigments.

We previously reported the construction of a vector (termed pX11; Figs. 1 and 4A), harboring the three betalain-related genes: CYP76AD1, BvDODA1, and cDOPA5GT. The introduction of pX11 into tobacco resulted in formation of red-pigmented plants (18). To explore the possibility of betalain production in edible plants, pX11 was introduced into three important food crops that do not naturally produce betalains, namely, tomato (in the cv. MicroTom background, i.e., pX11-Mt), potato (i.e., pX11-St), and eggplant (i.e., pX11-Sm). This resulted in the formation of entirely red-pigmented plants in all three species. Tomato and eggplant fruit and potato tubers exhibited strong red-violet coloration in flesh and skin (Figs. 2A and 3A). Liquid chromatography-mass spectrometry (LC-MS) analysis of tomato and eggplant fruit as well as potato tubers verified the occurrence of betalains, detecting predominantly the betacyanins betanin and isobetanin (Fig. 2B). While betanin was the major betalain found in all examined tissues, additional unexpected betacyanin compounds were detected, which are likely the result of betanin modification (e.g., acylation and glucosylation) through promiscuous activity of endogenous enzymes (Table S1), a phenomenon that commonly occurs following introduction of new metabolites or metabolic pathways into plants (20, 21). The betaxanthin profile varied among the three species. Several betaxanthins were identified in each one of the species; vulgaxanthin I (glutamine-betaxanthin), vulgaxanthin III (aspargine-betaxanthin), and valine-betaxanthin were predominantly detected in potato, while indicaxanthin (proline-betaxanthin) was the prominent betaxanthin detected in tomato. The major betaxanthin detected in eggplant (m/z 369.1) has not been previously reported in literature and remains currently unidentified (Table S1).

Fig. 1.

Representation of the betalain biosynthetic pathway in red beet. l-DOPA is formed from tyrosine by the cytochrome P450 enzymes CYP76AD1 or CYP76AD6. l-DOPA is then converted to cyclo-DOPA (cDOPA) or to betalamic acid, via enzymatic reactions involving CYP76AD1 or DOPA 4,5-dioxygenase (DOD), respectively. Betalamic acid next conjugates spontaneously with amino acids to form yellow betaxanthins, or with cDOPA to form betanidin, the aglycone precursor for red-violet betacyanins. Expression of the pX11 vector results mainly in production of betacyanins, while pX13 expression leads to formation of betaxanthins only, and pX12 expression directs flux toward the synthesis of both classes of pigments.

Fig. 4.

Engineering betalain production in flowers. (A) Schematic of the pX11, pX12, and pX13 overexpression vectors. nptII, kanamycin resistance marker; DODA1, B. vulgaris DOPA 4,5-dioxygenase; CYP76AD1/CYP76AD6, B. vulgaris cytochrome P450; cDOPA5GT, Mirabilis jalapa cyclo-DOPA-5-O-glucosyltransferase. (B) Introduction of the pX11, pX12, or pX13 vectors into tobacco results in formation of differently colored flowers, viewed from top (Top row), or magnified and viewed under bright-field (Middle row), or blue light (Bottom row), in which betaxanthins are typically fluorescent. (C) LC-MS analysis of pX11, pX12, and pX13 tobacco petals. Extracted ion chromatograms (XIC) of masses (M + H = 309.1, 331.1, 340.1, 551.1), respectively corresponding to proline-betaxanthin, tyramine-betaxanthin, glutamine-betaxanthin, and betanin/isobetanin. Vertical axes are linked. (D) Petunia × hybrida (cv. Mitchell) flowers, in wild type (Left) or a betalain-producing line, following expression of the pX11 vector (Right).

Fig. 2.

Engineering betalain production in transgenic eggplant fruit and potato tubers. (A) Transformation of the pX11 vector resulted in formation of violet-red pigmented plants in potato (S. tuberosum cv. Désirée) (Top) and eggplant (S. melongena line DR2) (Bottom). (B) Betanin and isobetanin were identified as the major betacyanins in potato tuber (pX11-St), tomato fruit (pX11-Mt), and eggplant fruit (pX11-Sm) by LC-MS analysis. Extracted ion chromatogram (XIC) of betanin/isobetanin corresponding mass (M + H = 551.1) and UV-VIS absorption of the betanin peak is shown for all three tissues.

Fig. 3.

Constitutive, fruit-specific, or anthocyanin coproduction of betalains in tomato. (A) Transformation of the pX11 vector resulted in formation of violet-red pigmented tomato plants (pX11-Mt). Unripe pX11-Mt fruit are shown (Left). pX11-Mt was crossed with large-fruited, commercial tomato (cv. M82). Ripe fruit are shown (Top Right). Introduction of the pX11(E8) vector into a cv. M82 tomato background resulted in pigmentation restricted to ripe fruit (Bottom Right). (B) Antioxidant capacity in ripe wild-type and pX11-Mt fruit analyzed by the TEAC assay. Average values of three biological replicates per genotype are shown, with error bars representing SEM. FW, fresh weight. ***P value < 0.001 (t test). (C) Crossing pX11 with the Del/Ros1 lines. (Top) Unripe fruit. (Bottom) Ripe fruit. (D) MALDI-MSI of ripe fruit of the four genotypes presented in C. Betanin (m/z 551.1513), petunidin (m/z 317.0661), lycopene (m/z 537.4460), chlorophyll a (m/z 892.5348), and naringenin chalcone (m/z 273.0758). (Scale bar, 2 mm.)

Table S1.

Betalains identified by LC-MS analysis

No.		Identified compound	Rt, min	[M + H], Da	λmax, nm	Elemental composition	Mass error, ppm	MS/MS fragments
pX11 tobacco flower
1		Glutamine-Bx	2.43	340.1148	469	C14H17N3O7	0.6	323.08 [M+H-NH3]
								277.08 [M+H-NH3-H2COO]
								231.07 [M+H-NH3-2xH2COO]
								194.04 [M+H-C5H10N2O3]
								185.07 [M+H-3xH2COO-NH3]
								150.05 [M+H-COO-C5H10N2O3]
								148.05 [M+H-H2COO-C5H10N2O3]
								130.05
2		Proline-Bx	7.76	309.1087	478
3		Betanin	8.30	551.1513	534	C24H26N2O13	0.0	389.10 [M+H-Hex]
								345.11 [M+H-Hex-COO]-
								343.09 [M+H-Hex-H2COO]
								297.09 [M+H-Hex-2xH2COO]
								150.1 [M+H-Hex-H2COO-C9H7NO4]
4		Isobetanin	9.04	551.1502	533	C24H26N2O13	2.0	389.10 [M+H-Hex]
								345.11 [M+H-Hex-COO]-
								343.09 [M+H-Hex-H2COO]
								297.09 [M+H-Hex-2xH2COO]
								150.1 [M+H-Hex-H2COO-C9H7NO4]
5		Tyramine-Bx	11.96	331.13	464	C17H18 N2O5	1.8	287.14 [M+H-COO]
								211.07 [M+H-C8H9O]
								121.06 [M+H-C9H10N2O4]
pX12 tobacco flower
1		Aspargine-Bx	1.59	326.0922	470
2		Glutamine-Bx	2.42	340.1148	470
3		Proline-Bx	7.76	309.1088	478
4		Betanin	8.30	551.1516	534
5		Isobetanin	9.04	551.1517	533
6		Tyramine-Bx	11.96	331.1298	464
pX13 tobacco flower
1		Aspargine-Bx	1.60	326.0984	470	C13H15N3O7	1.2	309.07 [M+H-NH3]
								194.04 [M+H-C4H8N2O3]
								166.05 [M+H-C4H8N2O3-CO]
								124.04
2		Glutamine-Bx	2.42	340.1146	470
3		Proline-Bx	7.77	309.1089	478
4		Tyramine-Bx	11.97	331.1306	465
pX11 potato tuber
1		Aspargine-Bx	1.57	326.0991	469
2		Glutamine-Bx	2.36	340.1146	470
3		Betanin	8.34	551.1503	534
4		Isobetanin	9.01	551.1510	533
5		Valine-Bx	10.57	311.1246	472	C14H18N2O6	1.0	267.11 [M+H-COO]
								219.13 [M+H-2xH2COO]
								193.13
								175.12 [M+H-COO-2xH2COO]
								150.05
6		Unidentified Bc	11.26	656.1711	533
pX11 eggplant fruit
1		Unidentified Bx	6.5	369.1299	479	C16H20N2O8	0.3	351.12 [M+H-H2O]
								237.09
								211.07
								150.05
3		Betanin	8.26	551.1508	533
4		Isobetanin	9.00	551.1509	534
5		Unidentified bc	11.27	656.1725	533	C30H29N3O14	0.5	612.18 [M+H-COO]
								568.19 [M+H-2xCOO]
								417.13 [betanidin+H+CH2CH2]
								389.10 [betanidin+H]
								345.10 [betanidin+H-COO]
								301.12 [betanidin+H-2xCOO] 268.08
6		Unidentified Bc	12.66	670.189	533	C31H31N3O14	0.9	626.20 [M+H-COO]
								582.21 [M+H-2xCOO]
								538.22 [M+H-3xCOO]
								389.10 [M+H-281.090 (C13H15NO6)]
								345.11 [M+H-281-COO]
								301.11 [M+H-281–2xCOO]
								282.09 [M+H-389.10 (Betanidin)]
								264.09 [M+H-betanidin-H2O]
								257.13 [M+H-281–3xCOO]
								247.08
								180.10
								138.06
7		Unidentified Bc	15.63	655.1771	533	C31H30N2O14	0.8	389.10 [M+H-266.0790 (C13H14O6)]
								345.10 [M+H-266-COO]
								343.10 [M+H-266-H2COO]
								301.12 [M+H-266–2xCOO]
								299.10 [M+H-266-H2COO-COO]
								196.06 [M+H-C9H7NO4]
								194.04 [M+H-C9H9NO4]
								150.05 [M+H-266.0790-H2COO-C9H7NO4]
pX11 tomato fruit
1		Proline-Bx	7.72	309.109	478	C14H16N2O6	1.0	263.10 [M+H-H2COO]
								217.10 [M+H-2xH2COO]
								106.07 [M+H-2COO-C5H9NO2]
2		Betanin	8.26	551.1517	534
3		Isobetanin	9.00	551.1510	534
4		Betanidin	9.78	389.0985	539
5		Unidentified bc	17.75	835.2209	524	C40H38N2O18	1.3	551.15 [betanin+H]
								478.11 [M+H-195.05 (C9H9NO4)-162.05 (Hex)]
								434.12 [M+H-195.05 (C9H9NO4)-162.05 (Hex)-COO] 389.10 [betanidin+H]
								314.07
								285.07 [C16H12O5+H]
pX11-BY2 cells
1		Unidentified Bx	2.01	362.0851	466	C12H15N3O10	4.1	200.03
2		Glutamine-Bx	2.41	340.1139	469
3		Alanine-Bx	6.49	283.0934	468	C12H14N2O6	1.4	237.09 [M+H-H2COO]
								191.08 [M+H-2xH2COO]
								165.10
								150.05 [M+H-C3H7NO4-COO]
								145.08 [M+H-3xH2COO]
4		Bougainvillein-r-I	7.72	713.2039	530	C30H36N2O18	0.3	551.15 [M+H-Hex]
								389.10 [M+H-2Hex]
5		Betanin	8.27	551.1520	533
6		Isobetanin	9.01	551.1515	535
pX13-BY2 cells
1		Glutamine-Bx	2.35	340.1148	470
2		Alanine-Bx	6.47	283.0931	468
pX11 Solanum nigrum fruit
1	Betanin		8.34	551.1514	535
2	2-Apiosyl-betanin		8.95	683.1938	535	C29H34N2O17	0.3	551.14 [M+H-C5H8O4 (Apiosyl)]
								389.09 [M+H-Apiosyl-Hex]
								345.11 [M+H-Apiosyl-Hex-COO]
								343.09 [M+H-Apiosyl-Hex-H2COO]
3	Isobetanin		9.08	551.1503	535
4	2-Apiosyl-isobetanin		9.66	683.1950	535	C29H34N2O17	2.0	551.14 [M+H-C5H8O4 (Apiosyl)]
								389.09 [M+H-Apiosyl-Hex]
								345.11 [M+H-Apiosyl-Hex-COO]
								343.09 [M+H-Apiosyl-Hex-H2COO]
5	Betanidin		9.89	389.0974	535	C18H16N2O8	2.8	345.11 [M+H-COO]
								343.09 [M+H-H2COO]
6	Unidentified Bc		10.36	667.1989	535			389.10 [betanidin+H]
								345.10 [betanidin+H-COO]
								343.09 [betanidin+H-H2COO]
7	Neobetanin		10.61	549.1353	465	C24H24N2O13	0.7	387.1 [M+H-Hex]
								341.0 [M+H-Hex-H2COO]
8	Isobetanidin		10.84	389.0986	535	C18H16N2O8	0.3
9	Unidentified Bc		11.06	637.1887	535	C28H32N2O15	0.9	389.10 [betanidin+H]
								345.10 [betanidin+H-COO]
								343.09 [betanidin+H-H2COO]
10	Unidentified Bc		11.23	700.1995	535	C32H33N3O15	0.7	389.10 [betanidin+H]
								350.60
								343.09 [betanidin+H-H2COO]
								294.10
								168.07
11	2-Decarboxy-hylocerenin		11.63	651.2054	535	C29H34N2O15	2.6	389.10 [betanidin+H]
								345.11 [betanidin+H-COO]
								343.09 [betanidin+H-H2COO]
12	Unidentified Bc		11.73	637.1890	535			389.10 [betanidin+H]
								345.10 [betanidin+H-COO]
								343.09 [betanidin+H-H2COO]
13	Glucosyl-salicyl-betanin		12.04	833.2260	535	C37H40N2O20	0.8	671.17 [M+H-Hex]
14	Unidentified Bc		12.25	863.2360	535	C38H42N2O21	0.2	701.18 [M+H-Hex]
								389.10 [betanidin+H]
								345.11 [betanidin+H-COO]
15	Unidentified Bc		12.38	718.2092	535	C32H35N3O16	0.6	389.10 [betanidin+H]
16	Glucosyl-salicyl-isobetanin		12.57	833.2263	535	C37H40N2O20	1.2	671.17 [M+H-Hex]
17	Unidentified Bc		12.66	723.2254	535	C32H38N2O17	0.7	389.10 [betanidin+H]
18	Unidentified Bc		12.77	670.1904	535
19	Unidentified Bc		12.73	863.2361	535			701.18
								389.10 [betanidin+H]
								345.11 [betanidin+H-COO]
20	Unidentified Bc		13.05	718.2103	535			389.10 [betanidin+H]
21	Unidentified Bc		13.20	670.1885	535
22	Unidentified Bc		13.20	723.2288	535			389.10 [betanidin+H]
23	Salicyl-betanin		14.07	671.1722	535	C31H30N2O15	0.3	389.10 [betanidin+H]
								345.11 [betanidin+H-COO]
								343.09 [betanidin+H-H2COO]
24	Unidentified Bc		14.35	701.1839	535	C32H32N2O16	1.3	389.09 [betanidin+H]
								345.11 [betanidin+H-COO]
								343.10 [betanidin+H-H2COO]
								196.0 [betanidin+H-C9H7NO4]
								194.04 [betanidin+H-C9H9NO4]
								150.05 [betanidin+H-H2CO2-C9H7NO4]
								106.0 [betanidin+H-H2CO2-C9H7NO4-CO2)]
25	Salicyl-isobetanin		14.57	671.1736	535	C31H30N2O15	1.8	389.10 [betanidin+H]
								345.11 [betanidin+H-COO]
								343.09 [betanidin+H-H2COO]
26	Unidentified Bc		14.78	701.1835	535	C32H32N2O16	0.7	389.09 [betanidin+H]
								345.11 [betanidin+H-COO]
								343.10 [betanidin+H-H2COO]
								196.0 [betanidin+H-C9H7NO4]
								194.04 [betanidin+H-C9H9NO4]
								150.05 [betanidin+H-H2CO2-C9H7NO4]
								106.0 [betanidin+H-H2CO2-C9H7NO4-CO2)]
27	Sinapoyl-betanin		16.33	757.2087	535	C35H36N2O17	0.7	551.15 [betanin+H]
								345.11 [betanidin+H-COO]
								343.10 [betanidin+H-H2COO]
								301.12 [betanidin+H-2xCOO]
								207.06 [sinapic acid+H-H2O]
								194.05 [betanidin+H-C9H9NO4]
								150.05 [betanidin+H-H2CO2-C9H7NO4]
28	Sinapoyl-isobetanin		16.55	757.2076	535	C35H36N2O17	2.1	551.15 [betanin+H]
								345.11 [betanidin+H-COO]
								343.09 [betanidin+H-H2COO]
								301.12 [betanidin+H-2xCOO]
								207.06 [sinapic acid+H-H2O]
								194.04 [betanidin+H-C9H9NO4]
								150.05 [betanidin+H-H2CO2-C9H7NO4]

Betalains identified by LC-MS analysis. Assigned elemental composition with mass error and fragmentation patterns are given once for each compound to avoid redundancy. All fragments were obtained in the MS/MS mode. Rt, retention time; UV/VIS, absorption maxima at UV/visible range; Bc, betacyanin; Bx, betaxanthin; Hex, hexose.

Transformation of the pX11 vector into the edible fruit-bearing plant Black Nightshade (Solanum nigrum; pX11-Sn lines) resulted in plants bearing intense purple-red colored fruit possessing a particularly rich repertoire of betalains, with more than 40 different peaks with typical betalain UV-visual spectroscopy (UV-VIS) absorption (Fig. S1). Out of all betalain metabolites detected in the pX11-Sn fruit, 28 were subsequently validated as betacyanins by MS/MS fragmentation, which showed fragments of betanin, betanidin, or both, for each of the compounds analyzed. Furthermore, to the best of our knowledge, 16 of the 28 analyzed compounds were never reported to occur in betalain-producing plants. These included sinapoylated betanin and isobetanin as well as 14 currently unidentified betacyanins that are possibly new to nature. Additional pX11-Sn betacyanins included, among others, betanin and isobetanin decorated with apsioyl and salicyl groups (Table S1). Eggplant fruit (of pX11-Sm) and potato tubers (of pX11-St) were assessed for total betacyanin content by spectrophotometric analysis, and were found to contain an estimate of 120 ± 6 mg⋅kg⁻¹ fresh weight (FW) and 65 ± 7 mg⋅kg⁻¹ FW (means ± SEM), respectively. Betalain content in juice extracted from pX11-Mt expressing tomato fruit was estimated at 248 ± 41 mg⋅L⁻¹.

Fig. S1.

Large diversity of betalains produced in engineered Solanum nigrum plants. (A) LC-MS/PDA analysis of ripe fruit of S. nigrum expressing the pX11 vector (pX11-Sn). Figure shows absorption at wavelengths of 450–550 nm, as detected by a photodiode array detector. Peaks marked with red asterisks denote betacyanin compounds, identified based on typical UV-VIS absorption (λmax ∼ 535 nm) and MS/MS fragmentation, producing fragments of betanin and/or betanidin. (B) pX11-Sn plants accumulate betalains in fruits, flowers, and vegetative tissues.

Betacyanins and betaxanthins are known to be strong antioxidants (6, 22). Antioxidant capacity of the betalain-producing pX11-Mt tomato was therefore assessed and compared with wild-type tomato using the Trolox equivalent antioxidant capacity (TEAC) assay. Significantly, pX11-Mt expressing tomato fruit extract showed a 60% increase in antioxidant capacity compared with wild-type fruit (Fig. 3B).

Fruit-Specific Betalain Production in Commercial Tomato.

To examine the possibility of betalain production in a commercial tomato variety, the pX11-Mt line was subsequently crossed with the processing-tomato cv. M82. pX11-Mt × M82 plants exhibited a similar visual phenotype to the one observed in pX11-Mt, in which fruit and vegetative tissues are pigmented, and fruit color changes from pink to dark red during ripening (Fig. 3A). Constitutive production of tyrosine-derived compounds such as betalains could pose a significant metabolic constraint on engineered plants in terms of precursor availability. We therefore generated an additional transformation vector, in which betalain accumulation would be restricted to ripening fruit by driving CYP76AD1 gene expression under the fruit-specific E8 promoter (23). Transformation of the modified pX11(E8) vector to tomato (cv. M82; pX11-M82-E8) indeed resulted in generation of plants with betalain pigmentation restricted to the ripening fruit stage (Fig. 3A). Betacyanin concentration in juice extracted from pX11-M82-E8 fruit was determined to be 50 ± 10 mg⋅L⁻¹ (mean ± SEM), fivefold lower than in pX11-Mt fruit. Notably, none of the transgenic plants reported above displayed an apparent developmental phenotype or growth retardation, despite constitutive accumulation of betalains at high quantities.

Combining Betalain and Anthocyanin Production in Tomato.

Engineering of a different class of pigments, namely anthocyanins, has been achieved previously via expression of the common snapdragon (Antirrhinum majus) transcription factors Delila (Del) and Rosea1 (Ros1) in tomato (24). Considering the health-beneficial qualities of betalains, anthocyanins, and carotenoids, it would be of interest to engineer edible crops that accumulate all three classes of pigments. To this end, pX11-Mt was crossed with a 35S::Del/PNH::Ros1 tomato line (in the cv. MicroTom background), which accumulates anthocyanins in fruit and vegetative tissues (25). The cross resulted in generation of plants that produce both anthocyanins and betalains, as could be seen by their distinct fruit pigmentation (Fig. 3C). MALDI mass spectrometry imaging (MALDI-MSI) was used for analyzing wild-type, pX11-Mt, Del/Ros1, and pX11 × Del/Ros1 fruit. Betanin was detected in the analyzed pX11 × Del/Ros1 fruit sections, together with the anthocyanidins petunidin and malvidin, and the carotenoids lycopene and phytofluene. Anthocyanidin signals were significantly higher than their respective glycosylated anthocyanin compounds, due to removal of the sugar moiety by the MALDI ionizing laser. While betanin and the identified carotenoids were generally found to be uniformly dispersed across the section, anthocyanins accumulated mostly around the skin area (Fig. 3D and Fig. S2). In addition, we also detected chlorophyll a and naringenin chalcone pigments in fruit of all four genotypes, indicating that five different pigment classes are in fact accumulated in ripe pX11 × Del/Ros1 fruit, including carotenoids, anthocyanins, betalains, chlorophylls, and chalcones (Fig. 3D and Fig. S2).

Fig. S2.

MALDI-MSI of wild-type, pX11, Del/Ros1, and pX11 × Del/Ros1 tomato fruit (cv. MicroTom) at three developmental stages detected pigments belonging to five different classes. Pigments detected include the anthocyanidins malvidin (m/z 331.0818) and petunidin (m/z 317.0656); the flavonoid naringenin chalcone (m/z 273.0758); the carotenoids lycopene (m/z 536.4377), phytofluene (m/z 542.4846) and phytoene (m/z 544.5002); the betalain betanin (m/z 551.1508) and chlorophyll a (m/z 892.5348). (Top) Immature green stage; (Middle) breaker stage; (Bottom) red ripe stage. (Scale bar, 2 mm.)

Variation in Tobacco Flower Color Obtained by Expressing Different Combinations of Betalain Pathway Genes.

All pX11-expressing plant species displayed dominant red-violet coloration due to the accumulation of high amounts of betacyanins. The pX11 vector included the CYP76AD1 gene, encoding an enzyme catalyzing both the hydroxylation of tyrosine to l-DOPA and the conversion of l-DOPA to cyclo-DOPA. A related enzyme in red beet, CYP76AD6, uniquely exhibits tyrosine 3-hydroxylation activity to form l-DOPA (18). Since cyclo-DOPA derivatives are required for the formation of betacyanins, expression of CYP76AD6 instead of CYP76AD1 was likely to result in the formation of betaxanthins but not betacyanins, as previously observed by transient expression in Nicotiana benthamiana (18). To explore the possibility of generating transgenic plants that accumulate only betaxanthin-type betalains, a plant transformation vector was generated for 35S driven expression of BvDODA1 and CYP76AD6 (i.e., pX13; Fig. 4A). An additional vector (termed pX12), was generated for expression of both cytochrome P450 genes CYP76AD1 and CYP76AD6, alongside BvDODA1 and cDOPA5GT (Fig. 4A). While pX13-expressing tobacco plants (pX13-Nt) produced only betaxanthins, resulting in formation of yellow-pigmented flowers, expression of pX12 generated plants (pX12-Nt) with flowers of an orange-pink hue (Fig. 4B). LC-MS analysis of petals derived from pX11-Nt, pX12-Nt, and pX13-Nt plants indicated that the different colors observed in flowers of the three lines are the result of varying betacyanin/betaxanthin ratios; pX11-Nt flower extracts predominantly contained betacyanins, pX13-Nt contained betaxanthins only, while pX12-Nt extracts contained both classes of betalains, with a lower betacyanin/betaxanthin ratio compared with pX11-Nt flowers (Fig. 4C).

Differences in betaxanthin versus betacyanin accumulation could also be observed by imaging under blue light (Fig. 4B), in which betaxanthins have a typical fluorescence (26). The flux toward biosynthesis of red-violet betacyanins or yellow betaxanthins can thus be manipulated via expression of CYP76AD1, CYP76AD6, or a combination of both (Fig. 1). To examine the possibility of altering flower color in a known ornamental plant, we transformed a white petunia variety (Petunia × hybrida cv. Mitchell) with the pX11 vector. As observed with other pX11-transformed species, petunia plants showed red-violet pigmentation in roots and vegetative tissue and produced flowers of a pale violet color (Fig. 4D). LC-MS analysis of pX11-Petunia petals validated the occurrence of betanin and isobetanin as the main betalains produced.

Betalain Production in Tobacco BY-2 Cell-Suspension Culture.

Biotechnological production of betalain pigments may provide new viable sources for natural colorants in the food, pharma, and cosmetics industries. To date, most research has primarily been focused on development of B. vulgaris hairy root culture or cell culture for the production of betalains (16). Metabolic engineering for heterologous betalain production could enable the development of numerous new sources for these pigments. One viable source may be the culture of a well-established plant cell line such as tobacco BY-2. Expression of the pX11 and pX13 vectors in tobacco BY-2 cells resulted in the generation of red-violet or yellow cells, respectively (Fig. 5 A and B). Interestingly, red and yellow pigmentation was observed within cells but not in the solid or liquid media in which they were grown, suggesting that betalains accumulate in the cells and are not secreted to the extracellular medium. LC-MS analysis of cell extract of the pX11-expressing cells showed betanin and isobetanin as the major betacyanins. Several betaxanthins were identified in both the pX11 and pX13 cell lines, including glutamine-betaxanthin and alanine-betaxanthin (Fig. 5C). Betacyanin or betaxanthin concentrations in pX11 or pX13 cell extracts were determined by spectrophotometric analysis to be 47 ± 1 mg⋅L⁻¹ and 100 ± 7 mg⋅L⁻¹ (means ± SEM), respectively.

Fig. 5.

Production of betalains in tobacco BY-2 cells. (A) Introduction of pX11 or pX13 into cells of the tobacco BY-2 line resulted in the formation of red-violet or yellow-orange calli, respectively. (B) Betalain production is observed in pX11 and pX13 cell lines grown in cell-suspension culture. (C) LC-MS analysis of pX11 and pX13 BY-2 cells. Extracted ion chromatograms (XIC) of masses (M + H = 283.1, 340.1, 551.1), respectively corresponding to alanine-betaxanthin, glutamine-betaxanthin, and betanin/isobetanin. Vertical axes are linked.

Betalains Confer Resistance to B. cinerea Infection in Tobacco Leaves.

It has previously been suggested that betalains may have evolved due to a presumed role in defense against pathogenic fungi (27). However, evidence for antifungal activity of betalains in scientific literature is exceedingly scarce. Heterologous betalain production in plants provides an excellent platform for studying the putative antifungal activities of betalains in planta. We therefore examined plant resistance toward B. cinerea infection in wild-type versus pX11-expressing tobacco plants. Droplets of B. cinerea spore suspension were applied on leaves of 4-wk-old plants and the extent of B. cinerea infection was estimated by measurements of the lesion area around the infection points. Plants expressing pX11 exhibited a 90% reduction in average lesion area versus wild-type plants 2 d postinfection (DPI). At 3 DPI, 45% of infected wild-type leaves and 4% of infected pX11 leaves were in a state of advanced necrosis. Average lesion area was determined in the remaining leaves and found to be approximately 60% smaller in pX11 versus wild-type leaves (Fig. 6 A and B). Fungal staining with aniline blue was applied on infected leaves to examine hyphae density in the lesion area; however, no clear differences between wild-type and pX11 leaves could be observed (Fig. 6A). Greening and depigmentation were frequently observed around infection sites in pX11 leaves. A similar effect could also be observed following injection of 30% H₂O₂ into pX11 tobacco leaves (Fig. 6C).

Fig. 6.

Gray mold resistance in tobacco plants engineered for betalain production. (A, Top row) Leaves of wild-type and pX11 tobacco plants were infected with droplets of B. cinerea conidial suspension and photographed 3 d postinfection. (Bottom row) Fungal staining with aniline blue, 3 d postinfection. (B) Wild-type and pX11 plants infected with a total of 500 B. cinerea spores per plant were scored for lesion size 2 and 3 d postinfection. Average lesion areas are shown with error bars representing SEM. ***P value < 0.001 (t test). (C) Red depigmentation was frequently detected around B. cinerea infection points in pX11 tobacco leaves (Left), similarly to the observed effect following injection of 30% H₂O₂ (Right).

Discussion

Betalains are highly nutritious plant pigments noted for their strong antioxidant properties (28). Here, we demonstrated that heterologous production of betalains in high quantities can be achieved in a variety of plant species, including important food crops tomato, potato, and eggplant. Betalain production not only provides attractive pigmentation but also serves to enhance nutritional quality. This is of particular interest due to the small number of betalain-producing edible plants (4) and thus the limited availability of betalains in a typical human diet. While betanin, typically accumulating in red beet, was the major betacyanin produced by engineered plants, different and unique betacyanins were identified in each of the transformed species, several of which were never reported to occur in natural betalain-producing plants.

Considering the occurrence of two classes of red or yellow betalains, we also explored the possibility of generating plants with additional colors to those observed in the pX11-expressing plants. Expression of betalain-related genes in different combinations consequently resulted in generation of tobacco plants displaying red-violet, yellow, or orange-pink pigmentation. Specifically, the ratio of betacyanin/betaxanthin production was manipulated by expression of CYP76AD1, CYP76AD6, or a combination of both genes. It is likely that an array of additional hues and colors can be obtained by regulating the ratio of expression levels between these two genes. Indeed, a variety of colors is displayed by natural betalain-producing plants such as Bougainvillea and Gomphrena, where betacyanin/betaxanthin ratios were also reported to be a major factor in determination of their inflorescence colors (29). Considering the possibility of engineering betalain production on the background of ornamental plants that have many existing varieties with diverse flower colors and patterns due to anthocyanin and/or carotenoid accumulation (e.g., petunia), a myriad of novel varieties with various flower colors can possibly be developed. The ubiquitous nature of the betalain precursor tyrosine and the relative simplicity of the betalain biosynthetic pathway also infer the feasibility of heterologous betalain production in many plant species in addition to those presented here.

Betacyanins and anthocyanins provide a similar range of colors and are both widely used today as natural colorants in the food industry. However, while numerous edible plant sources are exploited for anthocyanin recovery, red beet remains virtually the only commercially used source for production of betacyanins as food colorants (14). Despite its high betacyanin content, red beet extract has several drawbacks as a source of food colorants: it mainly produces betanin and thus has limited color variability; it carries adverse earthy flavors, due to the occurrence of geosmin and various pyrazines; and it holds the risk of carryover of soil-borne microbes (8). There are currently no natural sources in large-scale use for production of betaxanthins as food dyes. Yellow beet, for example, is not used, likely due to co-occurring phenolics that are easily oxidized and mask the yellow hue of betaxanthins (8). Evidently, it is of interest to develop alternative sources for betalain production, and particularly betaxanthins, as current solutions for natural, yellow, water-soluble pigments for commercial use in the food industry are limited. Heterologous production of betalains may provide numerous new viable sources for these pigments, such as edible plants, plant cell cultures, and yeast fermentation. Development of one such source, namely, plant cell-suspension culture, was demonstrated here as a possibility for production of either betacyanins or betaxanthins, by expression of CYP76AD1 or CYP76AD6, respectively.

Betalain-producing tobacco plants showed increased resistance toward leaf infection of the phytopathogenic “gray mold” fungus, B. cinerea, compared with control wild-type plants. Red depigmentation was frequently seen around the infection points, implying a possible mechanism for the increased fungal resistance, whereby betalains were degraded due to their scavenging activity of reactive oxygen species (ROS), thus delaying plant cell death and proliferation of the necrotrophic fungus. Interestingly, a similar mechanism of action was previously suggested for the increased B. cinerea resistance observed in transgenic anthocyanin-producing tomato fruit (30). The fact that both betalains and anthocyanins exhibit a similar function in protection against a phytopathogenic fungus contributes to the understanding of the intriguing evolutionary interplay between these two pigment classes, which are taxonomically distributed in plants in a mutually exclusive fashion. It is plausible that betalains could not have replaced anthocyanins in the Caryophyllales if they could not maintain roles other than pollinator attraction. Additional parallel physiological functions and properties of betalains and anthocyanins have been postulated (31, 32).

It will be of interest to further study the mechanism behind the antifungal activities of betalains and to examine whether heterologous betalain production can be implemented to confer resistance toward additional phytopathogens, including those infecting fruit, vegetative tissues, or roots. With the resistance they confer toward gray mold as well as their striking colors and nutritional qualities, betalain engineering holds the promise of creating new value for consumers, as well as producers and suppliers of food crops and ornamental plants.

Materials and Methods

Plant Material and Growth Conditions.

Solanum tuberosum, Solanum melongena, Solanum lycopersicum, Solanum nigrum, and Petunia × hybrida plants were soil grown in a greenhouse with long-day light conditions (25 °C). N. tabacum plants used for B. cinerea infection were soil grown in climate rooms (22 °C; 70% humidity; 18/6 h of light/dark).

Plant Transformation and Regeneration.

Agrobacteria-mediated plant transformation and regeneration was done according to the following methods: tobacco leaf discs (33), eggplant (34), tomato (35), potato (36), petunia (37), S. nigrum (18), and BY-2 cells (38). All plant species were transformed using agrobacteria GV3101 strain. Plant tissue culture and BY-2 cell suspension culture was carried out in climate rooms (22 °C; 70% humidity; 16/8 h of light/dark, 2,500 Lux light intensity). Tomato pX11 transformation was done in the cv. MicroTom background. The pX11 lines were crossed with S. lycopersicum cv. M82 (SP⁺) and with Del/Ros1 (in the cv. MicroTom background). Both crosses were obtained by transferring pX11 pollen to flowers of the respective plants.

Generation of DNA Constructs.

Gene sequences used in this study were cDOPA5GT (GenBank accession AB182643.1), BvDODA1 (HQ656027.1), CYP76AD1 (HQ656023.1), and CYP76AD6 (KT962274). Additional information is given in SI Materials and Methods.

LC-MS Analysis.

Samples were analyzed using a high-resolution UPLC/PDA-qTOF system comprised of a UPLC (Waters Acquity) connected on-line to an Acquity PDA detector (200–700 nm) and a qTOF detector (tandem quadrupole/time-of-flight mass spectrometer, XEVO, Waters) equipped with an electrospray ionization (ESI) source. Additional information on betalain extraction and LC-MS analysis of betalains is provided in SI Materials and Methods.

MALDI Mass-Spectrometry Imaging Analysis.

MSI measurements were performed using a 7T Solarix FT-ICR (Fourier transform ion cyclotron resonance) mass spectrometer (Bruker Daltonics). Additional information on MALDI-MSI is given in SI Materials and Methods.

Spectrophotometric Quantification.

Betacyanin or betaxanthin content of all samples was assessed by measuring absorption at 535 nm or 475 nm, respectively, subtracting the value of absorption at 600 nm, and calculated using a previously described method, applying a molar extinction coefficient of ε = 60,000 L·mol⁻¹⋅cm⁻¹ for betacyanins and ε = 48,000 L·mol⁻¹⋅cm⁻¹ for betaxanthins (39). Samples were diluted in double-distilled water (DDW) to obtain solutions of OD₅₃₅ < 2.0. Concentrations are given in milligrams/kilograms where extraction solvent was added to sample and in milligrams/liters for samples to which no extraction solvent was added.

Tobacco B. cinerea Infection.

Wild-type and pX11-expressing tobacco plants were screened by a leaf infection assay as previously described (40). Detailed information on infection experiments and analysis is provided in SI Materials and Methods.

TEAC Assay.

The total antioxidant capacity of wild-type and betalain-producing tomato was measured by the TEAC assay (41). Whole tomato fruit were ground. Following centrifugation, supernatant was used for the assay without addition of solvent. TEAC was measured by 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS^•+) decolorization using preformed ABTS^•+ radical cation, and calculated by absorbance at 734 nm, in comparison with Trolox, as described previously (42). All determinations were performed in triplicates, where each biological sample is a pool of five tomato fruit.

SI Materials and Methods

Generation of DNA Constructs.

M. jalapa cDOPA5GT and B. vulgaris CYP76AD1, BvDODA1, and CYP76AD6 transcripts were amplified from M. jalapa red petal or B. vulgaris red hypocotyl cDNA libraries as previously described (18). pX11, pX11(E8), pX12, and pX13 vectors were constructed using Goldenbraid cloning (43). BvDODA1, CYP76AD1, CYP76AD6, and cDOPA5GT were initially cloned into a pUPD vector. pX11, pX11(E8), and pX13 are 3α1 vectors. pX12 is a 3Ω1 vector. All vectors are based on a pCAMBIA backbone (44).

Betalain Extraction.

Betalain extraction from all plant tissues was performed as follows: Extraction solution (80% methanol and 0.1% formic acid in DDW) was added to frozen, ground plant tissue (0.1–0.25 g) in a ratio of 200 µL · 0.1 g⁻¹ tissue. Samples were incubated at room temperature for 30 min followed by 5 min of sonication, 5 min of centrifugation and filtration through 0.22-µm PVDF filters (Millipore). BY-2 cells were sampled from calli grown on solid media (approximately 0.5 g) for LC-MS analysis, or sampled from cell-suspension culture (0.25 mL packed cell volume) for spectrophotometric quantification. Cells were lysed by two freeze–thaw cycles with liquid nitrogen, followed by disruption with metal beads. Cell extract was centrifuged to remove cell debris and supernatant was collected and filtered through 0.22-µm PVDF filters (Millipore) before analysis.

LC-MS Analysis.

For betalain analysis, electro-spray ionization (ESI) was used in positive ionization mode at the m/z range from 50 to 1,600 Da. The following settings were used: capillary, 1 kV; cone, 27 V; and collision energy, 6 eV. For MS/MS or MS^E runs, collision energy ramp from 15 to 40 eV was used. Separation of compounds was performed on a UPLC HSS T3 column (Waters Acquity, 1.8 µm, 2.1 × 100 mm) using eluents A (1% acetonitrile, 0.1% formic acid) and B (100% acetonitrile, 0.1% formic acid) as follows: the first 3 min, isocratic elution at 100% A; 3.0–22.0 min, a linear gradient to 75% A; 22.0–22.5 min, a linear gradient to 100% B; 22.5–25.5 min, washing at 100% B; 25.5–26.0 min, return to 100% A; and 26.0–28.0 min, equilibration at 100% A. Column temperature was set to 35 °C; flow, 0.3 mL/min. Betanin and isobetanin were assigned using red beet leaf extract as reference. Other betalain compounds were putatively identified based on accurate mass, UV-VIS spectra, and MS/MS fragmentation (Table S1).

MALDI Mass-Spectrometry Imaging Analysis.

Fresh tomato fruits were embedded with M1 embedding matrix (Thermo Scientific) in Peel-A-Way disposable embedding molds (Peel-A-Way Scientific), and flash frozen in liquid nitrogen. The embedded tissues were transferred to a cryostat (Leica CM3050) and allowed to thermally equilibrate at −18 °C for 1 h. The frozen tissues were cut into 35-µm-thick sections. The sections were then thaw mounted onto Superfrost Plus slides (Fisher Scientific) and vacuum dried in a desiccator. An ImagePrep spray station (Bruker Daltonics) was used to coat the slide with DHB matrix (30 g⋅L⁻¹ in water/methanol vol/vol 50/50 containing 0.2% trifluoroacetic acid). The system settings were as follows: 50 spray cycles, 50% sprayer power, 20% modulation, 2 s spray, 2 s incubation, and 70 s dry time. MSI measurements were performed using a 7T Solarix FT-ICR (Fourier transform ion cyclotron resonance) mass spectrometer (Bruker Daltonics). MSI datasets were collected in positive mode using lock mass calibration (DHB matrix peak: m/z 409.055408) in the range of 150–3,000 m/z, with a spatial resolution of 65 µm. Each mass spectrum was obtained from a single scan of 100 laser shots at a frequency of 1 kHz and a laser power of 18%. The acquired spectra were processed using flexImaging software v. 4.0 (Bruker Daltonics). Datasets were normalized to RMS intensity and MALDI images were plotted at the observed m/z ± 0.001%, with pixel interpolation on.

Tobacco Botrytis cinerea Infection.

B. cinerea B05.10 conidial suspension was prepared in half-strength filtered (0.45 mM) grape juice (100% organic grape juice) and adjusted to 125 conidia μL⁻¹. Intact leaves of tobacco wild-type and pX11 plants were inoculated with 4-μL droplets of conidial suspension, resulting in a total dose of 500 spores per plant. Lesion area was measured 2 and 3 d postinoculation, using the image analysis software ASSESS 2.0 for plant disease quantification (APS Press). The experiment was repeated thrice using three independent betalain-producing pX11 tobacco lines, with similar results. For fungal staining, infected leaves were incubated with 0.2% aniline blue solution for 10 min and observed under a stereomicroscope.