Genetic engineering of yellow betalain pigments beyond the species barrier

Abstract

Betalains are one of the major plant pigment groups found in some higher plants and higher fungi. They are not produced naturally in any plant species outside of the order Caryophyllales, nor are they produced by anthocyanin-accumulating Caryophyllales. Here, we attempted to reconstruct the betalain biosynthetic pathway as a self-contained system in an anthocyanin-producing plant species. The combined expressions of a tyrosinase gene from shiitake mushroom and a DOPA 4,5-dioxygenase gene from the four-o'clock plant resulted in successful betalain production in cultured cells of tobacco BY2 and Arabidopsis T87. Transgenic tobacco BY2 cells were bright yellow because of the accumulation of betaxanthins. LC-TOF-MS analyses showed that proline-betaxanthin (Pro-Bx) accumulated as the major betaxanthin in these transgenic BY2 cells. Transgenic Arabidopsis T87 cells also produced betaxanthins, but produced lower levels than transgenic BY2 cells. These results illustrate the success of a novel genetic engineering strategy for betalain biosynthesis.

Figure 1. Betalain biosynthetic pathway.

Thick arrows show enzymatic reactions, dotted arrows show spontaneous reactions. I, enzyme with tyrosine-hydroxylating activity (LeTYR in this study); II, L-DOPA oxidase; III, DOPA 4,5-dioxygenase (MjDOD in this study); IV, betanidin 5-O-glucosyltransferase; V, cyclo-DOPA 5-O-glucosyltransferase. Enzymes used for betaxanthin engineering in this study are shown in red font.

Figure 2. Analyses of transgenic tobacco BY2 calli.

(a) Images of typical transgenic BY2 calli. WT, wild-type; VC, vector control (pIG121-Hm); TYR, 35Sp-LeTYR line no. 3; DOD, 35Sp-MjDOD line no. 3; TYR/DOD, 35Sp-LeTYR/35Sp-MjDOD line no. 3. (b) Northern blot analysis. Total RNAs were isolated from wild-type (WT) and transgenic BY2 including vector control (VC), and three independent transgenic lines 35Sp-LeTYR (TYR), 35Sp-MjDOD (DOD), and 35Sp-LeTYR/35Sp-MjDOD (TYR/DOD). Total RNAs (5 μg) were separated on an agarose gel and then transferred to a nylon membrane and hybridized with LeTYR or MjDOD probes. Ethidium bromide staining of rRNA bands shows quality and loading controls. (c) Tyrosine-hydroxylating activity and accumulation of LeTYR proteins in transgenic BY2 calli. The activity was determined by CE-MS analysis as described in Materials and methods. Western blot analysis was performed using anti-LeTYR antibody.

Figure 3. Betaxanthin accumulation in 35Sp-LeTYR/35Sp-MjDOD transgenic BY2 cells.

(a) Typical phenotypes of vector control (VC) and 35Sp-LeTYR/35Sp-MjDOD line no. 3 (TYR/DOD no. 3) calli after 4- and 8-weeks subculture. (b) Change in betaxanthin concentration over time in transgenic calli line no. 3 after subculture. Betaxanthins were extracted in 80% (v/v) methanol containing 0.04% (v/v) HCl, and concentration was calculated from absorbance at 480 nm. (c) Identification of betaxanthins by LC-TOF-MS in 35Sp-LeTYR/35Sp-MjDOD transgenic BY2 cells. Chromatography profiles of extracts from vector control (dashed line) and 35Sp-LeTYR/35Sp-MjDOD line no.3 (thick line). Peak 1, retention time 23.8 min, m/z = 340.114, λmax = 470 nm, Glutamine-Bx (Vulgaxanthin I); Peak 2, 27.4 min, m/z = 313.104, λmax = 470 nm, Threonine-Bx; Peak 3, 41.3 min, m/z = 309.1084, λmax = 480 nm, Proline-Bx (Indicaxanthin).

Figure 4. Analysis of 35Sp-LeTYR/35Sp-MjDOD transgenic Arabidopsis T87 cells.

(a) Analyses of LeTYR and MjDOD transcripts and LeTYR protein accumulation. VC, vector control pIG121Hm; TD1 and TD2, independent 35Sp-LeTYR/35Sp-MjDOD transgenic T87 cell lines. (b) Images of typical transgenic T87 calli. (c) Aqueous extracts from transgenic T87 calli.