FIGURE 3.

Silencing of CqDODA1 and CqCYP76AD1 inhibits betalain production in quinoa. (A) RT-qPCR analysis of CqDODA1 and CqCYP76AD1 transcripts in the uninoculated upper leaves of the inbred lines J056 and J131 inoculated with the indicated inocula. Data were normalized to quinoa polyubiquitin 10 (CqUBQ10) expression and are shown as means ± SD (n = 4). Different letters indicate significant differences by a Tukey’s HSD test (p < 0.05). (B) Representative images of quinoa plants at 28 dpi with mock buffer, ALSV-WT, ALSV-CqDODA1, and ALSV-CqCYP76AD1. Scale bars represent 2 cm. (C) Representative images of the uninoculated upper leaves of quinoa plants at 20 dpi with the indicated inocula. Scale bars represent 2 cm. (D) Representative images of stem cross-sections of plants at 28 dpi with the indicated inocula. Scale bars represent 1 mm. (E) Relative quantification of betacyanin content was assessed spectrophotometrically by light absorption measurements at 536 nm. Relative values are presented as means ± SD (n = 5). Betacyanin pigments were extracted from the uninoculated upper leaves of quinoa plants (inbred J056 and J131 lines) at 16 dpi inoculated with the indicated inocula. Representative images of aqueous extracts used for the measurements are shown. (F) High performance liquid chromatography analysis of betacyanin pigments in the aqueous extracts used in (E). The peak names of quinoa betacyanins composed of amaranthin, celosianin II, and a non-identified (N.I.) compound were identified based on the analysis of red-violet pigment extracted from hypocotyls of 14-day-old Kd plants (Imamura et al., 2019). No betacyanin peaks were detected in the uninoculated upper leaves of J082 inbred line plants inoculated with mock. The horizontal and vertical axes indicate the retention time (min) and signal intensity (mV), respectively.