Phenylalanine affects betalain biosynthesis and promotes ascorbic acid, α-tocopherol, and retinol accumulation in Amaranthus tricolor seedlings

Betalains are health-promoting plant pigments accumulated in non-anthocyanic plants. Owing to its limited distribution in nature, metabolic trade-offs of inhibiting them are poorly understood. The aim of this study was to investigate the effects of inhibition of betalains in Amaranthus tricolor seedlings to gain insights into the relationship of betalain biosynthesis with other biosynthetic pathways in betalain-accumulating plants. Phenylalanine (Phe; 12.5, 25, and 50 mmol L⁻¹) and 3-methyl-2-benzothiazolinone hydrazone (MBTH) (25 mmol L⁻¹) were treated to inhibit betalain biosynthesis in ten-day-old A. tricolor (red) seedlings. After two and six days of treatment, target (betalain-related metabolites) and non-target metabolites were analyzed. In two days, Phe content increased by 2.6, 8.5, and 17.4-fold in Phe (12.5, 25, and 50 mmol L⁻¹)-treated seedlings, respectively, compared to control, indicating the uptake of Phe by the seedlings. Phe treatment led to a 10.1–18% decrease in betacyanins, while MBTH caused an 18.3% decrease in two days. In both treatments, cyclo-DOPA formation, which is essential for betacyanin biosynthesis, seems to be inhibited, albeit through different mechanisms. Betalain biosynthetic precursors and intermediates, viz., tyrosine, L-DOPA, and dopamine decreased differentially. Ascorbic acid, α-tocopherol, and retinol contents increased in both treatments concomitant with the reduction in betalains, total phenols, and antioxidant enzymes. Therefore, Phe treatment is beneficial in enhancing antioxidant metabolites in betalain-accumulating plants. However, the mechanism of increasing ascorbic acid on inhibiting betalains needs further investigation in other betalain-producing plants also to understand if ascorbic acid is involved in regulating betalain biosynthesis.