Betalains are tyrosine-derived plant pigments present in several species of the Caryophyllales order. Betalains are classified in red betacyanins and yellow betaxanthins and are implicated in plant stress tolerance and visual attraction for pollinators. The compounds are used as natural colorants in many industries. Today, there is little information on betalain biosynthesis with several key enzymes that remain unknown on plants of the Caryophyllales order. Omic tools have proven to be very useful in gaining insights into various molecular mechanisms. In this study, we used suspension cells from fruits of the cactus Stenocereus queretaroensis. Two growing conditions were used to perform RNA-seq and differential expression analysis to help identify betalain biosynthesis-related genes. We found 98 differential expressed genes related to aromatic amino acids and betalain biosynthesis pathways. Interestingly, we found that only one gene of the betalain synthesis pathway was differentially expressed. The rest of the genes belong to the aromatic amino acid pathway, including hydroxy phenylpyruvate-related genes, suggesting the possibility of an alternative biosynthetic pathway similar to that observed in legumes.

Stenocereus queretaroensis (F.A.C.Weber ex Mathes.) Buxb is a cactus that has long been used as a source food in central and northern México. Its fruits, commonly called pitayas, biosynthesize high amounts of betalains. These molecules are water-soluble nitrogenous compounds; that compared to other pigments, such as anthocyanins or carotenoids, stand out for their physicochemical stability in industrial processes. Due to genetic and environmental factors involved in the biosynthesis and accumulation of secondary metabolites in plants, we tested different stress-inducing agents (elicitor, osmotic, salt, and temperature) to induce betalains accumulation in cell culture from fruits of Stenocereus queretaroensis. This work aimed to understand stress conditions that induce the metabolic pathways required for the accumulation of betalains. The results show how betacyanin concentration increases under high sugar conditions, thus affecting the expression of L-DOPA 4, 5 dioxygenase resulting in a strong dark red coloration. This suggests this enzyme is part of a rate-limiting step in betalain production. In addition, we found that betalains accumulation occurs under particular stress conditions. Cells that have a high level of betacyanins show better resistance to stress in the cell culture, as well as an overall different behavior including cell aggregation and alterations in nuclear size. Together the results shown here may provide new strategies to manipulate and mass produce the pigments from Stenocereus queretaroensis in cell culture.