Ectopic expression of snapdragon transcription factors facilitates the identification of genes encoding enzymes of anthocyanin decoration in tomato

Abstract

SUMMARYGiven the potential health benefits of polyphenolic compounds in the diet, there is a growing interest in the generation of food crops enriched with health-protective flavonoids. We undertook a series of metabolite analyses of tomatoes ectopically expressing the Delila and Rosea1 transcription factor genes from snapdragon (Antirrhinum majus), paying particular attention to changes in phenylpropanoids compared to controls. These analyses revealed multiple changes, including depletion of rutin and naringenin chalcone, and enhanced levels of anthocyanins and phenylacylated flavonol derivatives. We isolated and characterized the chemical structures of the two most abundant anthocyanins, which were shown by NMR spectroscopy to be delphinidin-3-(4 000 -O-trans-p-coumaroyl)-rutinoside-5-O-glucoside and petunidin-3-(4 000 -O-trans-p-coumaroyl)-rutinoside-5-O-glucoside. By performing RNA sequencing on both purple fruit and wild-type fruit, we obtained important information concerning the relative expression of both structural and transcription factor genes. Integrative analysis of the transcript and metabolite datasets provided compelling evidence of the nature of all anthocyanin biosynthetic genes, including those encoding species-specific anthocyanin decoration enzymes. One gene, SlFdAT1 (Solyc12g088170), predicted to encode a flavonoid-3-O-rutinoside-4 000 -phenylacyltransferase, was characterized by assays of recombinant protein and overexpression assays in tobacco. The combined data are discussed in the context of both our current understanding of phenylpropanoid metabolism in Solanaceous species, and evolution of flavonoid decorating enzymes and their transcriptional networks in various plant species.

Transcription Factor-Mediated Control of Anthocyanin Biosynthesis in Vegetative Tissues

“…MS analysis allowed the identification of seven anthocyanins ( Fig. 2C; Supplemental Table S1), all of which had been previously identified in tomato hypocotyls and ROS1/DEL fruit Tohge et al, 2015). Both tissues contained the same anthocyanins, but at different ratios and with different kinetics.…”

“…polyamine and nor-nicotine conjugates) was produced , whereas in tomato fruit, ROS1/DEL expression led to production of a set of complex anthocyanins and flavonoids (Butelli et al, 2008;Tohge et al, 2015). This suggests that the identity of ROS1/ DEL-regulated metabolites depends on the species or tissue, but at present there are no data available of the consequence of ROS1/DEL expression in tomato Figure 1.…”

“…In view of the observation that no anthocyanins could yet be detected at these stages, this would indicate that these genes are directly regulated by ROS1/DEL, and not by the presence of anthocyanins. Genes involved in converting Phe to polyphenols including anthocyanins mostly overlapped with those identified to be up-regulated in ROS1/DEL fruits, encoding enzymes and transporters from the pathway leading to anthocyanins, flavonoids, and chlorogenic acid (Butelli et al, 2008;Tohge et al, 2015). Interestingly, several genes from the Phe biosynthetic pathways were also activated after 3 h by ROS1/DEL, including shikimate kinase (SK1), a key enzyme of the shikimate pathway toward Phe, and cytosolic pyruvate kinase and acetyl coA carboxylase, involved in malonyl CoA biosynthesis (Table I and Fig.…”

“…Such studies have been done both on tomato seedlings in which anthocyanin formation is induced by nutrient stress and on purple ROS1/DEL fruits (Tohge et al, 2015). In these studies, networks of genes and metabolites were analyzed by combining data from different tissues and cell types.…”

“…These data provide insight into the processes that may accompany anthocyanin biosynthesis, including physiological and architectural changes in tomato vegetative tissues. be achieved by ectopically expressing two transcription factors (ROS1/DEL) from snapdragon (Antirrhinum; Butelli et al, 2008;Tohge et al, 2015). These studies use the fruit-specific promoter E8, which is regulated by ethylene and thereby is specifically activated during the breaker stage of fruit ripening.…”