Abstract
BackgroundUnderstanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism.ResultsThe cloned and sequenced tomato F3'5'H gene was named CYP75A31. The gene was inserted into the pYeDP60 expression vector and the corresponding protein produced in yeast for functional characterisation. Several putative substrates for F3'5'H were tested in vitro using enzyme assays on microsome preparations. The results showed that two hydroxylation steps occurred. Expression of the CYP75A31 gene was also tested in vivo, in various parts of the vegetative tomato plant, along with other key genes of the flavonoid pathway using real-time PCR. A clear response to nitrogen depletion was shown for CYP75A31 and all other genes tested. The content of rutin and kaempferol-3-rutinoside was found to increase as a response to nitrogen depletion in most parts of the plant, however the growth conditions used in this study did not lead to accumulation of anthocyanins.ConclusionsCYP75A31 (NCBI accession number GQ904194), encodes a flavonoid 3'5'-hydroxylase, which accepts flavones, flavanones, dihydroflavonols and flavonols as substrates. The expression of the CYP75A31 gene was found to increase in response to nitrogen deprivation, in accordance with other genes in the phenylpropanoid pathway, as expected for a gene involved in flavonoid metabolism.
Highlights
Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens
Information on substrate flow and regulation through the branch point between flavonol and anthocyanin synthesis is not fully elucidated, and for tomato the enzymes acting in the branch point have not been extensively characterised
Kaltenbach et al [15] isolated the flavonoid 3’5’-hydroxylase (F3’5’H) gene from C. roseus using heterologous screening with the CYP75 Hf1 cDNA from P. hybrida [16]. Both the C. roseus gene, named CYP75A8, and the petunia Hf1 were expressed in E. coli and found to accept flavones, flavanones, dihydroflavonols and flavonols as substrates, and both performed 3’- and 3’5’-hydroxylation
Summary
Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3’5’-hydroxylase (F3’5’H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). Experiments with expression of the snapdragon transcription factor genes Delila, a basic-helix-loop-helix (bHLH) transcription factor, and Rosea, a R2R3 MYBtype transcription factor, showed that F3’5’H expression is necessary for activation of anthocyanin synthesis in tomatoes [10]. Introduction of these transcription factors under control of the fruit-specific E8 promoter increased the expression of most of the structural genes in the biosynthetic pathway in the tomato fruit, including phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI) and F3’5’H. The genes encoding F3’5’H in grape have been shown to be expressed in different parts of the grape plant that accumulate flavonoids, especially in the skin of ripening berries where the highest levels of anthocyanins are synthesized [17]
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