Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase, isolated from Solanum lycopersicum

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.

Completion of Tricin Biosynthesis Pathway in Rice: Cytochrome P450 75B4 Is a Unique Chrysoeriol 5′-Hydroxylase

“…Recombinant CYP75A F3959H enzymes isolated from petunia (Petunia hybrida), Catharanthus roseus (Kaltenbach et al, 1999), and tomato (Solanum lycopersicum; Olsen et al, 2010) showed broad substrate specificity in vitro, producing 39,59-hydroxylated flavonoids, including flavones.…”

Quantitative trait loci affecting intensity of violet flower colour in potato

“…Furthermore, the red-skinned tubers of cultivar Desiree have been changed to violet after transforming the cultivar with the ''violet'' allele of the F3 0 ,5 0 H, thus confirming that locus P encodes this enzyme (Jung et al 2005). In tomato a gene encoding flavonoid 3 0 ,5 0 -hydroxylase was shown to accept flavones, flavanones, dihydroflavonols and flavonols as substrates as well as to respond transcriptionally to nitrogen deprivation (Olsen et al 2010).…”

ROS as Signaling Molecules and Enzymes of Plant Response to Unfavorable Environmental Conditions

“…Flavonoids play an important role in plant fertility and germination of pollen. They are involved in plant signaling with interaction with plant microbes (Olsen et al, 2010, Gill & Tuteja 2010. It has been proved that they are involved in plant responses to both, biotic or abiotic stresses such as wounding, drought and metal toxicity (Cle et al, 2008).…”

“…There are four flavonoid classes depending on their structure: flavonols, flavones, isoflavones and anthocyanines. Flavonoides belong to one of the most reactive secondary metabolites of plants (Olsen et al, 2010). Flavonoids play important role as ROS scavenger by locating and neutralizing radicals before they damage cell structure.…”