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


RESULTS
The 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.


CONCLUSIONS
CYP75A31 (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.
University of Stavanger, Centre for Organelle Research, Faculty of Science and Technology, N-4036 Stavanger, Norway. kristine.m.olsen@uis.no
Kristine M
KM
Olsen
Alain
A
Hehn
Hélène
H
Jugdé
Rune
R
Slimestad
Romain
R
Larbat
Frédéric
F
Bourgaud
Cathrine
C
Lillo
D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018744:DNA, Plant / Q000235:genetics; D005419:Flavonoids / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D018744:DNA, Plant; D005419:Flavonoids; D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; C084258:flavonoid 3',5'-hydroxylase; D009584:Nitrogen