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

<p>Abstract</p> <p>Background</p> <p>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 (<it>Petunia hybrida</it>), and potato (<it>Solanum tuberosum</it>). The present work involves the identification and characterisation of a <it>F3'5'H </it>gene from tomato (<it>Solanum lycopersicum</it>), and the examination of its putative role in flavonoid metabolism.</p> <p>Results</p> <p>The cloned and sequenced tomato <it>F3'5'H </it>gene was named <it>CYP75A31</it>. The gene was inserted into the <it>pYeDP60 </it>expression vector and the corresponding protein produced in yeast for functional characterisation. Several putative substrates for F3'5'H were tested <it>in vitro </it>using enzyme assays on microsome preparations. The results showed that two hydroxylation steps occurred. Expression of the <it>CYP75A31 </it>gene was also tested <it>in vivo</it>, 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 <it>CYP75A31 </it>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.</p> <p>Conclusions</p> <p><it>CYP75A31 </it>(NCBI accession number GQ904194), encodes a flavonoid 3'5'-hydroxylase, which accepts flavones, flavanones, dihydroflavonols and flavonols as substrates. The expression of the <it>CYP75A31 </it>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.</p

Spatial and Temporal Variation in Selection of Genes Associated with Pearl Millet Varietal Quantitative Traits In situ

Ongoing global climate changes imply new challenges for agriculture. Whether plants and crops can adapt to such rapid changes is still a widely debated question. We previously showed adaptation in the form of earlier flowering in pearl millet at the scale of a whole country over three decades. However, this analysis did not deal with variability of year to year selection. To understand and possibly manage plant and crop adaptation, we need more knowledge of how selection acts in situ. Is selection gradual, abrupt, and does it vary in space and over time? In the present study, we tracked the evolution of allele frequency in two genes associated with pearl millet phenotypic variation in situ. We sampled 17 populations of cultivated pearl millet over a period of 2 years. We tracked changes in allele frequencies in these populations by genotyping more than seven thousand individuals. We demonstrate that several allele frequencies changes are compatible with selection, by correcting allele frequency changes associated with genetic drift. We found marked variation in allele frequencies from year to year, suggesting a variable selection effect in space and over time. We estimated the strength of selection associated with variations in allele frequency. Our results suggest that the polymorphism maintained at the genes we studied is partially explained by the spatial and temporal variability of selection. In response to environmental changes, traditional pearl millet varieties could rapidly adapt thanks to this available functional variability