List of the accessions investigated in the study and their phylogenetic constitution.

<p>List of the accessions investigated in the study and their phylogenetic constitution.</p

Schematic representation of coumarin/furanocoumarin inheritance from the 4 ancestral taxa (pummelo, mandarin, citron, and papeda) in the cultivated <i>Citrus</i> species.

<p>Parental relations between species are illustrated by arrows. Thick arrows represent high chemotype similarities between hybrids and their ascendants. Same colors were used as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142757#pone.0142757.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142757#pone.0142757.g005" target="_blank">5</a> for representing <i>Citrus</i> species. Coumarins and furanocoumarins are indicated in the same color as the ancestral taxon that transmitted them to their descendants. The symbol “+” indicates the probable transmission of compounds in the secondary species, while the symbol “-”indicates their disappearance.</p

Heatmap displaying the coumarin and furanocoumarin profiles in the pulp of the 61 <i>Citrus</i> species investigated.

<p>A: heatmap; B: dendrogram of compounds; C: dendrogram of <i>Citrus</i> varieties. A red square highlights the presence of a given compound, while a yellow square shows its absence. Coumarins are represented in orange; furanocoumarins of the bergapten cluster, in blue; furanocoumarins of the xanthotoxin cluster, in red; and furanocoumarins of the isopimpinellin cluster, in green.</p

Coumarin and furanocoumarin quantities (mmol kg^-1 fresh weight) in the peel extracts of the 61 <i>Citrus</i> species investigated.

<p>Coumarins are represented in orange; furanocoumarins of the bergapten cluster, in blue; furanocoumarins of the xanthotoxin cluster, in red; and furanocoumarins of the isopimpinellin cluster, in green. Ancestral taxa and secondary species are highlighted in the bottom of the graph. Sweet mandarins are presented in bright red, and acidic mandarins are illustrated in deep red.</p

NJ analysis of the 23 citrus varieties belonging to the 4 ancestral taxa based on the coumarins and furanocoumarins contents in peel expressed as mg.kg^-1 fresh weight.

<p>Numbers in black represent the bootstrap probability values. The colors correspond to the phylogenetic constitution of the varieties and are indicated in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142757#pone.0142757.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142757#pone.0142757.g003" target="_blank">3</a>.</p

Heatmap displaying the coumarin and furanocoumarin profiles in the peel of the 61 <i>Citrus</i> species investigated.

<p>A: heatmap; B: dendrogram of compounds; C: dendrogram of <i>Citrus</i> varieties. A red square highlights the presence of a given compound, while a yellow square shows its absence. Coumarins are represented in orange; furanocoumarins of the bergapten cluster, in blue; furanocoumarins of the xanthotoxin cluster, in red; and furanocoumarins of the isopimpinellin cluster, in green.</p

Schematic representation of the coumarin and furanocoumarin pathways.

<p>Solid lines indicate direct enzymatic steps; dashed lines represent multiple enzymatic steps. Coumarins are framed in orange, furanocoumarins of the bergapten cluster are framed in blue, furanocoumarins of the xanthotoxin cluster are framed in red, and furanocoumarins of the isopimpinellin cluster are framed in green.</p

Coumarin and furanocoumarin quantities (mmol kg^-1 fresh weight) in the pulp extracts of the 61 <i>Citrus</i> species investigated.

<p>Coumarins are represented in orange; furanocoumarins of the bergapten cluster, in blue; furanocoumarins of the xanthotoxin cluster, in red; and furanocoumarins of the isopimpinellin cluster, in green. Ancestral taxa and secondary species are highlighted in the bottom of the graph. Sweet mandarins are presented in bright red, and acidic mandarins are illustrated in deep red.</p

Presentation_1_The CYP71AZ P450 Subfamily: A Driving Factor for the Diversification of Coumarin Biosynthesis in Apiaceous Plants.pptx

<p>The production of coumarins and furanocoumarins (FCs) in higher plants is widely considered a model illustration of the adaptation of plants to their environment. In this report, we show that the multiplication of cytochrome P450 variants within the CYP71AZ subfamily has contributed to the diversification of these molecules. Multiple copies of genes encoding this enzyme family are found in Apiaceae, and their phylogenetic analysis suggests that they have different functions within these plants. CYP71AZ1 from Ammi majus and CYP71AZ3, 4, and 6 from Pastinaca sativa were functionally characterized. While CYP71AZ3 merely hydroxylated esculetin, the other enzymes accepted both simple coumarins and FCs. Superimposing in silico models of these enzymes led to the identification of different conformations of three regions in the enzyme active site. These sequences were subsequently utilized to mutate CYP71AZ4 to resemble CYP71AZ3. The swapping of these regions lead to significantly modified substrate specificity. Simultaneous mutations of all three regions shifted the specificity of CYP71AZ4 to that of CYP71AZ3, exclusively accepting esculetin. This approach may explain the evolution of this cytochrome P450 family regarding the appearance of FCs in parsnip and possibly in the Apiaceae.</p