The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways

Abstract

Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the "grapefruit juice effect". Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in creating species devoid of these toxic compounds in future breeding programs.

Fig 1. Schematic representation of the coumarin and furanocoumarin pathways.

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.

Fig 2. Coumarin and furanocoumarin quantities (mmol kg^-1 fresh weight) in the peel extracts of the 61 Citrus species investigated.

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.

Fig 3. Coumarin and furanocoumarin quantities (mmol kg^-1 fresh weight) in the pulp extracts of the 61 Citrus species investigated.

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.

Fig 4. Heatmap displaying the coumarin and furanocoumarin profiles in the peel of the 61 Citrus species investigated.

A: heatmap; B: dendrogram of compounds; C: dendrogram of Citrus 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.

Fig 5. Heatmap displaying the coumarin and furanocoumarin profiles in the pulp of the 61 Citrus species investigated.

A: heatmap; B: dendrogram of compounds; C: dendrogram of Citrus 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.

Fig 6. 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.

Numbers in black represent the bootstrap probability values. The colors correspond to the phylogenetic constitution of the varieties and are indicated in Figs 2 and 3.

Fig 7. PCA representation of the citrus varieties belonging to the 3 ancestral taxa pummelos, mandarins and citrons and their respective hybrids based on the coumarins and furanocoumarins contents in peel expressed as mg.kg^-1 fresh weight.

A: Distribution of the observations (citrus varieties); B: Distribution of the variables (coumarins and furanocoumarins). Ancestral taxa were used as active individuals (circles) to build the PCA, while secondary species are supplementary individuals (squares). Quantitative data were transformed as log₁₀(1 + x). The colors correspond to the phylogenetic constitution of the varieties and are indicated in Figs 2 and 3.

Fig 8. Schematic representation of coumarin/furanocoumarin inheritance from the 4 ancestral taxa (pummelo, mandarin, citron, and papeda) in the cultivated Citrus species.

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 4 and 5 for representing Citrus 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.