Characterization, high-resolution mapping and differential expression of three homologous PAL genes in Coffea canephora Pierre (Rubiaceae)

Abstract

Phenylalanine ammonia lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway producing phenolics, widespread constituents of plant foods and beverages, including chlorogenic acids, polyphenols found at remarkably high levels in the coffee bean and long recognized as powerful antioxidants. To date, whereas PAL is generally encoded by a small gene family, only one gene has been characterized in Coffea canephora (CcPAL1), an economically important species of cultivated coffee. In this study, a molecular- and bioinformatic-based search for CcPAL1 paralogues resulted successfully in identifying two additional genes, CcPAL2 and CcPAL3, presenting similar genomic structures and encoding proteins with close sequences. Genetic mapping helped position each gene in three different coffee linkage groups, CcPAL2 in particular, located in a coffee genome linkage group (F) which is syntenic to a region of Tomato Chromosome 9 containing a PAL gene. These results, combined with a phylogenetic study, strongly suggest that CcPAL2 may be the ancestral gene of C. canephora. A quantitative gene expression analysis was also conducted in coffee tissues, showing that all genes are transcriptionally active, but they present distinct expression levels and patterns. We discovered that CcPAL2 transcripts appeared predominantly in flower, fruit pericarp and vegetative/lignifying tissues like roots and branches, whereas CcPAL1 and CcPAL3 were highly expressed in immature fruit. This is the first comprehensive study dedicated to PAL gene family characterization in coffee, allowing us to advance functional studies which are indispensable to learning to decipher what role this family plays in channeling the metabolism of coffee phenylpropanoids.

Fig. 1

Representation of the structure of the PAL gene family in Coffea canephora. Exons and untranslated regions (UTRs) are shown as boxes and introns are indicated as lines

Fig. 2

Phylogenetic relationships between the Coffea canephora PAL proteins and PAL proteins from spermatophytes (dicots, monocots and gymnosperms) and tracheophytes using Rhodotorula glutinis PAL protein sequence as an outgroup (see Supplementary Table S2)

Fig. 3

Relative expression of CcPAL1, CcPAL2, CcPAL3, three PAL family members and of HQT in different tissues and organs of a Coffea canephora variety (Robusta BP409). The gene expression relates to the constitutively expressed gene RPL39. Bean-SG small green-stage bean, Bean-LG large green-stage bean, Bean-Y yellow-stage bean, bean-R red-stage bean, Pericarp-SG small green-stage pericarp, Pericarp-LG large green-stage pericarp, Pericarp-Y yellow-stage pericarp, Pericarp-R red-stage pericarp

Fig. 4

Genetic mapping of CcPAL1, CcPAL2 and CcPAL3 on the coffee COSII “synteny” map. The letters A, B and F represent coffee linkage groups A, B and F. Shared regions of synteny between coffee and tomato are shown as colored blocks, and were deduced by COSII loci mapping in both species (Lefebvre-Pautigny et al. 2010). Each region sharing synteny with tomato is marked by a different color (modified from Wu et al. 2009). Shared regions of synteny with tomato chromosomes are indicated by: red for chromosome 11, blue for chromosome 9, black for chromosome 3, green for chromosome 2, pink for chromosome 1 and brown for chromosome 7