The R2R3-MYB gene family in Cicer arietinum: genome-wide identification and expression analysis leads to functional characterization of proanthocyanidin biosynthesis regulators in the seed coat.

We identified 119 typical CaMYB encoding genes and reveal the major components of the proanthocyanidin regulatory network. CaPARs emerged as promising targets for genetic engineering toward improved agronomic traits in C. arietinum. Chickpea (Cicer arietinum) is among the eight oldest crops and has two main types, i.e., desi and kabuli, whose most obvious difference is the color of their seeds. We show that this color difference is due to differences in proanthocyanidin content of seed coats. Using a targeted approach, we performed in silico analysis, metabolite profiling, molecular, genetic, and biochemical studies to decipher the transcriptional regulatory network involved in proanthocyanidin biosynthesis in the seed coat of C. arietinum. Based on the annotated C. arietinum reference genome sequence, we identified 119 typical CaMYB encoding genes, grouped in 32 distinct clades. Two CaR2R3-MYB transcription factors, named CaPAR1 and CaPAR2, clustering with known proanthocyanidin regulators (PARs) were identified and further analyzed. The expression of CaPAR genes correlated well with the expression of the key structural proanthocyanidin biosynthesis genes CaANR and CaLAR and with proanthocyanidin levels. Protein-protein interaction studies suggest the in vivo interaction of CaPAR1 and CaPAR2 with the bHLH-type transcription factor CaTT8. Co-transfection analyses using Arabidopsis thaliana protoplasts showed that the CaPAR proteins form a MBW complex with CaTT8 and CaTTG1, able to activate the promoters of CaANR and CaLAR in planta. Finally, transgenic expression of CaPARs in the proanthocyanidin-deficient A. thaliana mutant tt2-1 leads to complementation of the transparent testa phenotype. Taken together, our results reveal main components of the proanthocyanidin regulatory network in C. arietinum and suggest that CaPARs are relevant targets of genetic engineering toward improved agronomic traits.