Genomic analyses of caffeine metabolism and perspectives for gene editing on Coffea canephora

Abstract

Humans cultivate arboreal plants for food production and industrial raw material since the early days of our society. This co-evolutionary process led to the relationship we have today with the tree crops, but the domestication course is hard to track for these species due to frequent hybridization events and the tendency of clonal propagation performed by farmers to preserve the selected phenotypes and, for modern agriculture using arboreal plants, the biggest challenge is breeding. The longer juvenile phase of these plants impairs crossing-related activities, which in turns is the basis for gene introgressions, development of segregating populations for uncovering genetic basis of traits, construction of genetic maps, among others. In face of the climate changes and resources scarcity forecast, it is important to improve the ways of tailoring tree crops and one alternative is using gene editing technology for rational molecular design. Among these arboreal plants, coffee tree is one of the most explored crops, as coffee is worldwide appreciated. Due to the upper mentioned forecast, a diploid species and generally more adapted to higher temperatures and drought like C. canephora could be the focus of coffee breeding programs to sustain coffee production chain. An interesting perspective to this end should consider, initially, using CRISPR-Cas-based gene editing to modify biochemical characteristics associated with coffee beverage quality and surpass self-incompatibility on C. canephora. Among the aspects which influences metabolite accumulation, is the membrane transporters activity and here we identified 1,847 potentially transporter-coding genes through a comprehensive genomic strategy and pointed some of them as possibly related to diterpenes, chlorogenic acids and alkaloid accumulation by gene co-expression analyses using all the public transcriptomic data for coffee. This inventory is the first of its kind constructed for coffee as well as for any tree species. One determinant substance for coffee consumption and for the plant development is caffeine but, despite its importance, little is known about the genetic bases of its accumulation on cells or the regulation of its synthesis. By using field grown C. canephora plants, we performed caffeine quantification on two leaf development stages as well as six points of fruit maturation. We identified putative membrane transporters and transcription factor genes co-expressed with caffeine synthesis-related ones using public RNA-seq data and analyzed its gene expression through RT-qPCR on these caffeine content-contrasting samples. Due to its potential involvement on caffeine transport and synthesis regulation, some identified genes should be further explored and a vector for CRISPR-mediated gene editing was already developed. The analyses performed here help on expanding the knowledge on genetics of caffeine accumulation in coffee and this might be part of the basis for varied rational molecular design strategies, which are essential for preparing agriculture for the challenges we expect to come.