Identification Of MADS-Box Genes Involved In Regulation Of Caffeine And Chlorogenic Acid Accumulation In Coffee

Coffee is the world’s most widely consumed beverage and the second most internationally traded commodity after oil.The quality of coffee,with its complex variables,is significantly dependent on the fluctuating biochemical compositions of the coffee bean during its development.In this study we identified and analyzed the expression of coffee MIKCc-type genes from the MADS-box gene family.Identification revealed a total of 29 MIKCc-type MADS-box genes from C.canephora based on phylogeny.Based on transcriptome analysis as well as previously reported studies that support the role of MADS-box genes in regulating secondary metabolites,three MIKCc-type MADS-box genes,Cc AG2,Cc FUL and Cc AGL8,were further selected to conduct functional analysis.All three genes were successfully transiently overexpressed in coffee leaves and seeds,and caffeine and chlorogenic acid contents were determined using spectrometry in order to assess the role of these genes in the regulation of these secondary metabolites.Virus Induced Gene Silencing(VIGS)technique was also employed in coffee leaves.Overexpression of Cc AG2 in leaves and seeds resulted in a decrease in both caffeine and chlorogenic acid(CGA)accumulation,while an opposite trend was observed for both Cc FUL and Cc AGL8.Silencing Cc AG2 in leaves increased caffeine and CGA accumulation,while Cc FUL and Cc AGL8-silenced leaves decreased caffeine and CGA accumulation.Gene expression analysis by q RT-PCR in leaf tissues containing low levels of caffeine revealed that low-caffeine tissues had a lower expression of the major caffeine biosynthetic pathway genes,while expression analysis of the same genes in Cc AG2-silenced tissues resulted in high expression levels of the caffeine biosynthetic pathway genes.Gene expression analysis in the seeds revealed that tissues with high CGA content had a high expression of HQT the gene encoding the enzyme that is closely related to chlorogenic acid biosynthesis in coffee.These results suggest the roles of coffee MADS-Box genes,Cc AG2,Cc FUL and Cc AGL8,in controlling caffeine and chlorogenic acid accumulation.Our study will be helpful for genetic improvement of fruit quality in coffee breeding programs in the future.