Identification of CsAK as a critical caffeine-related upstream gene in tea accessions through genome-wide association study

Caffeine (CAF), a primary flavor component in tea, is one of the main reasons for the popularity of tea beverages. As an important secondary metabolite in tea plants, the CAF content varied greatly among different tea accessions. However, the genetic mechanisms underlying the CAF biosynthesis were still unclear. In this study, we performed a genome-wide association study (GWAS) on 359 tea accessions in the Guizhou Plateau to identify genetic variation associated with CAF content. A total of 19 significant single nucleotide polymorphisms (SNPs) and key gene (CsAK) involved in CAF biosynthesis were identified. Subcellular localization revealed that the CsAK-GFP fusion protein was located on the cell membrane. Antisense oligodeoxynucleotide (AsODN) targeting the CsAK gene to the buds and leaves revealed that the expression levels of the CsAK gene were significantly reduced, and the corresponding CAF content was also decreased in AsODN-treated tea plants. Overexpression of the CsAK gene in eukaryotic cells resulted in the accumulation of key intermediate product (L-methionine) during CAF biosynthesis process. These findings offered a theoretical foundation for future tea breeding programs aimed at cultivating excellent germplasm with high or low levels of CAF.