Promoter and coding sequence diversity of CsCCD1 may contribute to the differential accumulation of floral b-ionone in fresh tea leaves

The carotenoid-derived volatile d-ionone makes an important contribution to tea fragrance. Here, we qualitatively and quantitatively analysed 15 carotenoids in tea leaves of 13 cultivars by UHPLC-APCI-MS/MS. The 13 cultivars were divided into two groups by PCA (Principal Component Analysis) clustering analysis of their carotenoid content, and OPLS-DA (Orthogonal projections to latent structures) indicated that the levels of d-carotene (VIP = 2.89) and lutein (VIP = 2.30) were responsible for much of the variation between the two groups. Interestingly, the d-carotene to d-ionone conversion rates in Group 1 were higher than in Group 2, while the d-carotene content was significantly lower in Group 1 than in Group 2. The d-ionone content was significantly higher in Group 1. Pearson Correlation Coefficient calculation between the tran-scription level of candidate genes (CsCCD1 and CsCCD4) and the accumulation of d-ionone indicated that CsCCD1 may involve in the formation of d-ionone in 13 cultivars. Prokaryotic expression and in vitro enzyme activity assays showed that 'Chuanhuang 1' had an amino acid mutation in carotenoid cleavage dioxygenases 1 (CsCCD1) compared with 'Shuchazao', resulting in a significantly higher d-ionone content in 'Chuan-huang 1'. Sequence analysis showed that 'Chuanhuang 1' and 'Huangdan' had different CsCCD1 promoter sequences, leading to significantly higher CsCCD1 expression and d-ionone accumulation in 'Chuanhuang 1'. These results indicated that the promoter and coding sequence diversity of CsCCD1 might contribute to the differential accumulation of d-ionone in different tea cultivars.