Crocetin dialdehyde

Standards of lycopene (CAS, 502-65-8), β-carotene (CAS, 7235-40-7), zeaxanthin (144-68-3), and crocetin dialdehyde (CAS, 502-70-5) were purchased from Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, MO, USA). Phanta Max Super-Fidelity DNA Polymerase was purchased from Vazyme (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China). A ClonExpress II One Step Cloning Kit C112 was purchased from Vazyme. IPTG was purchased from Solarbio (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). All restriction endonucleases were purchased from Takara (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). T4 DNA Ligase was purchased from Takara. A DNA plasmid isolation kit and DNA gel extraction kit were purchased from Tiangen (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China). Tryptone and yeast extract were purchased from Thermo (Thermo Fisher Scientific Inc., Waltham, MA, USA). E. coli DH5α and E. coli BL21 (DE3) were purchased from Tiangen. Z/B/L represent the BL21 (DE3) harboring pACCAR25ΔcrtX, pACCAR16Δcrt, or pACCRT-EIB, respectively. All the chemical reagents utilized in this experiment were of analytical grade.

The following plant metabolites used in our study were ordered: (−)-caryophyllene oxide, (+)-nootkatone, zerumbone, β-amyrin, crocetin dialdehyde, saffron stigma (Sigma-Aldrich, St. Louis, MO, USA), α amyrin (Carl Roth, Karlsruhe, Germany) and taraxasterol acetate (Biorbyt, Cambridge, UK) (Figure 1). All compounds were dissolved in dimethyl sulfoxide (DMSO) in order to be used in the assays. Saffron threads were incubated in DMSO for 1 h on a rotary shaker (100 rpm); then, the supernatant was collected and used in the work. A C₇-C₃₀ saturated alkanes standard solution was also obtained from Sigma-Aldrich to calculate the GC retention indices of the sesquiterpenes formed by A. thaliana. The liquid standards used were β-elemene, ordered from Abcam plc (Cambridge, UK) and (−)-trans-caryophyllene (sum of enantiomers, purity: ≥98.0%) and (−)-α-cedrene (sum of enantiomers, purity: 96.4%) purchased from Sigma-Aldrich.