The reduction of vanillin in the biosynthetic pathway of capsiate, a non-pungent component of Capsicum fruits, is catalyzed by cinnamyl alcohol dehydrogenase

Plant materials

Annual capsicum CH-19 Sweet seeds were provided by Ajinomoto Co., Inc. (Tokyo, Japan). The CH-19 Sweet, red habanero (chinese pepper), and Himo (C. annual) were grown at the Josai University Experimental Farm (Saitama, Japan), while Yume-matsuri (C. annual), Yellow Moruga (C. chinense) and the Aji Dulce 2 strain (C. chinense) plants were grown at the Experimental Farm of Kyoto University (Kyoto, Japan).

Real-time quantitative PCR analyzes

Total RNA was extracted from placenta, pericarp and seeds of green and red fruits of CH-19 Sweet, habanero red, Himo, Yume-matsuri, Aji Dulce strain 2 and Moruga Yellow using the Fast Gene Kit RNA Premium (Nippon Genetics Co., Ltd., Tokyo, Japan) according to manufacturer’s instructions. The extracted total RNA (0.5 μg) served as template for cDNA synthesis using PrimeScript RT Master Mix (Takara Bio Inc., Otsu, Japan). The CAD, pAMTand Word game1 transcript levels were determined by qPCR with the LightCycler 96 instrument (Roche Diagnostics, Penzberg, Germany). β-actin (AY572427), CaUBIQUITIN (DQ975458.1) and elongation factor 1-alpha genes (AY496125) were used as internal reference control. The specificity of each primer set (Table S3) was confirmed by melting curve analysis. Each experiment was performed using RNA extracted from three different individual fruits (not = 3). The relative expression level of each gene was calculated using the comparative Ct method, with the expression level of each gene in CH-19 Sweet set at 1.

Preparation of placental extracts

Latin fruits at different stages of development (immature green fruits and mature red fruits) were dissected to isolate the placenta. Then, 100 mg of placenta were homogenized in 1 ml of 20 mM potassium phosphate buffer (PKB), pH 6.8. After centrifugation at 18,000 g at 4°C for 5 min, the supernatant was collected and filtered using a syringe filter (pore size 0.45 µm). The filtrate was dialyzed against 20 mM phosphate-buffered saline (PBS), pH 7.2, at 4°C for 2 h, then used as placental extract for enzyme analyses.

Cloning of the BODY CH-19 sweet and red habanero cDNA

Total RNA was extracted as described above, then cDNA was synthesized from 0.5 μg total RNA using PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio Inc. ). Each full-length cDNA fragment was amplified using the primers listed in Table S4. The PCR fragments were inserted into the T vector pMD20 (Takara Bio Inc.) and then sequenced.

Generation of recombinant CaCAD1

The complete cDNA of CaCAD1 with a His tag was transferred into the expression vector pET3c (Promega, Madison, WI, USA). Escherichia coli strain BL21 Star (DE3) (Thermo Fisher Scientific, Waltham, MA, USA) were transformed with the CaCAD1/pET3c recombinant plasmid. BL21 Star (DE3) cells harboring CaCAD1/pET3cwere then grown in 20 mL of LB culture medium containing 50 µg/mL carbenicillin at 37°C in a shaking incubator for 4 h. The culture was added to 250 ml of pre-warmed LB culture medium containing carbenicillin and then incubated at 37°C with shaking. When the absorbance at 600 nm reached 0.6, isopropyl β-D-thiogalactoside was added for a final concentration of 1 mM. After a 4 h incubation with shaking, the cells were harvested by centrifugation at 3200 g for 10 min. The cell pellet was resuspended in 10 ml of 50 mM Tris-HCl buffer (pH 8.0). Lysozyme was added for a final concentration of 0.2 mg/mL and the mixture was incubated at 37°C for 20 min then sonicated. The supernatant was collected after centrifugation (10,000 g for 10 min). The precipitate was resuspended in 10 mL of Triton X-100/50 mM 5% Tris-HCl buffer (pH 8.0), then the mixture was sonicated and incubated at 37°C for 30 min. The supernatant was collected after centrifugation (10,000 g for 10 min) and combined with the previous one for subsequent Ni–NTA Superflow column chromatography (Qiagen, Venlo, The Netherlands). Bound protein was eluted using 0.3 M imidazole/0.15 M NaCl/50 mM Tris–HCl buffer (pH 8.0).

Assay of vanillin-reducing activity of rCaCAD1 and placental extracts by HPLC

To assay vanillin-reducing activity, 5 μg rCaCAD1 in 100 μL reaction mix and 35 μL CH-19 sweet green fruit placental extract in 50 μL reaction mix were incubated with 4 mM of vanillin and 1 mM NADPH (final concentrations) in 20 mM PKB (pH 6.8) at 30°C. At each sampling time point (1–24 h), a 10 µL aliquot of the reaction mixture was removed and added to 30 µL of 0.2% trifluoroacetic acid (TFA) to terminate the reaction. Vanillyl alcohol and residual vanillin contents were measured using an HPLC system (Shimadzu Prominence LC-20A series; Shimadzu Co., Kyoto, Japan). A 5 µL aliquot of the completed reaction mixture was loaded onto a Wakopak Fluofix-II 120E (4.6 × 250 mm) column (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) equilibrated with 0.05% TFA at a flow rate of 1 mL/min. Elution involved a linear gradient of 5-10% methanol for the first 10 minutes, then a linear gradient of 10-60% methanol for the next 10 minutes. For fluorescence detection, the excitation and emission wavelengths were 280 nm and 320 nm, respectively. The amount of vanillyl alcohol synthesized was estimated based on the area of ​​the peak. Analysis of rCaCAD1 was also performed using NADPH-free reaction solution. The experiment using placental extracts was completed using samples from three different individuals.

Reactions between rCaCAD1 and coniferyl aldehyde, cinnamaldehyde or vanillin

Recombinant CaCAD1 (5 μg) and excess substrates (10 mM cinnamaldehyde, 1 mM coniferyl aldehyde or 4 mM vanillin) were added to a 100 μL reaction mixture comprising 20 mM PKB (pH 6.8) and 1 mM NADPH. After a 30 minute incubation at 30°C, a 10 µL aliquot of the reaction mixture was collected and added to 30 µL of 0.2% TFA to terminate the reaction. The products were analyzed by HPLC under the same conditions as those used for the analysis of vanillylic alcohol.

Heat inactivation of rCAD1 and placental extracts

Two sets of 5 μg of rCaCAD1 and 35 μL of CH-19 sweet green fruit placental extract in 20 mM PKB (pH 6.8) were prepared. One set was incubated at 100°C and the other was incubated at 30°C for 10 min. After cooling the reaction mixtures to room temperature, 4 mM vanillin and 1 mM NADPH (final concentrations) were added. After incubation for 30 minutes at 30°C, a 10 µL aliquot was removed and added to 30 µL of 0.2% TFA to terminate the reaction. The products were analyzed by HPLC. Analysis of placental extract was performed using samples from three different individual fruits.

Summary of OHPAS

As previously described, OHPAS was synthesized via the condensation of 2-tert-butoxy-2-oxoethylzinc bromide with 2-hydroxy-NOT -sulfinylaniline31. The structure of the product is characterized by 1H NMR and fast atom bombardment mass spectroscopy.

Inhibition of rCaCAD1 and placental extracts by OHPAS

Each reaction mix (100 µL) contained 10 µg rCaCAD1 or 20 µL CH-19 Sweet green or red fruit placental extract, 4 mM vanillin, 1 mM NADPH and 2, 2.5, 5, 10 or 20 mM (concentrations final) OHPAS in 20 mM PKB (pH 6.8). Reactions were incubated at 30°C for 30 min or 1 h. At each sampling instant, an aliquot of 10 μL of each mixture was added to 30 μL of 0.2% TFA to complete the reaction. The amount of vanillyl alcohol synthesized was analyzed by HPLC as described above. The rate of enzyme activity inhibition was estimated as a function of the peak area. Analysis of placental extract was performed using samples from three different individual fruits.

Inhibition of rCaCAD1 and placental extracts by EDTA

Each reaction mix (100 µL) contained 5 µg rCaCAD1 or 40 µL CH-19 sweet or red green habanero fruit placenta extract, 4 mM vanillin, and 12.5, 25, 50, or 100 mM (final concentrations) d EDTA in 20 mM PKB (pH 6.8). For the rCaCAD1 and CH-19 Sweet placental extract reaction mixtures, 1 mM NADPH was added as a proton donor. For habanero red placental extract reaction mixtures, 16 mM gamma-amino butyric acid was added as an amine donor. Reactions were incubated at 30°C for 30 min or 1 h. At each sampling instant, an aliquot of 10 μL of each mixture was added to 30 μL of 0.2% TFA to complete the reaction. The amount of vanillyl alcohol or vanillylamine synthesized was analyzed by HPLC as described above. The rate of enzyme activity inhibition was estimated as a function of the peak area. The analysis of the placental extract was carried out using samples from three different individuals.

Syntenic analysis

The genomic synteny around the C. annual Genoa CaCAD1 (NM001324580), pAMT (LC423555), and Word game1(LC423556) was analyzed using the C. annual, S.tuberosum, S. lycopersicum , N. attenuata, and I. trilobe genomes in plants together (https://plants.ensembl.org/) and Genomicus (https://www.genomicus.bio.ens.psl.eu/genomicus-plants-49.01/cgi-bin/search.pl) data base. The TBLASTN algorithm was used to screen the genome sequences of other species for sequences similar to the C. annualCAD1, pAMT and Pun1 sequences. Query coverage greater than 80% and sequence homology greater than 70% were defined as criteria for identifying CAD, pAMT, and Pun1 homologs for synteny analysis.

Ethics statement

The experimental methods were approved by the ethics committee of Josai University and carried out in strict accordance with the guidelines and regulations in force.