Cytochrome P450 CYP76F14 Mediates the Conversion of Its Substrate Linalool in Table Grape Berries

Abstract

Aroma composition serves as a pivotal quality determinant in table grapes (Vitis vinifera). While the cytochrome P450 enzyme CYP76F14 is implicated in aroma biosynthesis, its functional role in grape berries remains uncharacterized. A comparative analysis of three aroma-distinct cultivars—Muscat type ‘Irsai Oliver’, Neutral type ‘Yanhong’, and Berry-like type ‘Venus Seedless’—revealed cultivar-specific linalool accumulation patterns. ‘Irsai Oliver’ exhibited sustained linalool biosynthesis from the fruit set through to maturity (from Stage 1 to Stage 5), with concentrations peaking at Stage 3 (veraison phase) and remaining elevated until harvest, surpassing the other two cultivars. Transcriptional profiling demonstrated that the CYP76F14 expression exhibited a similar trend with the accumulation of linalool levels, showing a higher expression in ‘Irsai Oliver’ across the developmental stages. A structural analysis identified 12 divergent residues in the ‘Irsai Oliver’ CYP76F14 variant, including E378 and T380 within the conserved substrate recognition site. The site-directed mutagenesis of these residues (CYP76F14-E378G/T380A) reduced the catalytic efficiency by 68–72% compared to the wild-type (in vitro LC-MS/MS assays), confirming their functional significance. This work reveals that cytochrome P450 CYP76F14 mediates the conversion of its substrate linalool in table grape berries, especially of Muscat type grapes, and proposes the CYP76F14 polymorphic variants as molecular markers for aroma-type breeding. The identified catalytic residues (E378/T380) provide targets for enzymatic engineering to modulate the terpenoid profiles in Vitis species.

1. Introduction

Aroma serves as a critical sensory indicator of fruit quality in table grapes [1,2,3,4,5]. Cytochrome P450 is a superfamily of enzymes with heme as a secondary group involved in the biosynthesis of metabolites, such as fatty acids, plant sterols, plant hormones, and phenylpropanoids, and plays an important role in plant growth and development [6,7,8]. Recently, Kunert et al. identified two enzymes belonging to the CYP87A subfamily in the distant plants Digitalis purpurea and Calotropis procera. They act on cholesterol and the plant sterols (Brassinosteroids and β-sitosterols) to form pregnenolone, which is the first step in the biosynthesis of cardiac sterols [9]. The overexpression of these CYP87A enzyme encoding genes in Arabidopsis thaliana resulted in the ectopic accumulation of pregnenolone in transgenic plants, while silencing CYP87A in the leaves of Eucommia ulmoides through RNA interference led to a significant reduction in pregnenolone and cardiac sterols [9].

Unlike most plant monofunctional cytochrome P450 enzymes that catalyze a single substrate, the CYP76 subfamily of enzymes is a class of multifunctional monooxygenases that can catalyze multiple substrates [10]. Among them, the redox partner NADPH cytochrome P450 reductase (CPR) acts as an electron donor, promoting the catalytic activity of CYP76 [7,8,11,12]. In Arabidopsis, CYP76C1 can catalyze the conversion of linalool to various terpenoid compounds, such as 8-hydroxylinalool, 8-oxolinalool, and 8-carboxylinalool, and participate in the formation of syringaldehyde and syringanol [7,13]. In wine grapes (Vitis vinifera), enzymes from the CYP76F subfamily (VvCYP76F14) undergo a three-step enzymatic process (hydroxylation, dehydrogenation, and carboxylation) to catalyze the substrate linalool to produce 8-carboxylinalool [2,11,14,15,16], which is a precursor substance for the synthesis of bicyclic monoterpenoid lactones and participates in regulating the formation of wine’s aroma.

According to the different types and contents of aroma compounds, table grapes can be classified into three different aroma types, such as Muscat type, Neutral type, and Berry-like type. Among them, linalool and geraniol are the main aroma compounds of Muscat type grapes, and together with other volatile compounds form the characteristic aromatic profile of table grapes [1,2,17]. However, the molecular mechanism behind the aroma formation of Muscat type grapes is still unclear, and the enzymatic activity characteristics of CYP76F14 catalyzing linalool are still unknown. In this study, CYP76F14 genes were cloned from three different flavor type table grape fruits, and CYP76F14 proteins were expressed and purified in Escherichia coli (E. coli), and the key amino acid residues that determine CYP76F14 enzymatic activity were analyzed. This study provides a theoretical basis for studying the molecular mechanism of table grape aroma formation.

2. Materials and Methods

2.1. Table Grape Cultivars

Three table grape cultivars of ‘Irsai Oliver’ (Muscat type), ‘Yanhong’ (Neutral type), and ‘Venus Seedless’ (Berry-like type) were provided by the National Grape Germplasm Repository of the National Grape Industry System (Yantai, China). Five-year-old vines were grown under routine field management. Following the methods described by Song et al. [18], berries exhibiting similar maturity stages were harvested at the Stage 1 (cell division phase), Stage 2 (seed hardening phase), Stage 3 (veraison phage), Stage 4 (post-veraison, cell expansion phase), and Stage 5 (mature phase), respectively. Berries with consistent maturity and no damage or disease were randomly harvested and stored in a −80 °C freezer after liquid nitrogen treatment for subsequent analysis. Three biological replicates were performed, each with 20 individual clusters.

2.2. Determination of Linalool Content in Berries

Fresh berries were smashed and subjected to a vacuum freeze-drying treatment. A mixed solvent of ethanol and ethyl acetate with the ration of 6:1 (v/v) was added to the powdered material to accumulate linalool. The content of linalool in the table grape berries was determined using high-performance liquid chromatography combined with high-resolution mass spectrometry (HPLC-HRMS) (Waters, Milford, MA, USA) by Shanghai Bioprofile Technology Co. Ltd. (Shanghai, China), as described by Iln et al. [15] and Song et al. [2,16].

2.3. Isolation and Sequence Analysis of CYP76F14

Total RNA was extracted from table grape berries of different aroma types, and the first strand cDNA was synthesized using the PrimeScriptTM RT reagent Kit (TaKaRa, Dalian, China) as a template. The coding region (CDS) sequence of the CYP76F14 gene in wine grapes was used as a reference sequence [2,16,19]. The CYP76F14 gene was amplified from three table grape varieties with different aroma types using high fidelity polymerase Prime STARTM HS DNA (TaKaRa, Dalian, China) and sent to Biotechnology (Shanghai) Co., Ltd. (Shanghai, China) for sequencing verification. The amino acid sequence identity of the encoded CYP76F14 proteins was aligned via the help of the ClustalX 2.0.13 software.

2.4. Quantitative Real Time PCR (qRT-PCR)

The specific expression primers for CYP76F14 were designed using the NCBI/Primer BLAST online server (Forward: 5′-TGTATCCACACCATAT-3′, Reverse: 5′-TCCCAGCTTCCTCCATCACA-3′). Grape Ubiquitin (Forward: 5′-CCTCATCTTCGCTGGCAAAC-3′, Reverse: 5′-GGTGTAGGTCTTCTTCTTGCG-3′) was chosen as an internal reference [2,16,18,19,20]. The expression levels of CYP76F14 during the different fruit developmental stages were analyzed by labeling with the fluorescent dye SYBR Green (TaKaRa, Dalian, China) and using the ABI 7500 real-time fluorescence quantitative PCR instrument (ABI, New York, NY, USA). The PCR reaction procedure was as follows: first, pre denature at 95 °C for 30 s. Second, denaturation at 95 °C for 5 s, annealing at 60 °C for 34 s, 40 cycles. Finally, extend at 72 °C for 15 s. The corresponding Ct values were obtained using an ABI 7500 PCR instrument. The relative expression level was calculated using the 2^−ΔΔCt method [2,16,17,18,19,20,21] after normalization to the internal reference Ubiquitin from three independent biological repeats, each with four technical replicates.

2.5. Site-Directed Mutagenesis (SM)

To explore the catalytic activities of key amino acid residues in the CYP76F14 proteins with different aroma types, we employed the alanine-scanning method to generate alanine-substituted CYP76F14-SMs, using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Stratagene, New York, NY, USA), as previously described [2,16,19,21]. The CYP76F14 gene from the Muscat type ‘Irsai Oliver’ was cloned, and site-directed mutagenesis kits were used to obtain single amino acid mutagenesized mutants of the CYP76F14 protein, referred to as CYP76F14-SMs. The specific mutants obtained were CYP76F14-N46S, CYP76F14-T107R, CYP76F14-N111K, CYP76F14-R175Q, CYP76F14-L222V, CYP76F14-M264I, CYP76F14-S286N, CYP76F14-K325T, CYP76F14-E378G, CYP76F14-T380A, CYP76F14-E383G, and CYP76F14-T386A.

2.6. In Vitro Enzymatic Activity Assay

The crude protein of CYP76F14-MBP or CYP76F14-SM-MBP was purified by the NEBExpress^® MBP Fusion and Purification System (New England Biolabs, Hitchin, UK), using the affinity between MBP and amylose resin [2,16,19]. According to the method described by Song et al. [2] and Kunert et al. [9], table grape CPR1 was used as the electron transfer redox partner for the fresh grape CYP76F14 at an appropriate ratio of 2:1 (CYP76F14/CPR1), and linalool was used as the substrate. The purified recombinant enzymes of CYP76F14 or CYP76F14-SMs were subjected to an in vitro enzymatic activity assay. The VvCYP76F14 enzyme assays were performed in a total reaction volume of 5 mL with a 100 mM Na⁺/K⁺ phosphate buffer (pH 5.0), with varying substrate concentrations, 1 mM NADPH, and adjusted enzyme amounts. The reactions were carried out at 26 °C for 1 h with agitation. The reaction product was collected and analyzed using HPLC-HRMS (Waters, Milford, MA, USA), and boiled protein (nonfunctional) served as a control. The turnover number (K_cat) and affinity (K_m) were individually calculated, and the K_cat/K_m ratio represents the corresponding enzyme activity [2,16,19]. Each reaction undergoes 6 technical replicates for a total of three biological replicates.

2.7. Statistical Analysis

The statistical graphs were generated using the OriginPro 12.0 software (OriginLab Corporation, Northampton, MA, USA). Significant differences were analyzed using ANOVA Fisher’s LSD in the SPSS 13.0 software (SPSS, Chicago, IL, USA). The conditions for the applicability of LSD were checked and the ANOVA showed global significance.

3. Results

3.1. Determination of Linalool Content in Three Different Flavor Type Table Grape Varieties

Three different flavor type table grapes of ‘Irsai Oliver’ (Muscat type), ‘Yanhong’ (Neutral type), and ‘Venus Seedless’ (Berry-like type) were selected in this study. The results of the linalool content determination showed that the linalool content in the Muscat type ‘Irsai Oliver’ berries was significantly higher than that in the ‘Yanhong’ and ‘Venus Seedless’ fruits throughout the entire grape development period, from Stage 1 to Stage 5 (

Table 1. Linalool determination from the different fruit developmental stages of three distinct flavor type varieties.

Cultivar	Flavor Type	Content of Linalool (μg·g−1 FW)
		Stage 1	Stage 2	Stage 3	Stage 4	Stage 5
Irsai Oliver	Muscat	0.46 ± 0.04 a	1.07 ± 0.012 a	1.66 ± 0.14 a	1.68 ± 0.17 a	1.62 ± 0.13 a
Yanhong	Neutral	0.008 ± 0.0009 b	0.013 ± 0.0014 b	0.017 ± 0.0015 b	0.016 ± 0.0014 b	0.016 ± 0.0016 b
Venus Seedless	Berry-like	0.0031 ± 0.00043 c	0.0054 ± 0.00062 c	0.0091 ± 0.0011 c	0.0094 ± 0.0013 c	0.0093 ± 0.0014 c

Note: Stage 1, cell division phase. Stage 2, seed hardening phase. Stage 3, veraison phage. Stage 4, post-veraison (cell expansion phase). Stage 5, mature phase. Data were presented as the means ± SE (n = 3). Letters represent significant differences among the different flavor type cultivars at a significance level of p ≤ 0.01, as determined using ANOVA followed by Fisher’s LSD test.

). In addition, the linalool content in the fruits of all the three different flavor types of grapes continuously increased with the development of the grape fruits, reaching a peak during Stage 3 and continuing until Stage 5 (Table 1).

3.2. Cloning of the CYP76F14 Gene and Analysis of the Encoded Protein Sequence

Using the CDS sequence of the CYP76F14 gene from wine grapes as a reference [2,16,19], the homologous CYP76F14 genes were cloned from three flavor type grape varieties. The encoded products all contained the AGTDT (active domain) and FGAGRRICFG (HEME-binding domain) motifs, which are typical conserved domains of the cytochrome P450 family proteins (Figure 1). Amino acid sequence alignment showed that the amino acid sequence identity of the CYP76F14 proteins in the three different flavor type varieties was 99.20%. Compared with the CYP76F14 amino acid sequences of ‘Yanhong’ and ‘Venus Seedless’, the CYP76F14 amino acids in the Muscat type ‘Irsai Oliver’ showed mutations in the N46, T107, N111, R175, L222, M264, S286, K325, E378, T380, E383, and T386 residues (Figure 1).

3.3. Relative Expression of CYP76F14 Genes in Three Different Table Grape Varieties

qRT-PCR analysis showed that the expression level of the CYP76F14 gene in the ‘Irsai Oliver’ (Muscat type) berries was significantly higher than that in the ‘Yanhong’ (Neutral type) and ‘Venus Seedless’ (Berry-like type) berries throughout the entire fruit development period (Figure 2). The expression level of the CYP76F14 gene in the ‘Irsai Oliver’ berries continuously increased with the fruit’s development, reaching a peak during Stage 3 and continuing until Stage 5 (Figure 2). In particular, the expression trends of the CYP76F14 genes were closely aligned with the accumulation patterns of the linalool content during different developmental stages in the three table grape varieties, respectively.

3.4. Site-Directed Mutagenesis of CYP76F14 (CYP76F14-SMs) and Heterologous Expression of CYP76F14 Protein in E. coli

The CYP76F14 gene from the Muscat type ‘Irsai Oliver’ berries was cloned, and 12 site-directed mutant proteins (CYP76F14-SMs: CYP76F14-N46S, CYP76F14-T107R, CYP76F14-N111K, CYP76F14-R175Q, CYP76F14-L222V, CYP76F14-M264I, CYP76F14-S286N, CYP76F14-K325T, CYP76F14-E378G, CYP76F14-T380A, CYP76F14-E383G, and CYP76F14-T386A) were obtained and sequenced. The recombinant plasmids of pMAL-c6T-CYP76F14 or pMAL-c6T-CYP76F14-SMs were transformed into E. coli BL21 strains for expression and purification. The resulting CYP76F14-MBP recombinant proteins were subsequently used for the in vitro enzyme activity assays. The expression and purification of the CYP76F14-MBP recombinant proteins is exhibited in Figure 3.

3.5. In Vitro Enzyme Activity Assay of CYP76F14 and CYP76F14-SMs

According to the method described by Peng et al. [16] and Xia et al. [19], using linalool as the substrate, the purified recombinant enzymes CYP76F14-MBP or CYP76F14-SMs-MBP were subjected to the in vitro enzyme activity assays. The enzymatic kinetic analysis results showed that the enzyme activities of the CYP76F14-E378G and CYP76F14-T380A site-directed mutant proteins were significantly lower than those of the control protein CYP76F14. However, there were no significant differences in the enzyme activities of the other 10 amino acid residue site-directed mutant proteins CYP76F14-SMs (CYP76F14-N46S, CYP76F14-T107R, CYP76F14-N111K, CYP76F14-R175Q, CYP76F14-L222V, CYP76F14-M264I, CYP76F14-S286N, CYP76F14-K325T, CYP76F14-E383G, and CYP76F14-T386A) compared to the control protein (Figure 4 and

Table 2. In vitro determination of the ‘Irsai Oliver’ CYP76F14 and CYP76F14-SM mutants.

Protein	km (μM)	kcat (S−1)	Remaining Amount of Linalool (mmol∙min−1∙mg−1 Protein)
CYP76F14	69.20 ± 5.26 b	12.93 ± 1.42 a	8.02 ± 0.89 c
CYP76F14-N146S	70.52 ± 7.53 b	12.27 ± 1.33 a	8.43 ± 0.94 c
CYP76F14-T107I	76.84 ± 8.27 b	13.14 ± 1.48 a	7.79 ± 0.82 c
CYP76F14-N111K	68.24 ± 6.65 b	11.26 ± 1.21 a	7.80 ± 0.91 c
CYP76F14-R175Q	75.56 ± 8.21 b	13.45 ± 1.44 a	8.24 ± 0.78 c
CYP76F14-L222V	67.25 ± 6.23 b	12.24 ± 1.51 a	7.87 ± 0.83 c
CYP76F14-M264I	71.39 ± 6.96 b	11.78 ± 1.09 a	8.11 ± 0.74 c
CYP76F14-S286N	74.08 ± 7.24 b	12.15 ± 1.18 a	8.39 ± 0.90 c
CYP76F14-K325T	70.57 ± 8.16 b	11.08 ± 1.02 a	8.33 ± 0.76 c
CYP76F14-E378G	269.35 ± 25.18 a	8.35 ± 0.89 b	16.67 ± 1.85 b
CYP76F14-T380A	134.91 ± 14.67 a	7.15 ± 0.83 b	23.72 ± 0.94 a
CYP76F14-E383D	66.95 ± 7.02 b	11.85 ± 1.16 a	7.92 ± 0.81 c
CYP76F14-T386A	83.27 ± 9.12 b	13.24 ± 1.46 a	8.11 ± 0.85 c

Note: Different lowercase letters indicate extremely significant differences (p < 0.01) among the different recombinant proteins.

).

4. Discussion

Aroma is one of the important quality indicators of grape fruits, and the content of linalool varies among the different aroma types of table grapes [1,2,17]. In wine grapes, the enzyme CYP76F14 catalyzes the production of different terpene compounds from linalool, which are precursor substances that determine the aroma of wine [2,15,16,17]. The physiological function of CYP76F14 in regulating the formation of aromas in table grape fruits is still unclear.

The highly conserved monooxygenase region of the cytochrome P450 features a general activity characteristic motif A(G)G(A)XD(E)T [22,23]. In this study, the typical AGTDT activity domain and HEME binding domain were detected in the monooxygenase region of CYP76F14 proteins from three different aroma types of table grapes, consistent with previous reports in Arabidopsis [22] and yeast [24]. Notably, sequence differences in the CYP76F14 protein were found among different aroma types of table grapes, with multiple amino acid mutation sites detected only in the Muscat type ‘Irsai Oliver’ CYP76F14 protein. Similarly, amino acid sequence differences were observed among CYP76F14 proteins in different wine grape varieties, closely related to the enzyme activity of their in vitro expressions [16,19]. Moreover, similar reports of amino acid residue mutations in cytochrome P450 subfamily proteins exist in potatoes (CYP77A) [25] and Salvia miltiorrhiza (CYP76AK) [26].

In recent years, studies have utilized prokaryotic systems to express plant cytochrome P450 proteins and conduct in vitro enzymatic kinetic characteristic analyses. Such studies have been reported in Arabidopsis [9,22,23], potatoes [25], S. miltiorrhiza [26], and wine grapes [2,16,19]. In this study, site-directed mutagenesis of two amino acid residues (E378G and T380A) of the table grape CYP76F14 protein resulted in a significant decrease in in vitro CYP76F14 enzyme activity, suggesting that these two amino acid residues may be critical for CYP76F14 enzyme activity and may directly participate in the process of CYP76F14 catalyzing the production of other terpene compounds from the substrate linalool, warranting further ontological functional verification. These results again indicate that certain key amino acid residues in plant CYP76F14 proteins directly determine cytochrome P450 enzyme activity [2,15,18], and their specific biological functions and regulatory mechanisms merit further in-depth study.

In summary, this study provides gene resources for revealing the biological function of CYP76F14 in table grapes and lays a theoretical foundation for exploring the use of CYP76F14 as a fingerprint marker in breeding table grape varieties with different aroma types.

5. Conclusions

There is a significant difference in the content of linalool in the fruits of three different flavor-type table grape varieties. The Muscat type ‘Irsai Oliver’ exhibited sustained linalool accumulation from Stage 1 (cell division phase) to Stage 5 (mature phase), with concentrations peaking at Stage 3 (veraison phage) and remaining elevated until harvest, surpassing other two cultivars of the Neutral type ‘Yanhong’ and Berry-like aroma type ‘Venus Seedless’. Transcriptional profiling demonstrated that the CYP76F14 expression exhibited a similar trend with the linalool levels showing a higher expression in ‘Irsai Oliver’ across the developmental stages. A structural analysis identified 12 divergent residues in the ‘Irsai Oliver’ CYP76F14 variant. The site-directed mutagenesis of two residues (CYP76F14-E378G and CYP76F14-T380A) reduced catalytic efficiency by 68–72% compared to the wild-type. E378 and T380 may be two key amino acid residues determining the enzyme activity of ‘Irsai Oliver’ CYP76F14.