A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ-dependent manner in Catharanthus roseus

Abstract

Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.

Figure 1.

The position of tabersonine hydroxylation governs the biosynthesis of downstream MIAs. In aerial organs, tabersonine is hydroxylated at the C-16 position by T16H and subsequently metabolized by 16OMT, uncharacterized hydratase, NMT, D4H, and DAT to produce vindoline. In roots, tabersonine is hydroxylated at the C-19 position by T19H prior to acetylation catalyzed by MAT and additional reactions of reduction or epoxidation. Dashed lines represent uncharacterized enzymatic reactions.

Figure 2.

CYP71D351 and CYP71D12 catalyze the 16-hydroxylation of tabersonine. A, LC-MS results using selected ion monitoring (tabersonine, mass-to-charge ratio 337; 16-hydroxytabersonine, mass-to-charge ratio 353) of the reaction products of microsomes purified from yeast cell cultures expressing either CYP71D351 or CYP71D12 or containing the empty pYeDP60 vector. B, Partial HMBC spectrum for 16-hydroxytabersonine isolated from yeast culture expressing CYP71D351. The position of the new hydroxyl group was established from HMBC correlations through three bonds (H-14/C-12, H-14/C-16, H-14/C-18, H-15/C-13, H-15/C-17, H-17/C-13, and H-17/C-15) as indicated by arrows in the inset structure. [See online article for color version of this figure.]

Figure 3.

Analysis of CYP71D351 (T16H2) and CYP71D12 (T16H1) expression in C. roseus organs and cell cultures. CYP71D351 (light gray bars) and CYP71D12 (dark gray bars) transcript levels were determined by real-time RT-PCR analyses performed on total RNA extracted from C. roseus cells subjected to MeJa or mock treatment during 0.5, 8, 24, and 48 h (A) and from C. roseus organs (B). CYP71D351 and CYP71D12 transcript copy numbers were normalized using CrRPS9. R, Roots; S, stems; YL, young leaves; ML, mature leaves; FB, flower buds; Fl, flowers; Fr, fruits.

Figure 4.

CYP71D351 (T16H2) has a similar expression pattern to other vindoline biosynthetic genes. Relative expression of CYP71D12, CYP71D351, 16OMT, NMT, and DAT was determined by real-time RT-PCR analyses performed on total RNA extracted from various C. roseus organs. MAT, a root-specific hydroxylase, was also included for comparison. CrRPS9 was used as a reference gene. R, Roots; S, stems; YL, young leaves; ML, mature leaves; FB, flower buds; Fl, flowers; Fr, fruits.

Figure 5.

T16H activity and CYP71D351 (T16H2) transcripts are specifically located in epidermis of C. roseus leaves. A and B, Analysis of T16H enzymatic activity (A) and relative expression of CYP71D351 in leaf epidermis-enriched protein/transcript extracts produced by carborundum abrasion compared with that found in whole leaves (B). Cr60S was used as a reference gene. Ep, Epidermis; WL, whole leaf. C to F, Analysis of CYP71D351 and CYP71D12 transcript distribution performed by RNA in situ hybridization. Serial sections of young developing leaves were hybridized either with CYP71D12 antisense (AS) probes (C), CYP71D351 antisense probes (E), CYP71D12 sense (S) probes (D), or CYP71D351 sense probes (F) used as negative controls. ab, Abaxial epidermis; ad, adaxial epidermis. Bars = 100 µm.

Figure 6.

CYP71D351 (T16H2) is located to the ER. C. roseus cells were transiently transformed with CYP71D351-YFP-expressing vector (CYP-YFP; A and E) in combination with plasmids expressing a nucleus-CFP marker (“nuc”-CFP; B) or an ER-CFP marker (“ER”-CFP; F). Colocalization of the two fluorescence signals appears on the merged images (C and G). Cell morphology (D and H) was observed with differential interference contrast (DIC). Bars = 10 µm.

Figure 7.

Comparison of CYP71D351 (T16H2) and CYP71D12 (T16H1) transcript levels in low- and high-vindoline-accumulating C. roseus cultivars. CYP71D12 and CYP71D351 transcript levels were measured by real-time RT-PCR analyses performed on total RNA extracted from C. roseus leaves of the low-vindoline-accumulating cv Vinca Mediterranean DP Orchid (DP Orc.; dark gray bars) and of the high-vindoline-accumulating cv Little Delicata (Lit. Del.; light gray bars). Transcript copy numbers were normalized using the ribosomal 60S RNA.

Figure 8.

Down-regulation of CYP71D351 (T16H2) affects vindoline biosynthesis. A, CYP71D351 transcript down-regulation by VIGS. Relative expression of G10H (encoding an enzyme involved in secologanin biosynthesis), CYP71D351, D4H, and DAT was determined by real-time RT-PCR analyses performed on total RNA extracted from C. roseus leaves of CYP71D351-silenced plants (CYP-VIGS; white bars) or plants transformed with an empty vector control (gray bars). CrRPS9 was used as a reference gene. Data correspond to average values (n = 4) ± sd of independent transformant plants. B, Relative MIA content in CYP-VIGS plants (white bars) as compared with empty vector plants (gray bars). The accumulation of tabersonine and 16-hydroxytabersonine (first molecule; R = H and R = OH, respectively), deacetylvindoline and deacetylvindorosine (second molecule; R = OCH₃ and R = H, respectively), vindoline and vindorosine (last molecule; R = OCH₃ and R = H, respectively) was quantified by LC-MS. The amount of each MIA in CYP-VIGS plants was expressed relative to that measured in empty vector plants (normalized to 1). Asterisks denote statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test). The results shown are representative of eight CYP-VIGS plants and eight empty vector plants.