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

TY - JOUR

T1 - A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ-dependent manner in Catharanthus roseus

AU - Besseau, Sébastien

AU - Kellner, Franziska

AU - Lanoue, Arnaud

AU - Thamm, Antje M K

AU - Salim, Vonny

AU - Schneider, Bernd

AU - Geu Flores, Fernando

AU - Höfer, René

AU - Guirimand, Grégory

AU - Guihur, Anthony

AU - Oudin, Audrey

AU - Glevarec, Gaëlle

AU - Foureau, Emilien

AU - Papon, Nicolas

AU - Clastre, Marc

AU - Giglioli-Guivarc'h, Nathalie

AU - St-Pierre, Benoit

AU - Werck-Reichhart, Danièle

AU - Burlat, Vincent

AU - De Luca, Vincenzo

AU - O'Connor, Sarah E

AU - Courdavault, Vincent

PY - 2013/12

Y1 - 2013/12

N2 - 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.

AB - 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.

KW - Biocatalysis

KW - Biosynthetic Pathways

KW - Catharanthus

KW - Cytochrome P-450 Enzyme System

KW - DNA, Complementary

KW - Endoplasmic Reticulum

KW - Gene Expression Regulation, Enzymologic

KW - Gene Expression Regulation, Plant

KW - Gene Silencing

KW - Genes, Plant

KW - Hydroxylation

KW - Indole Alkaloids

KW - Kinetics

KW - Metabolome

KW - Molecular Sequence Data

KW - Organ Specificity

KW - Plant Epidermis

KW - Plant Proteins

KW - Quinolines

KW - RNA, Messenger

KW - Substrate Specificity

KW - Vinblastine

U2 - 10.1104/pp.113.222828

DO - 10.1104/pp.113.222828

M3 - Journal article

C2 - 24108213

SN - 0032-0889

VL - 163

SP - 1792

EP - 1803

JO - Plant Physiology

JF - Plant Physiology

IS - 4

ER -