Class II Cytochrome P450 Reductase Governs the Biosynthesis of Alkaloids

Abstract

Expansion of the biosynthesis of plant specialized metabolites notably results from the massive recruitment of cytochrome P450s that catalyze multiple types of conversion of biosynthetic intermediates. For catalysis, P450s require a two-electron transfer catalyzed by shared cytochrome P450 oxidoreductases (CPRs), making these auxiliary proteins an essential component of specialized metabolism. CPR isoforms usually group into two distinct classes with different proposed roles, namely involvement in primary and basal specialized metabolisms for class I and inducible specialized metabolism for class II. By studying the role of CPRs in the biosynthesis of monoterpene indole alkaloids, we provide compelling evidence of an operational specialization of CPR isoforms in Catharanthus roseus (Madagascar periwinkle). Global analyses of gene expression correlation combined with transcript localization in specific leaf tissues and gene-silencing experiments of both classes of CPR all point to the strict requirement of class II CPRs for monoterpene indole alkaloid biosynthesis with a minimal or null role of class I. Direct assays of interaction and reduction of P450s in vitro, however, showed that both classes of CPR performed equally well. Such high specialization of class II CPRs in planta highlights the evolutionary strategy that ensures an efficient reduction of P450s in specialized metabolism.

Recent advances in allelopathy for weed control: from knowledge to applications

“…These secondary metabolites are commonly used as defensive allelochemicals, as in many cases, herbivores or microorganisms are deterred, poisoned or inhibited . These compounds with a nitrogen atom can be biosynthesized in the cytoplasm, vesicles or organelles such as chloroplasts . Nevertheless, they can accumulate in specific cell types, such as a root‐hair (e.g.…”

“…[156][157][158] These compounds with a nitrogen atom can be biosynthesized in the cytoplasm, vesicles or organelles such as chloroplasts. 159 Nevertheless, they can accumulate in specific cell types, such as a root-hair (e.g. Lycoris chinensis), and more specifically in vacuoles due to the water solubility of alkaloids.…”

A BAHD acyltransferase catalyzing 19‐O‐acetylation of tabersonine derivatives in roots of Catharanthus roseus enables combinatorial synthesis of monoterpene indole alkaloids

“…While we were not able to confirm TAT functions using VIGS due to the inefficiency of this approach in roots (Figure S13), its high catalytic efficiency in vitro and its capacity to work in planta and in other distinct heterologous organisms (Table and Figure , Figure S13 and Figure S14) collectively provide strong evidence of a prominent role for TAT in MIA biosynthesis in C. roseus roots. Accordingly, TAT gene expression profile is very similar to T19H and follows tabersonine root metabolite accumulation (Figure (a, b)) in accordance with the fact that genes belonging to a same pathway often exhibit similar expression profiles correlated to MIA accumulation (Besseau et al ., ; Dugé Bernonville et al ., ; Parage et al ., ).…”

“…Protein expression was induced by cultivating the harvested yeast in 50 mL of YPGal medium (2% bacto peptone, 1% yeast extract, and 2% galactose) for 6 h as described in Parage et al . ().…”

“…UPLC‐MS analyses were conducted using equipment and condition described in Parage et al . (). Data collection was carried in selected ion monitoring (SIM) mode for the following compounds: tabersonine m/z 337 RT = 14.4 min, 16‐hydroxytabersonine m/z 353 RT = 8.4 min, tabersonine 2,3‐epoxide m/z 353 RT = 11.4 min, 19‐hydroxytabersonine m/z 353 RT = 6.5 min, 16‐methoxytabersonine m/z 367 RT = 12.03 min, 16‐methoxy‐19‐hydroxytabersonine m/z 383 RT = 7.66 min, 16‐methox‐19‐acetyltabersonine m/z 425 RT =14.6 min, vincadifformine m/z 339 RT = 11.40 min, minovincinine m/z 355 RT = 5.56 min, echitovenine m/z 397 RT = 10.12 min, vincadifformine 2,3‐epoxide m/z 355 RT = 11.67 min, ervinceine (syn: 16‐methoxyvincadifformine) m/z 369 RT = 12.5 min, 16‐methoxyechitovenine m/z 427 RT = 7.24 min, hörhammericine m/z 369 RT = 5.7 min, 19‐acetylhörhammericine m/z 411 RT = 12.2 min, 16‐methoxy‐19‐acetylhörhammericine m/z 441 RT = 6.1 min, 19‐acetyltabersonine m/z 395 RT = 15.6 min, 16‐methoxytabersonine epoxide/imine alcohol m/z 383 RT = 8.89 min, 16‐methoxyvincadifformine epoxide m/z 385 RT = 8.34 min, hörhammericine epoxide m/z 385 RT = 5.4, deacetylvindoline m/z 415 RT = 12.4, vindoline m/z 457 RT = 15.4, quercetin m/z 301 (es‐) RT = 11.1, naringenin m/z 271 (es‐) RT = 12.28, cyanidin m/z 287 (es‐) RT = 10.43, caffeic acid m/z 179 (es‐) RT = 3.87, spermidine m/z 146 (es‐) RT = 4.53.…”