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1.
Plant Physiol ; 172(3): 1563-1577, 2016 11.
Article in English | MEDLINE | ID: mdl-27688619

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.


Subject(s)
Alkaloids/biosynthesis , Biosynthetic Pathways , Catharanthus/enzymology , NADPH-Ferrihemoprotein Reductase/metabolism , Biocatalysis , Biosynthetic Pathways/genetics , Catharanthus/genetics , Cotyledon/metabolism , Endoplasmic Reticulum/metabolism , Gene Expression Regulation, Plant , Gene Silencing , Genes, Plant , Indole Alkaloids/metabolism , NADPH-Ferrihemoprotein Reductase/chemistry , NADPH-Ferrihemoprotein Reductase/genetics , Plant Leaves/metabolism , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Subcellular Fractions/enzymology
2.
Front Plant Sci ; 7: 509, 2016.
Article in English | MEDLINE | ID: mdl-27200002

ABSTRACT

Plants use monoterpenols as precursors for the production of functionally and structurally diverse molecules, which are key players in interactions with other organisms such as pollinators, flower visitors, herbivores, fungal, or microbial pathogens. For humans, many of these monoterpenol derivatives are economically important because of their pharmaceutical, nutraceutical, flavor, or fragrance applications. The biosynthesis of these derivatives is to a large extent catalyzed by enzymes from the cytochrome P450 superfamily. Here we review the knowledge on monoterpenol oxidative metabolism in plants with special focus on recent elucidations of oxidation steps leading to diverse linalool and geraniol derivatives. We evaluate the common features between oxidation pathways of these two monoterpenols, such as involvement of the CYP76 family, and highlight the differences. Finally, we discuss the missing steps and other open questions in the biosynthesis of oxygenated monoterpenol derivatives.

3.
BMC Genomics ; 16: 619, 2015 Aug 19.
Article in English | MEDLINE | ID: mdl-26285573

ABSTRACT

BACKGROUND: Transcriptome sequencing offers a great resource for the study of non-model plants such as Catharanthus roseus, which produces valuable monoterpenoid indole alkaloids (MIAs) via a complex biosynthetic pathway whose characterization is still undergoing. Transcriptome databases dedicated to this plant were recently developed by several consortia to uncover new biosynthetic genes. However, the identification of missing steps in MIA biosynthesis based on these large datasets may be limited by the erroneous assembly of close transcripts and isoforms, even with the multiple available transcriptomes. RESULTS: Secologanin synthases (SLS) are P450 enzymes that catalyze an unusual ring-opening reaction of loganin in the biosynthesis of the MIA precursor secologanin. We report here the identification and characterization in C. roseus of a new isoform of SLS, SLS2, sharing 97 % nucleotide sequence identity with the previously characterized SLS1. We also discovered that both isoforms further oxidize secologanin into secoxyloganin. SLS2 had however a different expression profile, being the major isoform in aerial organs that constitute the main site of MIA accumulation. Unfortunately, we were unable to find a current C. roseus transcriptome database containing simultaneously well reconstructed sequences of SLS isoforms and accurate expression levels. After a pair of close mRNA encoding tabersonine 16-hydroxylase (T16H1 and T16H2), this is the second example of improperly assembled transcripts from the MIA pathway in the public transcriptome databases. To construct a more complete transcriptome resource for C. roseus, we re-processed previously published transcriptome data by combining new single assemblies. Care was particularly taken during clustering and filtering steps to remove redundant contigs but not transcripts encoding potential isoforms by monitoring quality reconstruction of MIA genes and specific SLS and T16H isoforms. The new consensus transcriptome allowed a precise estimation of abundance of SLS and T16H isoforms, similar to qPCR measurements. CONCLUSIONS: The C. roseus consensus transcriptome can now be used for characterization of new genes of the MIA pathway. Furthermore, additional isoforms of genes encoding distinct MIA biosynthetic enzymes isoforms could be predicted suggesting the existence of a higher level of complexity in the synthesis of MIA, raising the question of the evolutionary events behind what seems like redundancy.


Subject(s)
Catharanthus/genetics , Cytochrome P-450 Enzyme System/genetics , Gene Expression Profiling/methods , Iridoid Glucosides/metabolism , Oxidoreductases Acting on CH-CH Group Donors/genetics , Alternative Splicing , Catharanthus/enzymology , Cytochrome P-450 Enzyme System/metabolism , Databases, Genetic , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/analysis , RNA, Plant/analysis
4.
Plant Physiol ; 160(3): 1407-19, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22961129

ABSTRACT

Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.


Subject(s)
Acyltransferases/chemistry , Acyltransferases/genetics , Evolution, Molecular , Multigene Family/genetics , Vitis/enzymology , Vitis/genetics , Acyltransferases/metabolism , Agrobacterium/metabolism , Amino Acids/genetics , Chromosomes, Plant/genetics , Gene Expression Regulation, Plant , Genes, Plant/genetics , Models, Molecular , Phylogeny , Stilbenes/chemistry , Stilbenes/metabolism , Stress, Physiological/genetics , Nicotiana/microbiology
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