Sébastien Grec

NpPDR1, a Pleiotropic Drug Resistance-Type ATP-Binding Cassette Transporter from Nicotiana plumbaginifolia, Plays a Major Role in Plant Pathogen Defense

Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family.

Natural Hypolignification Is Associated with Extensive Oligolignol Accumulation in Flax Stems

Flax (Linum usitatissimum) stems contain cells showing contrasting cell wall structure: lignified in inner stem xylem tissue and hypolignified in outer stem bast fibers. We hypothesized that stem hypolignification should be associated with extensive phenolic accumulation and used metabolomics and transcriptomics to characterize these two tissues. 1H nuclear magnetic resonance clearly distinguished inner and outer stem tissues and identified different primary and secondary metabolites, including coniferin and p-coumaryl alcohol glucoside. Ultrahigh-performance liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry aromatic profiling (lignomics) identified 81 phenolic compounds, of which 65 were identified, to our knowledge, for the first time in flax and 11 for the first time in higher plants. Both aglycone forms and glycosides of monolignols, lignin oligomers, and (neo)lignans were identified in both inner and outer stem tissues, with a preponderance of glycosides in the hypolignified outer stem, indicating the existence of a complex monolignol metabolism. The presence of coniferin-containing secondary metabolites suggested that coniferyl alcohol, in addition to being used in lignin and (neo)lignan formation, was also utilized in a third, partially uncharacterized metabolic pathway. Hypolignification of bast fibers in outer stem tissues was correlated with the low transcript abundance of monolignol biosynthetic genes, laccase genes, and certain peroxidase genes, suggesting that flax hypolignification is transcriptionally regulated. Transcripts of the key lignan genes Pinoresinol-Lariciresinol Reductase and Phenylcoumaran Benzylic Ether Reductase were also highly abundant in flax inner stem tissues. Expression profiling allowed the identification of NAC (NAM, ATAF1/2, CUC2) and MYB transcription factors that are likely involved in regulating both monolignol production and polymerization as well as (neo)lignan production.

Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco)iridoid pathway.

The geraniol-derived (seco)iridoid skeleton is a precursor for a large group of bioactive compounds with diverse therapeutic applications, including the widely used anticancer molecule vinblastine. Despite of this economic prospect, the pathway leading to iridoid biosynthesis from geraniol is still unclear. The first geraniol hydroxylation step has been reported to be catalyzed by cytochrome P450 enzymes such as CYP76B6 from Catharanthus roseus and CYP76C1 from Arabidopsis thaliana. In the present study, an extended functional analysis of CYP76 family members was carried-out to identify the most effective enzyme to be used for pathway reconstruction. This disproved CYP76C1 activity and led to the characterization of CYP76C4 from A. thaliana as a geraniol 9- or 8-hydroxylase. CYP76B6 emerged as a highly specialized multifunctional enzyme catalyzing two sequential oxidation steps leading to the formation of 8-oxogeraniol from geraniol. This dual function was confirmed in planta using a leaf-disc assay. The first step, geraniol hydroxylation, was very efficient and fast enough to outcompete geraniol conjugation in plant tissues. When the enzyme was expressed in leaf tissues, 8-oxogeraniol was converted into further oxidized and/or reduced compounds in the absence of the next enzyme of the iridoid pathway.

Dual Function of the Cytochrome P450 CYP76 Family from Arabidopsis thaliana in the Metabolism of Monoterpenols and Phenylurea Herbicides

Fast diversification and versatility of a subfamily of cytochrome P450 enzymes in Brassicaceae has been important in their metabolism of both monoterpenols and herbicides. Comparative genomics analysis unravels lineage-specific bursts of gene duplications related to the emergence of specialized pathways. The CYP76C subfamily of cytochrome P450 enzymes is specific to Brassicaceae. Two of its members were recently associated with monoterpenol metabolism. This prompted us to investigate the CYP76C subfamily genetic and functional diversification. Our study revealed high rates of CYP76C gene duplication and loss in Brassicaceae, suggesting the association of the CYP76C subfamily with species-specific adaptive functions. Gene differential expression and enzyme functional specialization in Arabidopsis thaliana, including metabolism of different monoterpenols and formation of different products, support this hypothesis. In addition to linalool metabolism, CYP76C1, CYP76C2, and CYP76C4 metabolized herbicides belonging to the class of phenylurea. Their ectopic expression in the whole plant conferred herbicide tolerance. CYP76Cs from A. thaliana. thus provide a first example of promiscuous cytochrome P450 enzymes endowing effective metabolism of both natural and xenobiotic compounds. Our data also suggest that the CYP76C gene family provides a suitable genetic background for a quick evolution of herbicide resistance.

Ectopic Lignification in the Flax lignified bast fiber1 Mutant Stem Is Associated with Tissue-Specific Modifications in Gene Expression and Cell Wall Composition

The cell walls of flax bast fibers contain high cellulose and low lignin levels, imparting tensile strength and flexibility. To learn more about the mechanisms responsible for this type of cell wall structure, a collection of ectopic lignin mutants was identified. Characterization of the lbf1 mutant provided key information on lignification in flax that is also relevant to other plants. Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

Cryptic polyadenylation sites within the coding sequence of three yeast genes expressed in tobacco.

Three yeast genes, MIP (mitochondrial DNA polymerase) and two genes, YCF1 (yeast cadmium factor 1) and PDR5 (pleiotropic drug resistance 5), conferring multidrug resistance, were provided with the cauliflower mosaic virus 35S transcription promoter and introduced into tobacco using an Agrobacterium tumefaciens T-DNA-derived vector. Transcripts of each gene much shorter than those expected were found in the transgenic plants. RT-PCR and S1 nuclease mapping of the PDR5 and MIP transcripts demonstrated the presence of one (PDR5), or several close (MIP), cryptic polyadenylation site(s) within the coding sequence of these yeast genes. Possible sequences involved in polyadenylation are discussed.

A "novel" protocol for the analysis of hydroxycinnamic acids in leaf tissue of chicory (Cichorium intybus L., Asteraceae).

A “novel” protocol is presented for easy and reliable estimation of soluble hydroxycinnamate levels in Cichorium intybus L. leaf tissue in large-scale experiments. Samples were standardized by punching 6 discs per leaf, and hydroxycinnamates were extracted by submerging the discs in 80% ethanol with 5% acetic acid for at least 48 h in the darkness at 4°C. Residual dry mass of the discs was used for a posteriori correction of compound levels. Chlorophyll was eliminated by chloroform, and the aqueous phases were transferred to microplates, dried, and dissolved in 50% methanol for HPLC analysis and storage. An HPLC program of 8 min was developed for the analysis of the extracts. Comparisons with extractions of liquid nitrogen powders indicated that the novel extraction method was reliable. No degradation of the major hydroxycinnamates—caftaric, chlorogenic, and chicoric acids—was observed, during maceration at ambient temperatures, or after storage for 1 year.