Laurent Legendre

Plant growth-promoting rhizobacteria and root system functioning

The rhizosphere supports the development and activity of a huge and diversified microbial community, including microorganisms capable to promote plant growth. Among the latter, plant growth-promoting rhizobacteria (PGPR) colonize roots of monocots and dicots, and enhance plant growth by direct and indirect mechanisms. Modification of root system architecture by PGPR implicates the production of phytohormones and other signals that lead, mostly, to enhanced lateral root branching and development of root hairs. PGPR also modify root functioning, improve plant nutrition and influence the physiology of the whole plant. Recent results provided first clues as to how PGPR signals could trigger these plant responses. Whether local and/or systemic, the plant molecular pathways involved remain often unknown. From an ecological point of view, it emerged that PGPR form coherent functional groups, whose rhizosphere ecology is influenced by a myriad of abiotic and biotic factors in natural and agricultural soils, and these factors can in turn modulate PGPR effects on roots. In this paper, we address novel knowledge and gaps on PGPR modes of action and signals, and highlight recent progress on the links between plant morphological and physiological effects induced by PGPR. We also show the importance of taking into account the size, diversity, and gene expression patterns of PGPR assemblages in the rhizosphere to better understand their impact on plant growth and functioning. Integrating mechanistic and ecological knowledge on PGPR populations in soil will be a prerequisite to develop novel management strategies for sustainable agriculture.

Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture

BackgroundSclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species.ResultsBased on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants.ConclusionsSsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.

Culture of Ciona intestinalis in closed systems

Improvements in closed‐system culturing methods for marine invertebrates are important prerequisites for the generalized use of transgenic lines. We discuss here the effects of several closed‐system conditions on the growth and survival of the solitary ascidian, Ciona intestinalis. In Shimoda, close to the sea, a small‐tank system was used to ensure that tanks and systems were reasonably equipped, water exchange was rapid, and animals separated to minimize the risk of infection. In Gif‐sur‐Yvette, an inland site, we tried to determine the optimal conditions to limit handling operations, and to save artificial seawater by avoiding water pollution. A mixture of at least two types of live algae was better than any single‐organism diet. With these maintenance protocols, we were able to obtain several generations of Ciona intestinalis, including several transgenic lines. Because these systems make it easier to rear Ciona intestinalis in laboratories, they increase the potentialities of this model organism for research. Developmental Dynamics 236:1832–1840, 2007.

A developmental staging table for Astyanax mexicanus surface fish and Pachón cavefish.

Every model species requires its own developmental table. Astyanax mexicanus, a teleost fish comprising both sighted river and blind cave populations, is becoming more and more important in the field of developmental and evolutionary biology. As such, a developmental staging table is increasingly necessary, particularly since comparative analysis of early developmental events is widely employed by researchers. We collected freshly spawned embryos from surface fish and Pachón cavefish populations. Embryos were imaged every 10-12 min during the first day of development, and less frequently in the following days. The results provide an illustrated comparison of selected developmental stages from one cell to hatching of these two populations. The two morphs show an essentially synchronous development regarding major events such as epiboly, neurulation, somitogenesis, heart beating, or hatching. We also present data on particular morphological characters appearing during larval development, such as eye size, yolk regression, swim bladder, and fin development. Some details about the development of F1 Pachón cave×surface hybrids are also given. Comparisons are made with Danio rerio (zebrafish) development.

Differences in chemosensory response between eyed and eyeless Astyanax mexicanus of the Rio Subterráneo cave

BackgroundIn blind cave-dwelling populations of Astyanax mexicanus, several morphological and behavioral shifts occurred during evolution in caves characterized by total and permanent darkness. Previous studies have shown that sensory systems such as the lateral line (mechanosensory) and taste buds (chemosensory) are modified in cavefish. It has long been hypothesized that another chemosensory modality, the olfactory system, might have evolved as well to provide an additional mechanism for food-searching in troglomorphic Astyanax populations.FindingsDuring a March 2013 cave expedition to the Sierra de El Abra region of San Luís Potosi, Mexico, we tested chemosensory capabilities of the Astyanax mexicanus of the Rio Subterráneo cave. This cave hosts a hybrid population presenting a wide range of troglomorphic and epigean mixed phenotypes. During a behavioral test performed in situ in the cave, a striking correlation was observed between the absence of eyes and an increased attraction to food extract. In addition, eyeless troglomorphic fish possessed significantly larger naris size than their eyed, nontroglomorphic counterparts.ConclusionsOur findings suggest that chemosensory capabilities might have evolved in cave-dwelling Astyanax mexicanus and that modulation of naris size might at least partially underlie this likely adaptive change.

One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L

The outermost floral whorl, composed of sepals, is generally thought to function in the protection of reproductive tissues. In the plant family Lamiaceae, sepals are fused into a tube that is densely covered by hairs for mechanical defence and contains secondary metabolites for chemical defence against insects and abiotic stresses. Despite the importance of this tissue in plant fitness, virtually no study has addressed the basic aspects of sepal development and functioning. Because of its large size and its impressive metabolic activity (both in terms of quantity and diversity of secondary metabolites), we have used clary sage calyx as a model system to generate the first high throughput sequencing of the transcriptome of an angiosperm calyx. We applied massive parallel 454 pyrosequencing technology to a normalized cDNA extract and unveiled potential candidate genes for all steps of secondary metabolite pathways (phenylpropanoids and terpenoids). It also proved efficient in predicting the expression of large numbers of transcription factors and, with the use of bioinformatics tools, it predicted in the same sequencing run the presence of a novel class of gene transcription regulatory elements, miRNAs, without the need to generate a separate miRNA library. In our clary sage EST library, 18 conserved miRNAs were predicted. Among them, 15 were present in most studied plant species while the others were only shared with limited or discrete plant lineages. A separate data mining of the same clary sage EST library suggested the presence of 19 potential target genes to the 18 predicted conserved miRNAs. These coded for only 6 transcription factors or F-box proteins, 11 metabolism or abiotic stress response related proteins and 2 products with no known predicted function. All in all, this study provides novel genomic information on an angiosperm calyx and an experimental framework to predict in a single step metabolic pathway enzymes and regulator genes including miRNAs.

Differential accumulation of volatile terpene and terpene synthase mRNAs during lavender (Lavandula angustifolia and L. x intermedia) inflorescence development

Despite the commercial importance of Lavandula angustifolia Mill. and L. x intermedia Emeric ex Loisel floral essential oils (EOs), no information is currently available on potential changes in individual volatile organic compound (VOC) content during inflorescence development. Calyces were found to be the main sites of VOC accumulation. The 20 most abundant VOCs could be separated into three sub-groups according to their patterns of change in concentration The three groups of VOCs sequentially dominated the global scent bouquet of inflorescences, the transition between the first and second groups occurring around the opening of the first flower of the inflorescence and the one between the second and third groups at the start of seed set. Changes in calyx VOC accumulation were linked to the developmental stage of individual flowers. Leaves accumulated a smaller number of VOCs which were a subset of those seen in preflowering inflorescences. Their nature and content remained constant during the growing season. Quantitative real time polymerase chain reaction assessments of the expression of two terpene synthase (TPS) genes, LaLIMS and LaLINS, revealed similar trends between their patterns of expression and those of their VOC products. Molecular and chemical analyses suggest that changes in TPS expression occur during lavender inflorescence development and lead to changes in EO composition. Both molecular data and terpene analysis support the findings that changes in biosynthesis of terpene occurred during inflorescence development.

Essential oils from wild populations of Algerian Lavandula stoechas L.: composition, chemical variability, and in vitro biological properties.

In an effort to develop local productions of aromatic and medicinal plants, a comprehensive assessment of the composition and biological activities of the essential oils (EOs) extracted from the aerial flowering parts of wild growing Lavandula stoechas L. collected from eleven different locations in northern Algeria was performed. The oils were characterized by GC‐FID and GC/MS analyses, and 121 compounds were identified, accounting for 69.88–91.2% of the total oil compositions. The eleven oils greatly differed in their compositions, since only 66 compounds were common to all oils. Major EO components were fenchone (2; 11.27–37.48%), camphor (3, 1.94–21.8%), 1,8‐cineole (1; 0.16–8.71%), and viridiflorol (10; 2.89–7.38%). The assessed in vitro biological properties demonstrated that the DPPH‐based radical‐scavenging activities and the inhibition of the β‐carotene/linoleic acid‐based lipid oxidation differed by an eight‐fold factor between the most and the least active oils and were linked to different sets of molecules in the different EOs. The eleven EOs exhibited good antimicrobial activities against most of the 16 tested strains of bacteria, filamentous fungi, and yeasts, with minimum inhibitory concentrations (MICs) ranging from 0.16 to 11.90 mg/ml.

Lens defects in Astyanax mexicanus Cavefish: Evolution of crystallins and a role for alphaA-crystallin

The fish Astyanax mexicanus presents, within the same species, populations of river‐dwelling surface fish (SF) and blind cave‐living fish. In cavefish (CF), the eyes develop almost normally during embryogenesis. But 40 h after fertilization, the lens enters apoptosis, triggering the progressive degeneration of the entire eye. Before apoptosis, the CF lens expresses early differentiation factors correctly. Here, we searched for possible late differentiation defects that would be causal in CF lens degeneration. We reasoned that crystallins, the major lens structural proteins, could be defective or misregulated. We surveyed the CF and SF transcriptomes and uncovered 14 Astyanax crystallins from the beta, gamma, lambda, mu, and zeta families. These proteins are less polymorphic and accumulate more fixed mutations, some at highly conserved positions, in CF than in SF, suggesting relaxed selection at these loci in CF. In situ hybridizations and qPCR show that crybb1c, crybgx, crygm5 are expressed at much lower levels or are not expressed in the CF lens. For the best crystallin candidates, we tested a potential causal role in CF lens apoptosis. Crybgx, crybb1c (not expressed in CF from very early on), and cryaa (previously shown to be faintly expressed in CF) failed to induce any defect when knocked‐down in zebrafish embryos. However, the anti‐apoptotic cryaa protected lens cells from apoptosis when reexpressed by transgenesis in CF, suggesting a cell‐autonomous effect of cryaa on lens cell survival. Altogether, these data suggest that crystallin sequence evolution and expression defects may contribute to the loss of eyes in CF.

Detection of flavokavins (A, B, C) in cultivars of kava (Piper methysticum) using high performance thin layer chromatography (HPTLC)

Kava (Piper methysticum) is used to prepare the traditional beverage of the Pacific islands. In Europe, kava has been suspected to cause hepatoxicity with flavokavin B (FKB) considered as a possible factor. The present study describes an HPTLC protocol for rapid screening of samples. The objectives are: to detect the presence of flavokavins in extracts and to compare the FKB levels in different cultivars. Overall, 172 samples originating from four cultivars groups (noble, medicinal, two-days and wichmannii), were analysed. Results indicate that the ratio FKB/kavalactones is much higher in two-days (0.39) and wichmannii (0.32) compared to nobles (0.09) and medicinal cultivars (0.10). For each group, the ratios flavokavins/kavalactones do not change significantly between roots, stumps or basal stems and among clones, indicating that they are genetically controlled. This protocol has good accuracy and is cost efficient for routine analysis. We discuss how it could be used for quality control.