Laurent Bonneau

Starvation-induced expression of autophagy-related genes in Arabidopsis

Background information. Autophagy is a catabolic process for degradation of cytoplasmic components in the vacuolar apparatus. A genome‐wide survey recently showed evolutionary conservation among autophagy genes in yeast, mammals and plants. To elucidate the molecular and subcellular machinery responsible for the sequestration and subsequent digestion of intracellular material in plants, we utilized a combination of morphological and molecular methods (confocal laser‐scanning microscopy, transmission electron microscopy and real‐time PCR respectively).

Combined phosphate and nitrogen limitation generates a nutrient stress transcriptome favorable for arbuscular mycorrhizal symbiosis in Medicago truncatula

Arbuscular mycorrhizal (AM) symbiosis is stimulated by phosphorus (P) limitation and contributes to P and nitrogen (N) acquisition. However, the effects of combined P and N limitation on AM formation are largely unknown. Medicago truncatula plants were cultivated in the presence or absence of Rhizophagus irregularis (formerly Glomus intraradices) in P-limited (LP), N-limited (LN) or combined P- and N-limited (LPN) conditions, and compared with plants grown in sufficient P and N. The highest AM formation was observed in LPN, linked to systemic signaling by the plant nutrient status. Plant free phosphate concentrations were higher in LPN than in LP, as a result of cross-talk between P and N. Transcriptome analyses suggest that LPN induces the activation of NADPH oxidases in roots, concomitant with an altered profile of plant defense genes and a coordinate increase in the expression of genes involved in the methylerythritol phosphate and isoprenoid-derived pathways, including strigolactone synthesis genes. Taken together, these results suggest that low P and N fertilization systemically induces a physiological state of plants favorable for AM symbiosis despite their higher P status. Our findings highlight the importance of the plant nutrient status in controlling plant-fungus interaction.

Polyamines and related enzymes in rice seeds differing in germination potential

In ungerminated rice seeds, (Japonica rice variety, CV Tapei 309), the content of free amines (putrescine, spermidine, spermine, tyramine) was higher in seed lots having a low germination frequency compared to those with high germination potential. Conversely, amine conjugates (di-feruloylputrescine, di-feruloylspermidine, diferuloyldiaminopropane and feruloyltyramine) decreased with loss of viability. Thus, these compounds appeared to constitute biochemical markers of seed viability. In seeds with high germination potential, conjugates decreased drastically during germination, with an early and rapid increase in free amines (putrescine, spermidine, tyramine). Arginine decarboxylase (ADC) activity was highest during the germination of high germination potential seeds, its activity gradually declining with loss of viability and being closely correlated with agmatine content. The polyamine biosynthetic inhibitors (α-DL-difluoromethylarginine, DFMA, a specific and irreversible inhibitor of ADC; α-DL-difluoromethylornithine, DFMO, a specific irreversible inhibitor of ornithine decarboxylase (ODC); cyclohexylammonium sulfate, CHA, inhibitor of spermidine synthase) neither depleted putrescine and spermidine levels nor inhibited germination in high germination potential seeds. In low germination potential seeds, the germination process was inhibited by DFMA or CHA. Application of agmatine resulted in a reversal of inhibition. DFMA inhibited ADC activity in both categories of seeds. In low germination potential seeds treated with CHA no ADC activity was found. These results suggest that amines are involved in the germination process of rice seeds. It appears that amine conjugates may serve as a storage form of amines which, upon enzymatic hydrolysis, could supply the cell with an additional amine reserve and influence cell division and/or cell elongation.

Control of in vitro somatic embryogenesis of the spindle tree (Euonymus europaeus L.) by the sugar type and the osmotic potential of the culture medium

Abstract In vitro somatic embryogenesis was achieved from zygotic embryo explants of a woody angiosperm species, the spindle tree, cultivated on various culture media differing in their sugar type and concentration, or in the applied osmotic potential. The highest frequency of somatic embryogenesis was obtained with a 350 mM sucrose, or a 89 mM glucose concentration in the culture medium. Experiments with culture media differing only in osmotic potential indicated that a minimal threshold osmotic potential is required to stimulate the emergence of somatic embryos. Elevated concentrations of glucose have an inhibitory effect, independent of their osmotic effect, while elevated concentrations of sucrose mainly act osmotically, stimulating the emergence of numerous somatic embryos.

Somatic embryogenesis and plant regeneration in a woody species: the European Spindle Tree (Euonymus europaeus L.)

Somatic embryogenesis and subsequent plant regeneration of Euonymus europaeus L (European Spindle Tree) were obtained from square pieces of mature zygotic embryos with an intervening callus phase. Callus and somatic embryos were induced using a Murashige and Skoogs semi-solid basal medium supplemented with several combinations of auxins and cytokinins. The greatest number of somatic embryos was obtained with a continuous exposure to 22.8 μM indoleacetic acid and 0.046 μM kinetin. The frequency of somatic embryogenesis from zygotic embryos depends on the cold conservation time of seeds. The embryos frequently germinated on the same medium. Further development of somatic embryos into plantlets was achieved on a medium devoid of growth regulators.

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of α-difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.

Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells.

The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of reactive oxygen species, together with an increase in both medium alkalinization and conductivity. Pharmacological inhibition studies suggest that these signaling events may be regulated by phosphorylations and free calcium. By conducting FRAP experiments using the di-4-ANEPPDHQ probe and spectrofluorimetry using the Laurdan probe, we provide evidence for a filipin-induced increase in PM viscosity that is also regulated by phosphorylations. We conclude that filipin triggers ligand-independent signaling responses in plant cells. The present findings strongly suggest that changes in PM sterol availability could act as a sensor of the modifications of cell environment in plants leading to adaptive cell responses through regulated signaling processes.

Polyamine metabolism during seedling development in rice

The main free amines identified during growth and development of rice seedlings were agmatine, putrescine, spermidine, diaminopropane and tyramine. Amine composition differed according to tissue and stages of development. Conjugated amines were only found in roots. We present evidence that arginine decarboxylase (ADC) regulates putrescine during the development of rice seedlings. When ADC action was blocked by DFMA (α-DL-difluoromethylarginine, a specific irreversible inhibitor of ADC), polyamine titers and seedling development were diminished; when agmatine or putrescine was added, normal polyamine titers and growth were restored. The effects of DFMA were concentration dependent. DFMO (α-DL-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase or ODC) promoted growth and development at concentrations below 2 mM. This effect was probably related to its unexplained, but consistently observed slight enhancement of rice ADC. When the increase in the concentration of spermidine was prevented by CHA (cyclohexylammonium sulfate), the number of roots increased and the increase in length of leaves and roots was strongly inhibited. The addition of exogenous spermidine at the time of treatment with CHA reversed the inhibition by CHA.

The Medicago truncatula hypermycorrhizal B9 mutant displays an altered response to phosphate and is more susceptible to Aphanomyces euteiches

Inorganic phosphate (Pi) plays a key role in the development of arbuscular mycorrhizal (AM) symbiosis, which is favoured when Pi is limiting in the environment. We have characterized the Medicago truncatula hypermycorrhizal B9 mutant for its response to limiting (P/10) and replete (P2) Pi. On P2, mycorrhization was significantly higher in B9 plants than in wild-type (WT). The B9 mutant displayed hallmarks of Pi-limited plants, including higher levels of anthocyanins and lower concentrations of Pi in shoots than WT plants. Transcriptome analyses of roots of WT and B9 plants cultivated on P2 or on P/10 confirmed the Pi-limited profile of the mutant on P2 and highlighted its altered response to Pi on P/10. Furthermore, the B9 mutant displayed a higher expression of defence/stress-related genes and was more susceptible to infection by the root oomycete pathogen Aphanomyces euteiches than WT plants. We propose that the hypermycorrhizal phenotype of the B9 mutant is linked to its Pi-limited status favouring AM symbiosis in contrast to WT plants in Pi-replete conditions, and discuss the possible links between the altered response of the B9 mutant to Pi, mycorrhization and infection by A. euteiches.

Environmental and Biochemical Factors Controlling the in Vitro Emergence of Somatic Embryos in European Spindle Tree (Euonymus europaeus L)

The European spindle tree (Euonymus europaeus L. Celastraceae) is a shrubby tree widely distributed in temperate regions of western European countries from the northern part of France to St. Petersburg and the western part of Russia. The Celastraceae comprises 55 genera, including at least 850 known species of trees and shrubs which are found in tropical and temperate regions. Various secondary metabolites are elaborated in the family including steroids, triterpenoids, sesquiterpene, peptide ore alkaloids. The spindle tree is commonly found in quickset hedges and small broad-leaved forests when the soil is deep, moist, clayey and chalky. The leaves are relatively small (3–4 cm long) simple and are finely dentate. The green bark of young branches is characterised by three to four parallel and longitudinal dark fine straight lines. It bears pink berries (capsules) each containing four orange-colored seeds (5–6 mm diameter). The fruits produce a toxic alkaloid (evonymin) and are poisonous to humans. In Burgundy, this shrubby tree occurs as a wild plant and is the only species of this genera. Ecotypes have never been reported. The spindle tree is naturally propagated by seeds.