The necrogenic bacterium Erwinia amylovora responsible for the fire blight disease causes cell death in apple tissues to enrich intercellular spaces with nutrients. Apple leaves contain large amounts of dihydrochalcones (DHCs), including phloridzin and its aglycone phloretin. Previous work showed an important decrease in the constitutive DHCs stock in infected leaves, probably caused by transformation reactions during the infection process. At least two flavonoid transformation pathways have been described so far: deglucosylation and oxidation. The aim of the present study was to determine whether DHCs are differentially converted in two apple genotypes displaying contrasted susceptibilities to the disease. Different analyses were performed: i) enzymatic activity assays in infected leaves, ii) identification/quantification of end-products obtained after in vitro enzymatic reactions with DHCs, iii) evaluation of the bactericidal activity of end-products. The results of the enzymatic assays showed that deglucosylation was dominant over oxidation in the susceptible genotype MM106 while the opposite was observed in the resistant genotype Evereste. These data were confirmed by LC-UV/Vis-MS analysis of in vitro reaction mixtures, especially because higher levels of o-quinoid oxidation products of phloretin were measured by using the enzymatic extracts of Evereste infected leaves. Their presence correlated well with a strong bactericidal activity of the reaction mixtures. Thus, our results suggest that a differential transformation of DHCs occur in apple genotypes with a potential involvement in the establishment of the susceptibility or the resistance to fire blight, through the release of glucose or of highly bactericidal compounds respectively.

Flavonoids, like other metabolites synthesized via the phenylpropanoid pathway, possess a wide range of biological activities including functions in plant development and its interaction with the environment. Dihydrochalcones (mainly phloridzin, sieboldin, trilobatin, phloretin) represent the major flavonoid subgroup in apple green tissues. Although this class of phenolic compounds is found in very large amounts in some tissues (≈200mg/g of leaf DW), their physiological significance remains unclear. In the present study, we highlight their tissue-specific localization in young growing shoots suggesting a specific role in important physiological processes, most notably in response to biotic stress. Indeed, dihydrochalcones could constitute a basal defense, in particular phloretin which exhibits a strong broad-range bactericidal and fungicidal activity. Our results also indicate that sieboldin forms complexes with iron with strong affinity, reinforcing its antioxidant properties and conferring to this dihydrochalcone a potential for iron seclusion and/or storage. The importance of localization and biochemical properties of dihydrochalcones are discussed in view of the apple tree defense strategy against both biotic and abiotic stresses.

Fire blight is a bacterial disease of Maloideae caused by Erwinia amylovora (Ea). This necrogenic enterobacterium uses a type III secretion system (T3SS) to inject type III effectors into the plant cells to cause disease on its susceptible hosts, including economically important crops like apple and pear. The expressions of marker genes of the salicylic acid (SA) and jasmonic acid (JA) defense regulation pathways were monitored by RT-qPCR in leaves of two apple genotypes, one susceptible and one resistant, challenged with a wild type strain, a T3SS-deficient strain or water. The transcriptional data taken together with hormone level measurements indicated that the SA pathway was similarly induced in both apple genotypes during infection by Ea. On the contrary, the data clearly showed a strong T3SS-dependent down-regulation of the JA pathway in leaves of the susceptible genotype but not in those of the resistant one. Accordingly, methyl-jasmonate treated susceptible plants displayed an increased resistance to Ea. Bacterial mutant analysis indicated that JA manipulation by Ea mainly relies on the type III effector DspA/E. Taken together, our data suggest that the T3SS-dependent down-regulation of the JA pathway is a critical step in the infection process of Malus spp. by Ea.

Climate change is projected to have a significant ecological impact on natural ecosystems, most notably through direct and indirect modifications of local precipitation regimes. In addition, anthropic activities such as the removal of vegetation, soil proofing due to building, the absence of storm drains and crop over-irrigation will all increase the occurrence of flooding. As a result, forest species, and more specifically trees, will increasingly be exposed to soil waterlogging. It is now well established that such flooding events can lead to changes in forest distribution and composition. For such reasons, it is becoming increasingly important to study forest ecosystems and more particularly the adaptive potential of tree species to better understand the ecological plasticity of forest communities to environmental modifications.

Nitric oxide (NO) is a gaseous free radical recognized as a ubiquitous signal transducer that contributes to various biological processes in animals. It exerts most of its effects by regulating the activities of various proteins including Ca2+ channels, protein kinases and transcription factors. In plants, studies conducted over the past 10 years revealed that NO also functions as an endogenous mediator in diverse physiological processes ranging from root development to stomatal closure. Its biological role as an intracellular plant messenger molecule, however, remains poorly understood. Here, we review the molecular basis of NO signalling in animals and discuss current knowledge of NO signalling in plants, focusing on its interplay with Ca2+, protein kinases and reactive oxygen species which are well established as widespread key regulators of signal transduction.

Soil flooding is an environmental constraint that is increasingly important for forest ecosystems, affecting tree growth and regeneration. As a result, selection pressure will alter forest diversity and distribution by favouring tree species tolerant of soil oxygen deprivation. Sessile and pedunculate oaks are the most abundant oak species and they exhibit a strong differential tolerance to waterlogging. In order to gain some understanding of the mechanisms of tolerance of both species to hypoxia, we undertook the characterization of the physiological, morphological, cellular and molecular responses of both species to flooding stress. Our results indicate that pedunculate oak, the more tolerant species, succeeded in maintaining its growth, water status and photosynthetic activity at a higher level than sessile oak. Furthermore, pedunculate oak developed aerenchyma in its root cortex as well as adventitious roots. The later exhibited a strong accumulation of class1 non-symbiotic haemoglobin localized by in situ hybridization in the protoderm and in some cortical cells. In conclusion, the higher tolerance of pedunculate oak to flooding was associated with an enhanced capacity to maintain photosynthesis and water homeostasis, coupled with the development of adaptive features (aerenchyma, adventitious roots) and with a higher expression of non-symbiotic haemoglobin in the roots.

Apple (Malus x domestica) leaf phenolics, in particular the three major dihydrochalcones, phloridzin, trilobatin and sieboldin, were studied for their potential contribution to the resistance to Erwinia amylovora, the causative agent of fire blight. The constitutive phenolic compositions of part of a progeny resulting from a cross between two apple genotypes, displaying contrasted susceptibilities to fire blight, were investigated by RP-HPLC-DAD. With regards to the major dihydrochalcones, offsprings were found to display the parent profile: either phloridzin alone or a combination of phloridzin, trilobatin and sieboldin. Despite a strong antioxidant activity that could interfere with the oxidative burst triggered by the bacteria, sieboldin did not segregate with resistance even though it was strongly associated with a high constitutive antioxidant capacity of leaf extracts. Several hydroxycinnamic acids and flavonols were identified in the progeny but profiles were very similar. However, concentrations of several flavonols positively correlated with resistance in the genotypes that possessed trilobatin and sieboldin. The major dihydrochalcones that represent more than 200 mg/g of leaf DW are not directly responsible for resistance to fire blight in this system. In addition, a high constitutive antioxidant activity is unlikely to be sufficient to block bacterial spread. (C) 2011 Elsevier B.V. All rights reserved.

We reported on the transit and survival of 6 potentially pathogenic bacterial populations in a schist aquifer beneath the Etueffont landfill (France). Total coliforms, Escherichia coli, Enterococci, Pseudomonas aeruginosa, Salmonella and Staphylococcus aureus were monitored for 15months in groundwater and leachate and coupled to tracer tests in an attempt to identify the source of contamination. The results showed the absence of S. aureus and Salmonella. The monitoring of piezometer 30 (PZ30) located downstream from the landfill highlighted leachate infiltrations into the substrate. Groundwater analysis showed high levels of faecal bacteria in the underground environment (20,000CFU100mL−1 for total coliforms, 15,199CFU100mL−1 for E. coli and 3290CFU100mL−1 for Enterococci). Data from tracer tests indicated that bacteria originated from the septic tank of the transfer station and part of these bacteria transited through waste. Bacterial density was lower in leachates than in groundwater sampled from PZ30, except for P. aeruginosa which seemed to take advantage of adverse environmental conditions. The landfill, closed since 2002, was not a source of faecal bacteria which appeared to be able to survive in the schist substrate, and may be considered as good markers of recent faecal contamination.

Flavonoids are a group of polyphenol compounds with known antioxidant activities. Among them, dihydrochalcones are mainly found in apple leaves (Malus domestica). Glycosylated dihydrochalcones were previously found in large amounts in leaves of two genotypes of Malus with contrasting resistance to fire blight, a bacterial disease caused by Erwinia amylovora. In the present study we demonstrate that soluble polyphenol patterns comprised phloridzin alone or in combination with two additional dihydrochalcones, identified as sieboldin and trilobatin. Presence of sieboldin in young leaves correlated well with a high 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. Moreover, these leaves displayed enhanced tolerance to paraquat, a photooxidative-stress generating herbicide. Interestingly, phloridzin had a high activity in the oxygen radical absorbance capacity (ORAC) assay, but its presence alone in leaves did not correlate with tolerance to paraquat. In order to further characterise the activity of these compounds, we tested their ability to prevent oxidative-dependent formation of advanced glycation end-products (AGEs) and phenylephrine-induced contraction of isolated rat mesenteric arteries. The antioxidant capacity of sieboldin was clearly demonstrated by showing that this compound (i) prevented vasoconstriction and (ii) inhibited AGEs formation. Both assays provided interesting information concerning a potential use of sieboldin as a therapeutic. Hence, our results strongly argue for a bioactivity of dihydrochalcones as functional antioxidants in the resistance of Malus leaves to oxidative stress. In addition, we demonstrate for the first time that sieboldin is a powerful multipotent antioxidant, effective in preventing physiopathological processes. Further work should aim at demonstrating the potential use of this compound as a therapeutic in treating free radical-involving diseases.

The cellular and molecular adaptations of non-model woody species to environmental changes are still poorly understood. We have cloned and characterised a novel non-symbiotic hemoglobin from oak roots (QpHb1) which exhibits a specific cellular distribution in the root. The QpHb1 gene is strongly expressed in the protoderm and the protoxylem cells in two Quercus species (Q. petraea and Q. robur) with contrasting adaptive potential to drought and flooding. The constitutive expression of QpHb1 in both oak species in specific root tissues combined with the reported presence of nitric oxide in the same tissues and its potential for protein S-nitrosylation could support a role for non-symbiotic hemoglobins in signalling changes in the root environment and/or in controlling some aspects of root development.

The cellular and molecular adaptations of non-model woody species to environmental changes are still poorly understood. We have cloned and characterised a novel non-symbiotic hemoglobin from oak roots (QpHb1) which exhibits a specific cellular distribution in the root. The QpHb1 gene is strongly expressed in the protoderm and the protoxylem cells in two Quercus species (Q. petraea and Q. robur) with contrasting adaptive potential to drought and flooding. The constitutive expression of QpHb1 in both oak species in specific root tissues combined with the reported presence of nitric oxide in the same tissues and its potential for protein S-nitrosylation could support a role for non-symbiotic hemoglobins in signalling changes in the root environment and/or in controlling some aspects of root development.

Plants are constantly exposed to changes in environmental conditions. During periods of stress, the cellular redox homeostasis is altered as a result of reactive oxygen species accumulation. The change in redox is responsible for the symptoms commonly observed during periods of stress and reflects the phytotoxic nature of oxygen radical accumulation. However, oxygen radicals have recently been identified as key actors in the response to stress and their role as secondary messengers is now clearly established. The identification of their role in gene regulation has allowed one to identify them as key regulators in the induction and execution of programmed cell death typically observed during developmental processes as well as during stress responses. This review presents recent advances in the characterisation of the role of reactive oxygen species in plants.

Under natural conditions, plants are frequently exposed to transient or permanent soil waterlogging. Flooding drastically influences the soil physico-chemical properties, most notably soil redox potential, pH and O2 level. Thus, conditions of hypoxia or anoxia are commonly encountered by plant root systems. These O2 restrictive conditions dramatically affect plant growth, development and survival. One of the best characterised plant responses to soil waterlogging is the metabolic switch from aerobic respiration to anaerobic fermentation. In fact, most proteins induced during hypoxic conditions are enzymes involved in the establishment of this fermentative pathway. Because the plant cells need to keep a continuous ATP supply, the use of alternative electron acceptors and/or alternative pathways may be key elements of survival under soil waterlogging. The plant response may also include a reduction in stomatal conductance and photosynthesis, as well as root hydraulic conductivity. These physiological modifications may in turn affect carbohydrate reserves and translocation. In fact, efficient use of carbohydrates may discriminate between tolerant and intolerant species. Other observed adaptations include morphological changes which comprise the formation of hypertrophied lenticels, the initiation of adventitious roots and/or the development of aerenchyma. Our knowledge of the basic adaptive mechanisms of plants to soil waterlogging has benefited from large scale genomic and proteomic approaches, however, the diversity of the adaptive responses involved underlines the difficulty when studying this stress. This update reviews our current comprehension of the metabolic, physiological and morphological responses and adaptations of plants to soil waterlogging.

This study presents the isolation and characterization of a novel nonsymbiotic Hb gene from sessile oak (Quercus petraea) seedlings, herein designated QpHb1. The cellular and tissue expression of QpHb1 was analysed by Northern blotting and in situ hybridization. The encoded protein was predicted to consist of 161 amino acid residues, and shares 71 and 51% amino acid sequence identity with the Arabidopsis class 1 and 2 nonsymbiotic Hb, respectively. Northern blot analysis revealed that QpHb1 was strongly expressed in roots. Spatial expression analysis of QpHb1 in the root apical region of sessile oak by in situ hybridization indicated that transcripts were mostly abundant in protoxylem cell initials, some cortical cells and the protoderm. In addition, when comparing the expression profile of QpHb1 in sessile and pedunculate oak (Quercus robur), two species with contrasted hypoxia tolerance, the transcript level of QpHb1 rose early in the most flood-tolerant species, pedunculate oak, during root submergence. The spatial-temporal expression of QpHb1 suggests that this gene could participate in perception and signalling during hypoxia.

Paradoxically, death is an integral part of life. Cell death is essential for growth and development of eukaryotes, by maintaining tissue and organ homeostasis in concert with cell proliferation, growth, and differentiation. Until recently, the wide variety of cell death types reported in the

Sessile oak is a species of great ecological and silvicultural importance in Europe; however, increased frequency and severity of flooding of forested areas pose a threat to its regeneration. We monitored water relations, root anatomical changes and the expression of two calmodulin genes (QpCaM) in sessile oak seedlings during a 14-day flooding treatment. The response followed two characteristic sequences. The first phase, in response to between 1 h and 3 days of flooding, was characterized by a dramatic but transient decline in water relations parameters followed by a recovery towards control values with no noticeable change in root cell morphology. During the second phase, in response to 3 to 14 days of flooding, water relations parameters gradually and continuously declined and hypertrophied lenticels developed at the base of the shoot. Concurrently, root cortical cells became larger and less spherical and the root cortex more porous. These cellular changes were accompanied by a transient rise in root transcript levels of QpCaM-2. We conclude that sessile oak seedlings are capable of withstanding a 3-day period of flooding without significant morphological alterations. In contrast, exposure to flooding for more than 3 days resulted in anatomical and morphological changes in the root system. These changes are, however, insufficient to provide sessile oak with long-term tolerance to flooding.

Nitric oxide (NO) and hydrogen peroxide (H2O2) are regulatory molecules in various developmental processes and stress responses. Tobacco (Nicotiana tabacum) leaves exposed to moderate high light dramatically potentiated NO-mediated cell death in catalase-deficient (CAT1AS) but not in wild-type plants, providing genetic evidence for a partnership between NO and H2O2 during the induction of programmed cell death. With this experimental model system, the specific impact on gene expression was characterized by either NO or H2O2 alone or both molecules combined. By means of genome-wide cDNA-amplified fragment length polymorphism analysis, transcriptional changes were compared in high light-treated CAT1AS and wild-type leaves treated with or without the NO donor sodium nitroprusside. Differential gene expression was detected for 214 of the approximately 8,000 transcript fragments examined. For 108 fragments, sequence analysis revealed homology to genes with a role in signal transduction, defense response, hormone interplay, proteolysis, transport, and metabolism. Surprisingly, only 16 genes were specifically induced by the combined action of NO and H2O2, whereas the majority were regulated by either of them alone. At least seven transcription factors were mutually up-regulated, indicating significant overlap between NO and H2O2 signaling pathways. These results consolidate significant cross-talk between NO and H2O2, provide new insight into the early transcriptional response of plants to increased NO and H2O2 levels, and identify target genes of the combined action of NO and H2O2 during the induction of plant cell death.

We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species (ROS)- mediated lipid peroxidation from 9- and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H2O2-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H2O2 plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H2O2 and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H2O2 and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.