Hayat El-Maarouf-Bouteau

The Mechanisms Involved in Seed Dormancy Alleviation by Hydrogen Cyanide Unravel the Role of Reactive Oxygen Species as Key Factors of Cellular Signaling during Germination

The physiological dormancy of sunflower (Helianthus annuus) embryos can be overcome during dry storage (after-ripening) or by applying exogenous ethylene or hydrogen cyanide (HCN) during imbibition. The aim of this work was to provide a comprehensive model, based on oxidative signaling by reactive oxygen species (ROS), for explaining the cellular mode of action of HCN in dormancy alleviation. Beneficial HCN effect on germination of dormant embryos is associated with a marked increase in hydrogen peroxide and superoxide anion generation in the embryonic axes. It is mimicked by the ROS-generating compounds methylviologen and menadione but suppressed by ROS scavengers. This increase results from an inhibition of catalase and superoxide dismutase activities and also involves activation of NADPH oxidase. However, it is not related to lipid reserve degradation or gluconeogenesis and not associated with marked changes in the cellular redox status controlled by the glutathione/glutathione disulfide couple. The expression of genes related to ROS production (NADPHox, POX, AO1, and AO2) and signaling (MAPK6, Ser/ThrPK, CaM, and PTP) is differentially affected by dormancy alleviation either during after-ripening or by HCN treatment, and the effect of cyanide on gene expression is likely to be mediated by ROS. It is also demonstrated that HCN and ROS both activate similarly ERF1, a component of the ethylene signaling pathway. We propose that ROS play a key role in the control of sunflower seed germination and are second messengers of cyanide in seed dormancy release.

Oxidative signaling in seed germination and dormancy

Reactive Oxygen Species (ROS) play a key role in various events of seed life. In orthodox seeds, ROS are produced from embryogenesis to germination, i.e. in metabolically active cells, but also in quiescent dry tissues during after ripening and storage, owing various mechanisms depending on the seed moisture content. Although ROS have been up to now widely considered as detrimental to seeds, recent advances in plant physiology signaling pathways has lead to reconsider their role. ROS accumulation can therefore be also beneficial for seed germination and seedling growth by regulating cellular growth, ensuring a protection against pathogens or controlling the cell redox status. ROS probably also act as a positive signal in seed dormancy release. They interact with abscisic acid and gibberellins transduction pathway and are likely to control numerous transcription factors and properties of specific protein through their carbonylation.

DspA/E, a Type III Effector Essential for Erwinia amylovora Pathogenicity and Growth In Planta, Induces Cell Death in Host Apple and Nonhost Tobacco Plants

Erwinia amylovora is responsible for fire blight, a necrotic disease of apples and pears. E. amylovora relies on a type III secretion system (TTSS) to induce disease on hosts and hypersensitive response (HR) on nonhost plants. The DspA/E protein is essential for E. amylovora pathogenicity and is secreted via the TTSS in vitro. DspA/E belongs to a type III effector family that is conserved in several phytopathogenic bacteria. In E. amylovora, DspA/E has been implicated in the generation of an oxidative stress during disease and the suppression of callose deposition. We investigated the fate of DspA/E in planta. DspA/E delivered artificially to apple or tobacco cells by agroinfection induced necrotic symptoms, indicating that DspA/E was probably injected via the TTSS. We confirmed that DspA/E acts as a major cell-death inducer during disease and HR, because the dspA/E mutant is severely impaired in its ability to induce electrolyte leakage in apple and tobacco leaves. Expression of the defense marker gene PR1 was delayed when dspA/E was transiently expressed in tobacco, suggesting that DspA/E-mediated necrosis may be associated with an alteration of defense responses.

Release of sunflower seed dormancy by cyanide: cross-talk with ethylene signalling pathway

Freshly harvested sunflower (Helianthus annuus L.) seeds are considered to be dormant because they fail to germinate at relatively low temperatures (10 °C). This dormancy results mainly from an embryo dormancy and disappears during dry storage. Although endogenous ethylene is known to be involved in sunflower seed alleviation of dormancy, little attention had been paid to the possible role of cyanide, which is produced by the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene, in this process. The aims of this work were to investigate whether exogenous cyanide could improve the germination of dormant sunflower seeds and to elucidate its putative mechanisms of action. Naked dormant seeds became able to germinate at 10 °C when they were incubated in the presence of 1 mM gaseous cyanide. Other respiratory inhibitors showed that this effect did not result from an activation of the pentose phosphate pathway or the cyanide-insensitive pathway. Cyanide stimulated germination of dormant seeds in the presence of inhibitors of ethylene biosynthesis, but its improving effect required functional ethylene receptors. It did not significantly affect ethylene production and the expression of genes involved in ethylene biosynthesis or in the first steps of ethylene signalling pathway. However, the expression of the transcription factor Ethylene Response Factor 1 (ERF1) was markedly stimulated in the presence of gaseous cyanide. It is proposed that the mode of action of cyanide in sunflower seed dormancy alleviation does not involve ethylene production and that ERF1 is a common component of the ethylene and cyanide signalling pathways.

Role of relative humidity, temperature, and water status in dormancy alleviation of sunflower seeds during dry after-ripening

The effect of various combinations of temperature and relative humidity on dormancy alleviation of sunflower seeds during dry after-ripening was investigated. The rate of dormancy alleviation depended on both temperature and embryo moisture content (MC). Below an embryo MC of 0.1 g H2O g−1 dw, dormancy release was faster at 15 °C than at higher temperatures. This suggests that dormancy release at low MC was associated with negative activation energy, supported by Arrhenius plots, and low Q10 values. At higher MC, the rate of dormancy alleviation increased with temperature, correlating well with the temperature dependence of biochemical processes. These findings suggests the involvement of two distinct cellular mechanisms in dormancy release; non-enzymatic below 0.1 g H2O g−1 dw and associated with active metabolism above this value. The effects of temperature on seed dormancy release above the threshold MC were analysed using a population-based thermal time approach and a model predicting the rate of dormancy alleviation is provided. Sunflower embryo dormancy release was effective at temperatures above 8 °C (the base temperature for after-ripening, TbAR, was 8.17 °C), and the higher the after-ripening temperature above this threshold value, the higher was the rate of dormancy loss. Thermodynamic analyses of water sorption isotherms revealed that dormancy release was associated with less bound water and increased molecular mobility within the embryonic axes but not the cotyledons. It is proposed that the changes in water binding properties result from oxidative processes and can, in turn, allow metabolic activities.

DNA alteration and programmed cell death during ageing of sunflower seed

Sunflower (Helianthus annuus L.) seed viability is affected by moisture content (MC) during ageing and is related to accumulation of hydrogen peroxide and changes in energy metabolism. The aim of the present work was to investigate the effect of ageing on DNA alteration events by RAPD (random amplification of polymorphic DNA) analysis and to determine whether loss of seed viability might correspond to a controlled programmed cell death (PCD). Ageing of sunflower seeds was carried out at 35 °C for 7 d at different MCs. The higher the MC, the lower was the seed viability. RAPD analysis showed that DNA alterations occurred during ageing especially in seeds containing a high MC. In addition, PCD, as revealed by DNA fragmentation and TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick-end labelling) assay, was detected in aged seeds at MCs which resulted in ∼50% seed viability. At the cellular level, TUNEL assay and propidium iodide staining showed that cell death concerns all the cells of the embryonic axis. The quantification of the adenylate pool highlights mitochondrial dysfunction in aged seeds containing a high MC. The involvement of oxidative burst, mitochondria dysfunction, and PCD in seed loss of viability is proposed.

Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination

Sunflower (Helianthus annuus L.) seed dormancy is regulated by reactive oxygen species (ROS) and can be alleviated by incubating dormant embryos in the presence of methylviologen (MV), a ROS-generating compound. Ethylene alleviates sunflower seed dormancy whereas abscisic acid (ABA) represses germination. The purposes of this study were to identify the molecular basis of ROS effect on seed germination and to investigate their possible relationship with hormone signalling pathways. Ethylene treatment provoked ROS generation in embryonic axis whereas ABA had no effect on their production. The beneficial effect of ethylene on germination was lowered in the presence of antioxidant compounds, and MV suppressed the inhibitory effect of ABA. MV treatment did not alter significantly ethylene nor ABA production during seed imbibition. Microarray analysis showed that MV treatment triggered differential expression of 120 probe sets (59 more abundant and 61 less abundant genes), and most of the identified transcripts were related to cell signalling components. Many transcripts less represented in MV-treated seeds were involved in ABA signalling, thus suggesting an interaction between ROS and ABA signalling pathways at the transcriptional level. Altogether, these results shed new light on the crosstalk between ROS and plant hormones in seed germination.

Post-transcriptional regulation of GORK channels by superoxide anion contributes to increases in outward-rectifying K⁺ currents.

· Ion fluxes are ubiquitous processes in the plant and animal kingdoms, controlled by fine-tuned regulations of ion channel activity. Yet the mechanism that cells employ to achieve the modification of ion homeostasis at the molecular level still remains unclear. This is especially true when it comes to the mechanisms that lead to cell death. · In this study, Arabidopsis thaliana cells were exposed to ozone (O₃). Ion flux variations were analyzed by electrophysiological measurements and their transcriptional regulation by RT-PCR. Reactive oxygen species (ROS) generation was quantified by luminescence techniques and caspase-like activities were investigated by laser confocal microscopy. · We highlighted the delayed activation of K(+) outward-rectifying currents after an O₃ -induced oxidative stress leading to programmed cell death (PCD). Caspase-like activities are detected under O₃ exposure and could be decreased by K(+) channel blocker. Molecular experiments revealed that the sustained activation of K(+) outward current could be the result of an unexpected O₂ ·⁻ post-transcriptional regulation of the guard cell outward-rectifying K(+) (GORK) channels. · This consists of a likely new mode of regulating the processing of the GORK mRNA, in a ROS-dependent manner, to allow sustained K(+) effluxes during PCD. These data provide new mechanistic insights into K(+) channel regulation during an oxidative stress response.

Arabidopsis thaliana cells: a model to evaluate the virulence of Pectobacterium carotovorum.

Pectobacterium carotovorum are economically important plant pathogens that cause plant soft rot. These enterobacteria display high diversity world-wide. Their pathogenesis depends on production and secretion of virulence factors such as plant cell wall-degrading enzymes, type III effectors, a necrosis-inducing protein, and a secreted virulence factor from Xanthomonas spp., which are tightly regulated by quorum sensing. Pectobacterium carotovorum also present pathogen-associated molecular patterns that could participate in their pathogenicity. In this study, by using suspension cells of Arabidopsis thaliana, we correlate plant cell death and pectate lyase activities during coinfection with different P. carotovorum strains. When comparing soft rot symptoms induced on potato slices with pectate lyase activities and plant cell death observed during coculture with Arabidopsis thaliana cells, the order of strain virulence was found to be the same. Therefore, Arabidopsis thaliana cells could be an alternative tool to evaluate rapidly and efficiently the virulence of different P. carotovorum strains.

A diffusible signal from germinating Orobanche ramosa elicits early defense responses in suspension-cultured Arabidopsis thaliana.

In plant/parasitic plant interaction, little is known about the host plant response before the establishment of the parasite within the host. In the present work, we focused on host responses to parasitic plant, O. ramosa in the early stage of infection. We used a co-culture system of A. thaliana suspension cells and O. ramosa germinated-seeds to avoid parasite attachment. We showed that O. ramosa induced H2O2 generation and camalexin synthesis by A. thaliana followed by a drastic increase in cell death. We further demonstrated that a heat sensitive diffusible signal is responsible for this cell death. These data indicate that recognition of O. ramosa occurs before the attachment of the parasite and initiates plant defence responses.