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Regulatory role of hydrogen cyanide in dormancy removal of sunflower (Helianthus annuus L.) embryos

Abstract : Embryonic dormancy of freshly harvested sunflower (Helianthus annuus L.) seeds is expressed by their poor germination at low temperatures (10-15 °C). The research topic of the present work concerns the involvement of different endogenous factors such as hydrogen cyanide (HCN), reactive oxygen species (ROS), ethylene (C2H4) and abscisic acid (ABA) in the regulation of dormancy of sunflower embryos. The aims of the present work were: (i) to identify the effect of gaseous HCN on breaking of sunflower embryo dormancy, (ii) to determine whether HCN improving action is related with the ethylene synthesis or signaling pathways, and (iii) to investigate the mechanisms of action of HCN, particularly whether ROS are involved in this phenomenon. A short pre-treatment (3 hours) of dormant sunflower embryos by gaseous HCN released their dormancy and allowed their subsequent germination at 10-15 °C, temperatures which prevented germination of dormant embryos. This stimulatory effect did not result from an activation of the pentose phosphate or the cyanide-insensitive pathways, since other respiratory inhibitors such as sodium azide (NaN3) and salicylhydroxamic acid (SHAM), did not allow the germination of dormant embryos at 10-15 °C. In addition, b-cyanoalanine synthase (b-CAS) activity measurements excluded the role of HCN in stimulating amino acids synthesis (as alanine, serine, asparagine) during dormancy alleviation, by improving the activity of b-CAS, since no significant difference in the activity of this enzyme was observed in axes isolated from control non-treated and HCN or MV treated embryos. To identify the process by which dormancy of sunflower (non-cyanogenic) embryos is broken by HCN, the present work was focussed on its possible interaction with reactive oxygen species in this phenomenon. After HCN treatment, ROS (H2O2 - hydrogen peroxide and O2 .- - superoxide anions) progressively accumulated in cells of embryonic axis. However, application of HCN had only a slight effect on the in vitro activity of the main PDF stworzony przez wersje demonstracyjna pdfFactory Pro www.pdffactory.pl/ Summary in English 8 enzymes involved in the antioxidant defence system, such as catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), since these enzymes were potentially active. Imbibition of embryos in the presence of methylviologen (MV), as well as menadione (MD), ROS generating compounds, had the same stimulatory effect on germination as HCN, suggesting that HCN probably improved germination through accumulation of ROS. Increase in H2O2 and O2 .- concentration in the embryonic axis occurred concomitantly with an oxidation (carbonylation) of specific proteins. Obtained data allow us to propose a novel explanation for cyanide mode of action in alleviation of sunflower embryo dormancy, which involves ROS production and targets changes in protein oxidation patterns. In the group of carbonylated proteins specifically associated with breaking of dormancy by HCN and MV, some are identified as epoxide hydrolase and alcohol dehydrogenase (Oracz et al. 2007). In order to determine whether the stimulatory effect of HCN during germination of sunflower embryo might be related to ethylene biosynthesis and ethylene signaling pathway, transcription of genes involved in these proceses was studied simultaneously to HCN and MV treatments. The stimulatory effect of HCN and MV on germination of sunflower embryos, is not associated with a modification of transcription of genes involved in ethylene production such as ACS (ACC synthase) and ACO (ACC oxidase), and does not result from a stimulation of ethylene production. It was also demonstrated, that HCN and MV markedly increased the transcription of ERF1 (ethylene response factor 1) but not that of ETR2 (ethylene response 2) and CTR1 (constitutive triple response 1). This suggests that the transduction pathway of cyanide and MV, initiates the transcription factor ERF1, but through different mechanism of that involved by ethylene. The putative molecular mode of action of HCN on sunflower embryo germination is discussed, and its possible relationships with ROS and ethylene synthesis and signaling pathways are also evoked. The first time are presented the direct experimental data concerning HCN effect on the expression of genes related to (i) ROS production [NADPHox (NADPH oxidase), AO1 and AO2 (amine oxidase 1 and 2), POX (peroxidase)], (ii) ROS signaling [Ser/ThrPK (Ser/Thr protein kinase), MAPK6 (mitogen activated protein kinase 6), PTP (protein tyrosine phosphatase), CaM (calmodulin)], (iii) ethylene formation (ACS, ACO) and (iv) signal transduction induced by ethylene (ETR2, CTR1, ERF1). It is demonstrated, that HCN did not change the transcription of genes involved in ROS production (NADPHox, AO1, AO2, POX), and also did not seem to require the typical elements of ROS signaling pathway (MAPK6, SerThrPK, PTP, CaM), but can induce the physiological response, using some elements of other signal transduction pathways (e.g. ERF1 in ethylene signaling pathway). This work proposes HCN as a signal molecule, which via ROS as a second messenger, regulates many pathways leading in consequence to alleviation of embryo dormancy and stimulation of germination. These pathways probably do not operate independently, but rather are linked together in a network of interactions.
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  • HAL Id : tel-00812705, version 1

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Krystyna Oracz. Regulatory role of hydrogen cyanide in dormancy removal of sunflower (Helianthus annuus L.) embryos. Plant breeding. Université Pierre et Marie Curie - Paris VI, 2008. English. ⟨NNT : 2008PA066082⟩. ⟨tel-00812705⟩

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