Renata Bogatek

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.

Allelopathic effects of sunflower extracts on mustard seed germination and seedling growth

The allelopathic potential of two cultivars of sunflower (Helianthus annuus L.) Lech and Ogrodowy was studied. Leaf aqueous extracts of sunflower at 2.5, 5, and 10 % (m/v) concentrations were applied to determine their effect on mustard (Sinapis alba) seed germination and seedlings growth under laboratory conditions. Increasing concentration of aqueous extracts of sunflower inhibited seed germination, up to almost complete failure of germination, observed in the presence of 10 % extract from sunflower cv. Ogrodowy. The decrease in germinability was well correlated with increased membrane deterioration, assayed as electrical conductivity and enhanced lipid peroxidation, detected as increased malondialdehyde content.

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.

ROS and Phytohormons in Plant-Plant Allelopathic Interaction

Allelopathy refers to plant-plant interference mediated mostly by plant released products of secondary metabolism. It was recently suggested that allelochamicals may influence growth of neighboring plants by induction of oxidative stress. We have focused on the role of reactive oxygen species (ROS) and phytohormons (ABA and ethylene) in the biochemical and molecular regulation of plant response to sunflower phytotoxins.

Impact of Sunflower (Helianthus annuus L.) Extracts Upon Reserve Mobilization and Energy Metabolism in Germinating Mustard (Sinapis alba L.) Seeds

One commonly observed effect of phytotoxic compounds is the inhibition or delay of germination of sensitive seeds. Mustard (Sinapis alba L.) seeds were incubated with aqueous extracts of sunflower (Helianthus annuus L.) leaves. Although sunflower phytotoxins did not influence seed viability, extracts completely inhibited seed germination. Inhibition of germination was associated with alterations in reserve mobilization and generation of energy in the catabolic phase of germination. Degradation of lipids was suppressed by sunflower foliar extracts resulting in insufficient carbohydrate supply. The lack of respiratory substrates and decrease in energy (ATP) generation resulted in suppression of the anabolic phase of seed germination and ultimately growth inhibition.

Citral Induces Auxin and Ethylene-Mediated Malformations and Arrests Cell Division in Arabidopsis thaliana Roots

Citral is a linear monoterpene which is present, as a volatile component, in the essential oil of several different aromatic plants. Previous studies have demonstrated the ability of citral to alter the mitotic microtubules of plant cells, especially at low concentrations. The changes to the microtubules may be due to the compound acting directly on the treated root and coleoptile cells or to indirect action through certain phytohormones. This study, performed in Arabidopsis thaliana, analysed the short-term effects of citral on the auxin content and mitotic cells, and the long-term effects of these alterations on root development and ethylene levels. The results of this study show that citral alters auxin content and cell division and has a strong long-term disorganising effect on cell ultra-structure in A. thaliana seedlings. Its effects on cell division, the thickening of the cell wall, the reduction in intercellular communication, and the absence of root hairs confirm that citral is a strong phytotoxic compound, which has persistent effects on root development.

Allelochemicals as Bioherbicides — Present and Perspectives

Since the first implementation of synthetic herbicides in crop protection systems, weeds have continuously developed resistance. As a main reason of such evolution, long-lasting exploi‐ tation of herbicides with one target site in plants is considered. This has been the case with the first widely-used triazine herbicides, photosynthesis inhibitors, which have effectively eliminated a wide range of weeds. Unfortunately, inappropriate adjustment of herbicides to weed species occupying fields, application of herbicides at the incorrect developmental stage and in unsuitable weather conditions have contributed to the accumulation of active com‐ pounds in the soil, accumulation of weed species and acceleration evolution of resistant biotypes [1]. To date, there have been 211 species and 393 biotypes of herbicide resistant weeds identified [2]. Most of them are resistant to B, C1 and A groups of herbicides, inhibitors of: acetolactate synthase (ALS), photosystem II and acetyl CoA carboxylase, respectively. Ten species pose the biggest threat for crops due to causing yield losses, including the most important herbicide-resistant species which are characterized by multiple resistances: rigid ryegrass (Lolium rigidum Gaud.), wild oat (Avena fatua L.) and redroot pigweed (Amaranthus retroflexus L.).

Dormancy alleviation by NO or HCN leading to decline of protein carbonylation levels in apple (Malus domestica Borkh.) embryos.

Deep dormancy of apple (Malus domestica Borkh.) embryos can be overcome by short-term pre-treatment with nitric oxide (NO) or hydrogen cyanide (HCN). Dormancy alleviation of embryos modulated by NO or HCN and the first step of germination depend on temporary increased production of reactive oxygen species (ROS). Direct oxidative attack on some amino acid residues or secondary reactions via reactive carbohydrates and lipids can lead to the formation of protein carbonyl derivatives. Protein carbonylation is a widely accepted covalent and irreversible modification resulting in inhibition or alteration of enzyme/protein activities. It also increases the susceptibility of proteins to proteolytic degradation. The aim of this work was to investigate protein carbonylation in germinating apple embryos, the dormancy of which was removed by pre-treatment with NO or HCN donors. It was performed using a quantitative spectrophotometric method, while patterns of carbonylated protein in embryo axes were analyzed by immunochemical techniques. The highest concentration of protein carbonyl groups was observed in dormant embryos. It declined in germinating embryos pre-treated with NO or HCN, suggesting elevated degradation of modified proteins during seedling formation. A decrease in the concentration of carbonylated proteins was accompanied by modification in proteolytic activity in germinating apple embryos. A strict correlation between the level of protein carbonyl groups and cotyledon growth and greening was detected. Moreover, direct in vitro carbonylation of BSA treated with NO or HCN donors was analyzed, showing action of both signaling molecules as protein oxidation agents.

Loss of Gravitropism in Farnesene-Treated Arabidopsis Is Due to Microtubule Malformations Related to Hormonal and ROS Unbalance

Mode of action of farnesene, a volatile sesquiterpene commonly found in the essential oils of several plants, was deeply studied on the model species Arabidopsis thaliana. The effects of farnesene on the Arabidopsis root morphology were evaluated by different microscopic techniques. As well, microtubules immunolabeling, phytohormone measurements and ROS staining helped us to elucidate the single or multi-modes of action of this sesquiterpene on plant metabolism. Farnesene-treated roots showed a strong growth inhibition and marked modifications on morphology, important tissue alterations, cellular damages and anisotropic growth. Left-handed growth of farnesene-treated roots, reverted by taxol (a known microtubule stabilizer), was related to microtubule condensation and disorganization. As well, the inhibition of primary root growth, lateral root number, lateral root length, and both root hairs length and density could be explained by the strong increment in ethylene production and auxin content detected in farnesene-treated seedlings. Microtubule alteration and hormonal unbalance appear as important components in the mode of action of farnesene and confirm the strong phytotoxic potential of this sesquiterpene.

Phytotoxic cyanamide affects maize (Zea mays) root growth and root tip function: from structure to gene expression

Cyanamide (CA) is a phytotoxic compound produced by four Fabaceae species: hairy vetch, bird vetch, purple vetch and black locust. Its toxicity is due to complex activity that involves the modification of both cellular structures and physiological processes. To date, CA has been investigated mainly in dicot plants. The goal of this study was to investigate the effects of CA in the restriction of the root growth of maize (Zea mays), representing the monocot species. CA (3mM) reduced the number of border cells in the root tips of maize seedlings and degraded their protoplasts. However, CA did not induce any significant changes in the organelle structure of other root cells, apart from increased vacuolization. CA toxicity was also demonstrated by its effect on cell cycle activity, endoreduplication intensity, and modifications of cyclins CycA2, CycD2, and histone HisH3 gene expression. In contrast, the arrangement of microtubules was not altered by CA. Treatment of maize seedlings with CA did not completely arrest mitotic activity, although the frequency of dividing cells was reduced. Furthermore, prolonged CA treatment increased the proportion of endopolyploid cells in the root tip. Cytological malformations were accompanied by an induction of oxidative stress in root cells, which manifested as enhanced accumulation of H2O2. Exposure of maize seedlings to CA resulted in an increased concentration of auxin and stimulated ethylene emission. Taken together, these findings suggested that the inhibition of root growth by CA may be a consequence of stress-induced morphogenic responses.