Urszula Krasuska

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.

Polyamines and Nitric Oxide Link in Regulation of Dormancy Removal and Germination of Apple (Malus domestica Borkh.) Embryos

Polyamines (PAs) belong to plant growth regulators and in complex with classical phytohormones take part in regulation of seed dormancy and germination. Although the impact of reactive oxygen (ROS) and nitrogen (RNS) species on seed germination is well described, the cross talk of PAs with ROS/RNS has never been analyzed. Due to the close connection of PAs and ethylene biosynthetic pathways to arginine (Arg)-dependent NO biosynthesis we investigated production of nitric oxide (NO), peroxynitrite (ONOO−) and the level of O2•− or H2O2 in apple embryos, germination of which was PA regulated. PAs: putrescine (Put) and spermidine (Spd) in contrast to spermine (Spm) stimulated germination of apple embryos. Among amino acids, stimulation of germination was observed in Arg and ornithine (Orn) only. Dormancy removal of embryos by PAs was associated with increased accumulation of H2O2 and O2•− in embryonic axes. At the same stage of completion of sensu stricto germination the stimulatory effect of PAs (Put and Spd) and amino acids, mainly Arg and Orn, was accompanied by enhanced NO and ONOO− production in embryonic axis. The beneficial effect of PAs (Put and Spd) and their precursors on germination of apple embryos was removed by NO scavenging, suggesting a crucial role of NO in termination of embryo germination and radicle growth. Moreover, activity of polyamine oxidase in embryo axes was greatly enhanced by embryo fumigation with NO. Our data demonstrate the interplay of RNS/ROS with PAs and point to NO action as an integrator of endogenous signals activating germination.

Allelopathic Compounds as Oxidative Stress Agents: Yes or NO

Allelopathy phenomenon describes both positive and negative interactions between plants through chemicals released into environment. Inhibition of growth of various organs and delay or restriction of seed germination are most frequently observed effects of toxicity of allelochemicals, although some of them, when applied in minimal concentrations, lead to the effect of hormesis (stimulation of seed germination or seedling growth). Plants subjected to some allelopathic compounds respond by induction of oxidative stress, manifested as overproduction of reactive oxygen species (ROS), alterations in cellular antioxidant system (activity of catalase, superoxide dismutase, and enzymes of Halliwell–Asada–Foyer cycle, the level of ascorbate and glutathione). Due to differences in plant sensitivity to allelochemicals, and considering a wide spectrum of concentration of already tested compounds, it is hardly to believe that induction of oxidative stress is a global and uniform reaction to all allelopathins. There are several examples of allelopathic compounds that act as antioxidant rather then prooxidant agents. In the chapter we present the data describing induction of alterations in ROS production or/and metabolism in acceptor plants by various allelochemicals or extracts of other plants releasing allelochemicals into environment. Special attention is paid on oxidative deterioration induced by essential oils. Accumulation of ROS and modification of growth and/or development of organs of acceptor plants as induced by allelotoxins were correlated with “stress induced morphogenic responses” (SIMRs), modification of phytohormonal balance and factors crucial for cell wall formation. It was proposed a link between phytotoxicity of allelochemicals and activation of ROS-mediated transduction pathways, involving also calcium dependent intracellular signaling.

Canavanine Alters ROS/RNS Level and Leads to Post-translational Modification of Proteins in Roots of Tomato Seedlings.

Canavanine (CAN), a structural analog of arginine (Arg), is used as a selective inhibitor of inducible NOS in mammals. CAN is incorporated into proteins’ structure in the place of Arg, leading to the formation of aberrant compounds. This non-protein amino acid is found in legumes, e.g., Canavalia ensiformis (L.) DC. or Sutherlandia frutescens (L.) R.Br. and acts as a strong toxin against herbivores or plants. Tomato (Solanum lycopersicum L.) seedlings were treated for 24–72 h with CAN (10 or 50 μM) inhibiting root growth by 50 or 100%, without lethal effect. We determined ROS level/production in root extracts, fluorescence of DAF-FM and APF derivatives corresponding to RNS level in roots of tomato seedlings and linked CAN-induced restriction of root growth to the post-translational modifications (PTMs) of proteins: carbonylation and nitration. Both PTMs are stable markers of nitro-oxidative stress, regarded as the plant’s secondary response to phytotoxins. CAN enhanced H2O2 content and superoxide radicals generation in extracts of tomato roots and stimulated formation of protein carbonyl groups. An elevated level of carbonylated proteins was characteristic for the plants after 72 h of the culture, mainly for the roots exposed to 10 μM CAN. The proteolytic activity was stimulated by tested non-protein amino acid. CAN treatment led to decline of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives. Short-term exposure of tomato seedlings to CAN lowered the protein nitration level. Activity of peroxidase, polyamine oxidase and NADPH oxidase, enzymes acting as modulators of H2O2 concentration and governing root architecture and growth were determined. Activities of all enzymes were stimulated by CAN, but no strict CAN concentration dependence was observed. We conclude, that although CAN treatment led to a decline in the nitric oxide level, PTMs observed in roots of plants exposed to CAN are linked rather to the formation of carbonyl groups than to nitration, and are detected particularly after 24 h. Thus, oxidative stress and oxidative modifications of proteins seems to be of significant importance in the rapid response of plants to CAN.

“Nitrosative Door” in Seed Dormancy Alleviation and Germination

Seed germination is the first phase of plant ontogeny, therefore it determinates “to be or not to be” of the plant organism. As other physiological processes, it is regulated by environmental and endogenous signals. Seed germination is usually discussed in the context of seed dormancy, as although of their opposite nature, both processes are difficult to separate. Nitric oxide (NO)—gas naturally occurring in the environment is commonly known as dormancy breaking agent but also as a molecule synthesized in active, germinating embryos. The aim of our work is to review the recent knowledge of the role of NO in regulation of seed dormancy alleviation and germination. We have focused mostly on NO biosynthesis in germinating seeds, its potential mode of action and cross-talk with classical phytohormones and plant growth regulators that determine seed dormancy and germination. In our discussion, we refer to the model of “nitrosative door” describing the signaling function of NO and other reactive nitrogen species (RNS) in seed physiology. NO is suggested to play the role of “golden key,” which placed into the keyhole, allows to unlock “doors” existing in quiescent embryos and enable their transition from dormant to germination state.

Nitric oxide-polyamines cross-talk during dormancy release and germination of apple embryos

Nitric oxide (NO) and polyamines (PAs) belong to plant growth and development regulators. These compounds play a key role in numerous physiological processes e.g. seed germination. Based on the suggestion of overlapping of NO and PAs biosynthetic pathways, we demonstrated a cross-talk of NO and PAs in regulation of embryonic dormancy release. The aim of the work was to investigate an impact of PAs (Put, Spd and Spm) or NO short-term fumigation on nitrite, urea, Arg and ornithine (Orn) content, NO synthase-like (NOS-like) and arginase activity in axes of apple (Malus domestica Borkh.) embryos during dormancy alleviation and at the stage of termination of germination sensu stricto. NO, Put/Spd induced dormancy breakage and germination of apple embryos corresponded to stimulation of urea cycle and high free Arg pool in seedlings roots. After two days of the culture Put and Spd stimulated Arg dependent NO formation, inhibition of which was observed after Spm application. Put or Spd application as well as NO short-term pretreatment of apple embryos influenced level of ubiquitin-conjugated proteins. Higher abundance of such modified proteins correlated well to the declined content of nitrated proteins, suggesting their important role in regulation of embryo germination. NO led to stimulation of embryos germination by increasing level of free PAs (mostly Put). While transcriptomic approach showed down regulation of Spm synthesis and up-regulation of Spm degradation by NO, confirming negative role of Spm over-accumulation in embryo dormancy removal. Our data clearly indicate positive relationship of NO-Put/Spd acting as dormancy removing factors.

Switch from heterotrophy to autotrophy of apple cotyledons depends on NO signal

AbstractMain conclusionNO accelerates transition of germinated embryos from heterotrophy to autotrophy by stimulation of chloroplasts maturation. NO-mediated autotrophy of apple seedlings correlates to increased content of RuBisCO small subunit and improvement of parameters of chlorophyllafluorescence. Nitric oxide (NO) acts as signaling molecule involved in regulation of various physiological processes in plants, although its involvement in cotyledons greening is poorly recognized. To identify the importance of NO signal for plant growth and development we investigated the effects of short-term application of NO at various developmental stages of seedlings of apple (Malus domestica Borkh.) on cotyledons’ chlorophyll a to b ratio, chlorophyll a fluorescence, photosynthetic activity, carbohydrates and RuBisCO both subunits content. NO-dependent biochemical alterations were linked to cytological observation of developing plastids in cotyledons of apple plants. Abnormal plantlets developing from dormant apple embryos are characterized by anatomical malformations of cotyledons. Short-term pre-treatment with NO of isolated embryos or seedlings with developmental anomalies resulted in formation of plants with cotyledons of equal size and chlorophyll content; these responses were blocked by NO scavenger. NO independently of time point of application accelerated embryos transition from heterotrophy to autotrophy by stimulation of photosynthetic activity, improvement of parameters of chlorophyll a fluorescence (Fv/Fm, Fv/F0) and increased content of RuBisCO small subunit. Further analysis showed that NO application modified glucose and hydrogen peroxide concentration in cotyledons. Beneficial effect of NO on development of seedlings without any abnormalities was manifested at ultrastructural level by decline in amount of proplastids and induction of formation and maturation of chloroplasts. Our data suggest that progress of autotrophy of young seedlings is governed by NO acting as stimulator of chloroplast-to-nucleus signaling.