Do Reactive Oxygen and Nitrogen Species Have a Similar Effect on Digestive Processes in Carnivorous Nepenthes Plants and Humans?

Carnivorous plants attract animals, trap and kill them, and absorb nutrients from the digested bodies. This unusual (for autotrophs) type of nutrient acquisition evolved through the conversion of photosynthetically active leaves into specialised organs commonly called traps. The genus Nepenthes (pitcher plants) consists of approximately 169 species belonging to the group of carnivorous plants. Pitcher plants are characterised by specialised passive traps filled with a digestive fluid. The digestion that occurs inside the traps of carnivorous plants depends on the activities of many enzymes. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) also participate in the digestive process, but their action is poorly recognised. ROS and RNS, named together as RONS, exhibit concentration-dependent bimodal functions (toxic or signalling). They act as antimicrobial agents, participate in protein modification, and are components of signal transduction cascades. In the human stomach, ROS are considered as the cause of different diseases. RNS have multifaceted functions in the gastrointestinal tract, with both positive and negative impacts on digestion. This review describes the documented and potential impacts of RONS on the digestion in pitcher plant traps, which may be considered as an external stomach.

Is the Phytotoxic Effect of Digestive Fluid of Nepenthes x ventrata on Tomato Related to Reactive Oxygen Species?

The digestive fluid of pitcher plants is a rich source of enzymes and secondary metabolites, but its impact on higher plant growth and development remains unknown. The aim of the study was to determine the phytotoxicity of the digestive fluid of the pitcher plant (Nepenthes x ventrata) on the germination of tomato (Solanum lycopersicum L.) seeds, elongation growth and cell viability of roots of tomato seedlings. The digestive fluid was collected from pitchers before feeding and four days after feeding; the pH and electrical conductivity of the fluid were determined. Undiluted and 50% fluids were used in the study. An inhibition of germination of tomato seeds, by around 30% and 55%, was respectively observed in 50% and 100% digestive fluids collected before and after feeding. Digestive fluid did not affect the root growth of tomato seedlings; a slight (6%) inhibition was only observed after the application of 100% digestive fluid from an unfed trap. The roots of the tomato seedlings treated with undiluted fluid were characterized by reduced cell viability. Reactive oxygen species (H2O2 and O2•-) were mainly localized in the root apex regardless of the used phytotoxic cocktail, and did not differ in comparison to control plants.

Nitric Oxide in Seed Biology.

Nitric oxide (NO) has been recognized as a gasotransmitter in the mainstream of plant research since the beginning of the 21st century. It is produced in plant tissue and the environment. It influences plant physiology during every ontogenetic stage from seed germination to plant senescence. In this review, we demonstrate the increased interest in NO as a regulatory molecule in combination with other signalling molecules and phytohormones in the information network of plant cells. This work is a summary of the current knowledge on NO action in seeds, starting from seed pretreatment techniques applied to increase seed quality. We describe mode of action of NO in the regulation of seed dormancy, germination, and aging. During each stage of seed physiology, NO appears to act as a key agent with a predominantly beneficial effect.

Nitric oxide-an antidote to seed aging modifies meta-tyrosine content and expression of aging-linked genes in apple embryos.

Short-term (3 h) treatment of embryos isolated from accelerated aged apple seeds (Malus domestica Borkh.) with nitric oxide (NO) partially reduced the effects of aging. The study aimed to investigate the impact of the short-term NO treatment of embryos isolated from apple seeds subjected to accelerated aging on the expression of genes potentially linked to the regulation of seed aging. Apple seeds were artificially aged for 7, 14, or 21 days. Then, the embryos were isolated from the seeds, treated with NO, and cultured for 48 h. Progression of seed aging was associated with the decreased transcript levels of most of the analyzed genes (Lea1, Lea2a, Lea4, Hsp70b, Hsp20a, Hsp20b, ClpB1, ClpB4, Cpn60a, Cpn60b, Raptor, and Saur). The role of NO in the mitigation of seed aging depended on the duration of the aging. After 7 and 14 days of seed aging, a decreased expression of genes potentially associated with the promotion of aging (Tor, Raptor, Saur) was noted. NO-dependent regulation of seed aging was associated with the stimulation of the expression of genes encoding chaperones and proteins involved in the repair of damaged proteins. After NO application, the greatest upregulation of ClpB, Pimt was noted in the embryos isolated from seeds subjected to 7-day long accelerated aging, Hsp70b, Hsp70c, and Cpn in the embryos of seeds aged for 14 days, and Lea2a in the embryos of seeds after 21 days of aging. We also demonstrated the increased meta-tyrosine concentration depending or in respect the progression of artificial aging, and the NO-induced increased phenylalanine content in seeds artificially aged for 21 days. In the NO-treated embryos of seeds aged for 7 and 21 days, the level of tyrosine was almost doubled compared to the aged tissue. Our data confirmed the usage of meta-tyrosine as a marker of seed aging and indicated that the increased meta-tyrosine/tyrosine ratio could be related to the loss of seed viability.

Toxicity of meta-Tyrosine.

L-Tyrosine (Tyr) is one of the twenty proteinogenic amino acids and also acts as a precursor for secondary metabolites. Tyr is prone to modifications, especially under conditions of cellular redox imbalance. The oxidation of Tyr precursor phenylalanine leads to the formation of Tyr non-proteinogenic isomers, including meta-Tyr (m-Tyr), a marker of oxidative stress. The aim of this review is to summarize the current knowledge on m-Tyr toxicity. The direct m-Tyr mode of action is linked to its incorporation into proteins, resulting in their improper conformation. Furthermore, m-Tyr produced by some plants as an allelochemical impacts the growth and development of neighboring organisms. In plants, the direct harmful effect of m-Tyr is due to its modification of the proteins structure, whereas its indirect action is linked to the disruption of reactive oxygen and nitrogen species metabolism. In humans, the elevated concentration of m-Tyr is characteristic of various diseases and ageing. Indeed, m-Tyr is believed to play an important role in cancer physiology. Thus, since, in animal cells, m-Tyr is formed directly in response to oxidative stress, whereas, in plants, m-Tyr is also synthesized enzymatically and serves as a chemical weapon in plant-plant competition, the general concept of m-Tyr role in living organisms should be specified.

ROS Metabolism Perturbation as an Element of Mode of Action of Allelochemicals.

The allelopathic interaction between plants is one of the elements that influences plant communities. It has been commonly studied by applying tissue extracts onto the acceptors or by treating them with isolated allelotoxins. Despite descriptive observations useful for agricultural practice, data describing the molecular mode of action of allelotoxins cannot be found. Due to the development of -omic techniques, we have an opportunity to investigate specific reactive oxygen species (ROS)-dependent changes in proteome or transcriptome that are induced by allelochemicals. The aim of our review is to summarize data on the ROS-induced modification in acceptor plants in response to allelopathic plants or isolated allelochemicals. We present the idea of how ROS are involved in the hormesis and plant autotoxicity phenomena. As an example of an -omic approach in studies of the mode of action of allelopatic compounds, we describe the influence of meta-tyrosine, an allelochemical exudated from roots of fescues, on nitration-one of nitro-oxidative posttranslational protein modification in the roots of tomato plants. We conclude that ROS overproduction and an induction of oxidative stress are general plants' responses to various allelochemicals, thus modification in ROS metabolisms is regarded as an indirect mode of action of allelochemicals.

Nitric Oxide as a Remedy against Oxidative Damages in Apple Seeds Undergoing Accelerated Ageing.

Seed ageing is associated with a high concentration of reactive oxygen species (ROS). Apple (Malus domestica Borkh.) seeds belong to the orthodox type. Due to a deep dormancy, they may be stored in dry condition at 5 °C for a long time, without viability loss. In the laboratory, artificial ageing of apple seeds is performed by imbibition in wet sand at warm temperature (33 °C). The aim of the work was to study nitric oxide (NO) as a seed vigour preservation agent. Embryos isolated from apple seeds subjected to accelerated ageing for 7, 14, 21 or 40 days were fumigated with NO. Embryo quality was estimated by TTC and MDA tests. ROS level was confirmed by NBT staining. We analysed the alteration in transcript levels of CAT, SOD and POX. NO fumigation of embryos of seeds aged for 21 days stimulated germination and increased ROS level which correlated to the elevated expression of RBOH. The increased total antioxidant capacity after NO fumigation was accompanied by the increased transcript levels of genes encoding enzymatic antioxidants, that could protect against ROS overaccumulation. Moreover, post-aged NO application diminished the nitro-oxidative modification of RNA, proving NO action as a remedy in oxidative remodelling after seeds ageing.

Canavanine Increases the Content of Phenolic Compounds in Tomato (Solanum lycopersicum L.) Roots.

Canavanine (CAN) is a nonproteinogenic amino acid, and its toxicity comes from its utilization instead of arginine in many cellular processes. As presented in previous experiments, supplementation of tomato (Solanum lycopersicum L.) with CAN led to decreased nitric oxide (NO) level and induced secondary oxidative stress. CAN improved total antioxidant capacity in roots, with parallel inhibition of enzymatic antioxidants. The aim of this work was to determine how CAN-dependent limitation of NO emission and reactive oxygen species overproduction impact content, localization, and metabolism of phenolic compounds (PCs) in tomato roots. Tomato seedlings were fed with CAN (10 and 50 µM) for 24 or 72 h. Inhibition of root growth due to CAN supplementation correlated with increased concentration of total PCs; CAN (50 µM) led to the homogeneous accumulation of PCs all over the roots. CAN increased also flavonoids content in root tips. The activity of polyphenol oxidases and phenylalanine ammonia-lyase increased only after prolonged treatment with 50 µM CAN, while expressions of genes encoding these enzymes were modified variously, irrespectively of CAN dosage and duration of the culture. PCs act as the important elements of the cellular antioxidant system under oxidative stress induced by CAN.

Effect of Nitrogen Reactive Compounds on Aging in Seed.

Reactive nitrogen species (RNS) are universal compounds that are constantly present in plant cells. RNS function depends on their actual level (the "nitrosative door" concept), duration of plant exposure to RNS and the context of the exposure. RNS are involved in the nitration of nucleic acids and fatty acids, posttranslational protein modifications (nitration and S-nitrosylation), and modulation of reactive oxygen species metabolism. RNS are regulatory molecules of various physiological processes in plants, including seed formation, maturation, dormancy and germination. The free radical theory of aging, well documented for animals, indicated that RNS participate in the regulation of the life span. Some data point to RNS contribution in preservation of seed vigor and/or regulation of seed longevity. Seed aging is a problem for biologists and agriculture, which could be solved by application of RNS, as a factor that may potentially expand seed vitality resulting in increased germination rate. The review is focused on RNS, particularly nitric oxide contribution to regulation of seed aging.

Peroxynitrite induced signaling pathways in plant response to non-proteinogenic amino acids.

MAIN CONCLUSION: Nitro/oxidative modifications of proteins and RNA nitration resulted from altered peroxynitrite generation are elements of the indirect mode of action of canavanine and meta-tyrosine in plants Environmental conditions and stresses, including supplementation with toxic compounds, are known to impair reactive oxygen (ROS) and reactive nitrogen species (RNS) homeostasis, leading to modification in production of oxidized and nitrated derivatives. The role of nitrated and/or oxidized biotargets differs depending on the stress factors and developmental stage of plants. Canavanine (CAN) and meta-tyrosine (m-Tyr) are non-proteinogenic amino acids (NPAAs). CAN, the structural analog of arginine, is found mostly in seeds of Fabaceae species, as a storage form of nitrogen. In mammalian cells, CAN is used as an anticancer agent due to its inhibitory action on nitric oxide synthesis. m-Tyr is a structural analogue of phenylalanine and an allelochemical found in root exudates of fescues. In animals, m-Tyr is recognized as a marker of oxidative stress. Supplementation of plants with CAN or m-Tyr modify ROS and RNS metabolism. Over the last few years of our research, we have collected the complex data on ROS and RNS metabolism in tomato (Solanum lycopersicum L.) plants exposed to CAN or m-Tyr. In addition, we have shown the level of nitrated RNA (8-Nitro-guanine) in roots of seedlings, stressed by the tested NPAAs. In this review, we describe the model of CAN and m-Tyr mode of action in plants based on modifications of signaling pathways induced by ROS/RNS with a special focus on peroxynitrite induced RNA and protein modifications.

Canavanine-Induced Decrease in Nitric Oxide Synthesis Alters Activity of Antioxidant System but Does Not Impact S-Nitrosoglutathione Catabolism in Tomato Roots.

Canavanine (CAN) is a nonproteinogenic amino acid synthesized in legumes. In mammalians, as arginine analogue, it is an inhibitor of nitric oxide synthase (NOS) activity. The aim of this study was to investigate the impact of CAN-induced nitric oxide level limitation on the antioxidant system and S-nitrosoglutathione (GSNO) metabolism in roots of tomato seedlings. Treatment with CAN (10 or 50 µM) for 24-72 h led to restriction in root growth. Arginine-dependent NOS-like activity was almost completely inhibited, demonstrating direct effect of CAN action. CAN increased total antioxidant capacity and the level of sulphydryl groups. Catalase (CAT) and superoxide dismutase (SOD) activity decreased in CAN exposed roots. CAN supplementation resulted in the decrease of transcript levels of genes coding CAT (with the exception of CAT1). Genes coding SOD (except MnSOD and CuSOD) were upregulated by CAN short treatment; prolonged exposition to 50-µM CAN resulted in downregulation of FeSOD, CuSOD, and SODP-2. Activity of glutathione reductase dropped down after short-term (10-µM CAN) supplementation, while glutathione peroxidase activity was not affected. Transcript levels of glutathione reductase genes declined in response to CAN. Genes coding glutathione peroxidase were upregulated by 50-µM CAN, while 10-µM CAN downregulated GSHPx1. Inhibition of NOS-like activity by CAN resulted in lower GSNO accumulation in root tips. Activity of GSNO reductase was decreased by short-term supplementation with CAN. In contrast, GSNO reductase protein abundance was higher, while transcript levels were slightly altered in roots exposed to CAN. This is the first report on identification of differentially nitrated proteins in response to supplementation with nonproteinogenic amino acid. Among nitrated proteins differentially modified by CAN, seed storage proteins (after short-term CAN treatment) and components of the cellular redox system (after prolonged CAN supplementation) were identified. The findings demonstrate that due to inhibition of NOS-like activity, CAN leads to modification in antioxidant system. Limitation in GSNO level is due to lower nitric oxide formation, while GSNO catabolism is less affected. We demonstrated that monodehydroascorbate reductase, activity of which is inhibited in roots of CAN-treated plants, is the protein preferentially modified by tyrosine nitration.

Nitric Oxide-Induced Dormancy Removal of Apple Embryos Is Linked to Alterations in Expression of Genes Encoding ABA and JA Biosynthetic or Transduction Pathways and RNA Nitration.

Short-term (3 h) treatment of embryos isolated from dormant apple (Malus domestica Borkh.) seeds with NO donors stimulates their transition from dormancy to germination. Seed dormancy is maintained by ABA, while germination is controlled mainly by gibberellins (GAs) and jasmonic acid (JA). NO-induced dormancy removal correlates with low ABA concentration in embryonic axes and reduced embryo sensitivity to ABA. We analyzed the expression of genes encoding key enzymes of ABA degradation (CYP707A1, CYP707A2), biosynthesis (NCED3, NCED9), and elements of the ABA transduction pathway (PYL1, PYL2, RCAR1, RCAR3, PP2CA, ABI1, ABI2, SNRK2, ABI5, AREB3, ABF). A role for JA in the regulation of germination led us to investigate the expression of genes encoding enzymes of JA biosynthesis (AOS1, JMT, JAR1) and the transduction pathway (COI1, MYC2, JAZ3, JAZ12). The expression profiles of the genes were estimated in embryonic axes isolated from dormant or NO fumigated apple embryos. The analyzed genes were differentially regulated during dormancy alleviation, the main modifications in the transcription level were detected for NCED3, NCED9, CYP707A2, RCAR1, ABF, AOS1, JMT, JAR1 and JAZ3. A regulatory role of NO in the removal of seed dormancy is associated with the stimulation of expression of genes related to ABA degradation, down-regulation of genes responsible for ABA synthesis, an increase of expression level of genes engaged in JA synthesis and modification of the expression of genes engaged in signaling pathways of the hormones. To confirm a signaling role of NO during dormancy breakage, an increased RNA nitration level in embryonic axes was demonstrated.

Dormancy removal by cold stratification increases glutathione and S-nitrosoglutathione content in apple seeds.

S-nitrosoglutathione (GSNO), an integral metabolite of nitric oxide (NO) biochemistry is reduced by S-nitrosoglutathione reductase (GSNOR) (EC 1.2.1.46), leading to formation of glutathione in oxidised form (GSSG), further reduced to GSH by glutathione reductase (GR). GSH as a vital antioxidant has a significant role for seed quality and during seed germination. Since early 50th of 20th century it is known that deep dormancy of apple (Malus domestica Borkh.) embryos is removed by 90 days of cold stratification. Our previous studies demonstrated that similar effect is observed after short term (3 h) exposition of isolated embryos to nitric oxide (NO) donors. The aim of our work was to verify the differences in GSNO level and GSNOR activity in embryonic axes isolated after initiation of germination (24 h of imbibition) from dormant embryos (the control) and from 90 days cold stratified seeds. Our data indicated that seed dormancy breakage is accompanied by increased GSNO content and the decrease of GSNOR activity. The abundance of GSNOR protein is similar in both non-dormant and dormant embryonic axes during first hours of water uptake, while GSNOR transcript level increases in non-dormant tissue. Furthermore, in non-dormant embryonic axes we noticed a higher glutathione pool, mostly in its reduced form. These results are linked to the increase of cytosolic GR transcript level and increased enzyme activity in embryonic axes isolated from stratified seeds.

Destabilization of ROS metabolism in tomato roots as a phytotoxic effect of meta-tyrosine.

meta-Tyrosine (m-Tyr) is a non-protein amino acid produced in both plants and animals. Primary mode of action of this phenylalanine analog is its incorporation into protein structure leading to formation of aberrant molecules. An increased level of m-Tyr in animal cells is detected under oxidative stress and during age-related processes characterized by overproduction of reactive oxygen species (ROS). The aim of this study was to link m-Tyr physiological action to disturbances in ROS metabolism in tomato (Solanum lycopersicum L.) seedlings roots. Treatment of tomato seedlings with m-Tyr (50 or 250 µM) for 24-72 h led to inhibition of root growth without a lethal effect. Toxicity of m-Tyr after 72 h was connected with an increase in hydrogen peroxide concentration in roots and ROS leakage into the surrounding medium. On the contrary, membrane permeability and lipid peroxidation in roots were the same as for the control. This was accompanied by a decrease in total antioxidant activity and an increased accumulation of phenolic compounds. Catalase (CAT) activity declined in roots exposed to 50 µM m-Tyr after 24 h while after 72 h activity of this enzyme was inhibited in both treated and non-treated samples. Activities of different superoxide dismutase (SOD) isoforms were similar in m-Tyr stressed roots and in the control. Prolonged culture resulted in decrease of transcript level of genes coding CAT and SOD with the exception of FeSOD. Moreover, m-Tyr increased the level of protein carbonyl groups indicating induction of oxidative stress as a non-direct mode of action.

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.

meta-Tyrosine induces modification of reactive nitrogen species level, protein nitration and nitrosoglutathione reductase in tomato roots.

A non-protein amino acid (NPAA) - meta-Tyrosine (m-Tyr), is a harmful compound produced by fescue roots. Young (3-4 days old) tomato (Solanum lycopersicum L.) seedlings were supplemented for 24-72 h with m-Tyr (50 or 250 µM) inhibiting root growth by 50 or 100%, without lethal effect. Fluorescence of DAF-FM and APF derivatives was determined to show reactive nitrogen species (RNS) localization and level in roots of tomato plants. m-Tyr-induced restriction of root elongation growth was related to formation of nitrated proteins described as content of 3-nitrotyrosine. Supplementation with m-Tyr enhanced superoxide radicals generation in extracts of tomato roots and stimulated protein nitration. It correlated well to increase of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives corresponding respectively to NO and ONOO- formation. Alterations in RNS formation induced by m-Tyr were linked to metabolism of nitrosoglutathione (GSNO). Activity of nitrosoglutatione reductase (GSNOR), catalyzing degradation of GSNO was enhanced by long term plant supplementation with m-Tyr, similarly as protein abundance, while transcripts level were only slightly altered by tested NPAA. We conclude, that although in animal cells m-Tyr is considered as a marker of oxidative stress, its secondary mode of action in tomato plants involves perturbation in RNS formation, alteration in GSNO metabolism and modification of protein nitration level.

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.

Modification of the endogenous NO level influences apple embryos dormancy by alterations of nitrated and biotinylated protein patterns.

MAIN CONCLUSION: NO donors and Arg remove dormancy of apple embryos and stimulate germination. Compounds lowering NO level (cPTIO, L -NAME, CAN) strengthen dormancy. Embryo transition from dormancy state to germination is linked to increased nitric oxide synthase (NOS)-like activity. Germination of embryos is associated with declined level of biotin containing proteins and nitrated proteins in soluble protein fraction of root axis. Pattern of nitrated proteins suggest that storage proteins are putative targets of nitration. Nitric oxide (NO) acts as a key regulatory factor in removal of seed dormancy and is a signal necessary for seed transition from dormant state into germination. Modulation of NO concentration in apple (Malus domestica Borkh.) embryos by NO fumigation, treatment with NO donor (S-nitroso-N-acetyl-D,L-penicillamine, SNAP), application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), N ω-nitro-L-arginine methyl ester (L-NAME), canavanine (CAN) or arginine (Arg) allowed us to investigate the NO impact on seed dormancy status. Arg analogs and NO scavenger strengthened embryo dormancy by lowering reactive nitrogen species level in embryonic axes. This effect was accompanied by strong inhibition of NOS-like activity, without significant influence on tissue NO2 (-) concentration. Germination sensu stricto of apple embryos initiated by dormancy breakage via short term NO treatment or Arg supplementation were linked to a reduced level of biotinylated proteins in root axis. Decrease of total soluble nitrated proteins was observed at the termination of germination sensu stricto. Also modulation of NO tissue status leads to modification in nitrated protein pattern. Among protein bands that correspond to molecular mass of approximately 95 kDa, storage proteins (legumin A-like and seed biotin-containing protein) were identified, and can be considered as good markers for seed dormancy status. Moreover, pattern of nitrated proteins suggest that biotin containing proteins are also targets of nitration.

Toxicity of canavanine in tomato (Solanum lycopersicum L.) roots is due to alterations in RNS, ROS and auxin levels.

Canavanine (CAN) is non-proteinogenic aminoacid and a structural analog of arginine (Arg). Naturally, CAN occurs in legumes e.g. jack bean and is considered as a strong allelochemical. As a selective inhibitor of inducible nitric oxide synthase in mammalians, it could act as a modifier of nitric oxide (NO) concentration in plants. Modifications in the content of endogenous reactive nitrogen species (RNS) and reactive oxygen species (ROS) influence root structure and architecture, being also under hormonal control. The aim of the work was to investigate regulation of root growth in tomato (Solanum lycopersicum L. cv. Malinowy Ozarowski) seedling by application of CAN at concentration (10 and 50 µM) leading to 50% or 100% restriction of root elongation. CAN at higher concentration led to slight DNA fragmentation, increased total RNA and protein level. Decline in total respiration rate after CAN supplementation was not associated with enhanced membrane permeability. Malformations in root morphology (shorter and thicker roots, limited number of lateral roots) were accompanied by modification in NO and ONOO(-) localization; determined mainly in peridermal cells and some border cells. Although, CAN resulted in low RNS production, addition of exogenous NO by usage of NO donors did not reverse its negative effect, nor recovery effect was detected after roots imbibition in Arg. To build up a comprehensive view on mode of action of CAN as root growth inhibitor, it was shown an elevated level of auxin. To summarize, we demonstrated several secondary mode of action of CAN, indicating its toxicity in plants linked to restriction in RNS formation accompanied by simultaneous overaccumulation of ROS.

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

MAIN CONCLUSION: NO 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 chlorophyll a fluorescence. 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 (F v/F m, F v/F 0) 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.