The industrial application and environmental release of nickel oxide NPs (NiO NPs) is increasing, but the details of their relationship with plants are largely unknown. In this work, the cellular, tissue, organ, and molecular level responses of three ecotypes of Ni hyperaccumulator Odontarrhena lesbiaca grown in the presence of high doses of NiO NP (250 mg/L and 500 mg/L) were studied. All three ecotypes showed a similar accumulation of Ni in the presence of nano Ni, and in the case of NiO NPs, the root -to -shoot Ni translocation was slighter compared to the bulk Ni. In all three ecotypes, the walls of the root cells effectively prevented internalization of NiO NPs, providing cellular defense against Ni overload. Exposure to NiO NP led to an increase in cortex thickness and the deposition of lignin-suberin and pectin in roots, serving as a tissue -level defense mechanism against excessive Ni. Exposure to NiO NP did not modify or cause a reduction in some biomass parameters of the Ampeliko and Loutra ecotypes, while it increased all parameters in Olympos. The free salt form of Ni exerted more negative effects on biomass production than the nanoform, and the observed effects of NiO NPs can be attributed to the release of Ni ions. Nitric oxide and peroxynitrite levels were modified by NiO NPs in an ecotype -dependent manner. The changes in the abundance and activity of S-nitrosoglutathione reductase protein triggered by NiO NPs suggest that the enzyme is regulated by NiO NPs at the post -translational level. The NiO NPs slightly intensified protein tyrosine nitration, and the slight differences between the ecotypes were correlated with their biomass production in the presence of NiO NPs. Overall, the Odontarrhena lesbiaca ecotypes exhibited tolerance to NiO NPs at the cellular, tissue, organ/organism and molecular levels, demonstrating various defense mechanisms and changes in the metabolism of reactive nitrogen species metabolism and nitrosative protein modification.

The S-nitrosoglutathione reductase (GSNOR), a key regulator of nitric oxide (NO) signaling, requires zinc (Zn) cofactors for its catalytic activity. Possible changes in GSNOR function and NO signaling due to limited Zn supply and participation of GSNOR in response to Zn deficiency have not yet been examined. The wild-type Arabidopsis thaliana seedlings (Col-0) and the overproducer of GSNOR (35S::FLAG-GSNOR1) were grown in agar medium supplemented with 15 µM (full Zn), 1.5 µM (Zn/10) and 0 µM (Zn0) zinc sulfate. Limited availability of Zn in the nutrient medium during the experimental period resulted in a significantly reduced Zn content in the roots and shoots of Arabidopsis and altered the expression of Zn deficiency marker genes; however, the Zn content did not reflect Zn deficiency in the seedlings. GSNOR has no effect on the Zn limitation-induced reduction in the in planta Zn content. Suboptimal Zn supply results in a smaller but more active GSNOR protein pool in Col-0 A. thaliana. In 35S::FLAG-GSNOR1, overproduction possibly compensates for the negative effects of reduced Zn supply, resulting in unchanged GSNOR expression, protein abundance, and activity. Suboptimal Zn supply alters NO but did not modify peroxynitrite and hydrogen peroxide levels and exerts a pronitrant effect in the plant proteome. Overall, this is the first report to show that limited Zn supply affects the GSNOR enzyme at the gene and protein levels but not at the activity level and modifies reactive species levels and physiological nitroproteome in Arabidopsis.

•Nitric oxide (NO) signaling might be involved in plant responses to suboptimal zinc (Zn) supply•The rate of excess Zn/nano-ZnO-caused nitro-oxidative stress correlates with plant tolerance•Exogenous NO decreases Zn accumulation in Zn/nano ZnO-stressed plants•Exogenous NO up-regulates antioxidant defence in Zn/nano ZnO-stressed plants

Despite being a worldwide problem, responses of crops to zinc (Zn) deficiency is largely unknown. This study examines the effects of limited Zn supply in Brassica napus. The decreased Zn content of the nutrient solution caused reduced tissue Zn concentrations and reduced K, Ca, Mg levels were detected in the leaves. For Ca, Zn, Fe, Mo inhomogeneous distribution was induced by Zn limitation. Suboptimal Zn supply decreased the shoot and root biomass and altered the root structure. The levels of sugars and sugar phosphates (e.g. glucose, glucose-6-phosphate etc.) were decreased suggesting disturbance in carbon metabolism. Limited Zn availability induced organ-dependent superoxide anion and hydrogen peroxide production and triggered altered NADPH oxidase, superoxide dismutase activities, ascorbate peroxidase abundance and ascorbate and glutathione contents indicating oxidative stress. Moreover, nitric oxide, peroxynitrite and S-nitrosoglutathione (GSNO) levels and GSNO reductase gene expression, protein level and activity were modified by Zn limitation. As a consequence, protein tyrosine nitration and protein carbonylation were intensified in B. napus grown with suboptimal Zn supply. Collectively, these results provide the first evidence for Zn deficiency-induced imbalances in nutrient status, sugar contents, reactive oxygen and nitrogen species metabolisms and for the secondary nitro-oxidative stress in sensitive Brassica napus.

Hydrogen sulfide (H2S) has been known for centuries as a toxic gas for animals and human, especially at higher concentrations. Similar to nitric oxide (NO) it has been also demonstrated that lower amounts of H2S may have beneficial effects on plants exposed to several abiotic stress factors such as salinity, drought, heat, or heavy metal stress. In addition, both appear to have multiple physiological functions during plants' life cycles, from seed germination through stomatal movements to senescence. Several studies have tried to assess the relationship between them but there are still many questions to be answered, such as the up- or downstream role in signaling pathways or crosstalk with other molecules (reactive oxygen species, polyamines, etc.). In this chapter we try to give an overview of the cooperation of these remarkably "popular" gasotransmitters in plants under abiotic or biotic stress.

Nitric oxide (NO), strigolactones (SLs) and karrikins (KARs) are bioactive signal molecules controlling root morphology. Our knowledge regarding the signal interplay of NO with SLs or KARs is limit...

Since ZnO nanoparticles (ZnO NPs) are extensively applied engineered nanomaterials, e.g. in cosmetics or therapeutics due to their anticancer or antimicrobial effect, their emission to the environment has grown. Although there are several examples for “green” synthesis of ZnO NPs applying various plants or plant extracts, numerous studies exhibited that plants (including crops) also can be the target of these potentially hazardous agents. The presence of ZnO NPs may have both negative or positive effects on plant growth, physiological and/or biochemical processes depending on concentration or exposure time. Here, we attempt to give an overview of the experimental data published until now about the influence of ZnO NPs on plant physiological and biochemical procedures, and morphological features as well.

Both nitric oxide (NO) and strigolactone (SL) are growth regulating signal components in plants; however, regarding their possible interplay our knowledge is limited. Therefore, this study aims to provide new evidence for the signal interplay between NO and SL in the formation of root system architecture using complementary pharmacological and molecular biological approaches in the model Arabidopsis thaliana grown under stress-free conditions. Deficiency of SL synthesis or signaling (max1-1 and max2-1) resulted in elevated NO and S-nitrosothiol (SNO) levels due to decreased S-nitrosoglutathione (GSNO) reductase (GSNOR) protein abundance and activity indicating that there is a signal interaction between SLs and GSNOR-regulated levels of NO/SNO. This was further supported by the down-regulation of SL biosynthetic genes (CCD7, CCD8 and MAX1) in GSNOR-deficient gsnor1-3. Based on the more pronounced sensitivity of gsnor1-3 to exogenous SL (rac-GR24, 2 µM), we suspected that functional GSNOR is needed to control NO/SNO levels during SL-induced primary root (PR) elongation. Additionally, SLs may be involved in GSNO-regulated PR shortening as suggested by the relative insensitivity of max1-1 and max2-1 mutants to exogenous GSNO (250 µM). Collectively, our results indicate a connection between SL and GSNOR-regulated NO/SNO signals in roots of A. thaliana grown in stress-free environment. As this work used max2-1 mutant and rac-GR24 exerting unspecific effects to both SL and karrikin signaling, it cannot be ruled out that karrikins are partly responsible for the observed effects, and this issue needs further clarification in the future.

Similar to animals, it has recently been proven that nitro-fatty acids such as nitro-linolenic acid and nitro-oleic acid (NO2-OA) have relevant physiological roles as signalling molecules also in plants. Although NO2-OA is of great therapeutic importance, its presence in plants as a free fatty acid has not been observed so far. Since Brassica napus (oilseed rape) is a crop with high oleic acid content, the abundance of NO2-OA in its tissues can be assumed. Therefore, we quantified NO2-OA in B. napus seeds and differently developed seedlings. In all samples, NO2-OA was detectable at nanomolar concentrations. The seeds showed the highest NO2-OA content, which decreased during germination. In contrast, nitric oxide (•NO) levels increased in the early stages of germination and seedling growth. Exogenous NO2-OA treatment (100 µM, 24 h) of Brassica seeds resulted in significantly increased •NO level and induced germination capacity compared to untreated seeds. The results of in vitro approaches (4-Amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) fluorescence, •NO-sensitive electrode) supported the •NO liberating capacity of NO2-OA. We observed for the first time that Brassica seeds and seedlings contain free NO2-OA which may be involved in germination as an •NO donor as suggested both by the results of exogenous NO2-OA treatment of seeds and in vitro approaches. Due to their high NO2-OA content, Brassica sprouts can be considered as a good source of dietary NO2-OA intake.

Due to their release into the environment, zinc oxide nanoparticles (ZnO NPs) may come in contact with plants. In elevated concentrations, ZnO NPs induce reactive oxygen species (ROS) production, but the metabolism of reactive nitrogen species (RNS) and the consequent nitro-oxidative signalling has not been examined so far. In this work, Brassica napus and Brassica juncea seedlings were treated with chemically synthetized ZnO NPs (∼8 nm, 0, 25 or 100 mg/L). At low dose (25 mg/L) ZnO NP exerted a positive effect, while at elevated concentration (100 mg/L) it was toxic to both species. Additionally, B. juncea was more tolerant to ZnO NPs than B. napus. The ZnO NPs could enter the root cells due to their small (∼8 nm) size which resulted in the release of Zn2+ and subsequently increased Zn2+ content in the plant organs. ZnO NPs disturbed superoxide radical and hydrogen peroxide homeostasis and modulated ROS metabolic enzymes (NADPH oxidase, superoxide dismutase, ascorbate peroxidase) and non-enzymatic antioxidants (ascorbate and glutathione) inducing similar changes in oxidative signalling in both Brassica species. The homeostasis of RNS (nitric oxide, peroxynitrite and S-nitrosoglutathione) was also altered by ZnO NPs; however, changes in nitrosative signalling proved to be different in the examined species. Moreover, ZnO NPs triggered changes in protein carbonylation and nitration. These results suggest that ZnO NPs induce changes in nitro-oxidative signalling which may contribute to ZnO NP toxicity. Furthermore, difference in ZnO NP tolerance of Brassica species is more likely related to nitrosative than to oxidative signalling.

Despite of its essentiality, nickel (Ni) in excess is toxic for plants partly due to the overproduction of reactive oxygen species (ROS) and the consequent increase in oxidative stress signalling. However, in Ni-stressed plants little is known about the signal transduction of reactive nitrogen species (RNS) and protein tyrosine nitration as the protein-level consequence of increased RNS formation. Our experiments compared the nickel accumulation and tolerance, the redox signalling and the protein nitration in the agar-grown Arabidopsis thaliana and Brassica juncea exposed to Ni (50 μM nickel chloride). Studying GUS-tagged Arabidopsis lines (ARR5::GUS, ACS8::GUS and DR5::GUS) revealed that Ni-increased lateral root (LR) emergence, and concomitantly reduced LR initiation were accompanied by elevated levels of auxin, cytokinin, and ethylene in the LRs or in upper root parts, whereas Ni-induced primary root shortening is related to decreased auxin, and increased cytokinin and ethylene levels. These suggest the Ni-induced disturbance of hormonal balance in the root system. Results of the comparative study showed that weaker Ni tolerance of A. thaliana was coupled with a Ni-induced increase in RNS, ROS, and hydrogen sulfide levels, as well as with an increase in redox signalling and consequent increment of protein nitration. However, in relative Ni tolerant B. juncea, redox signalling (except for peroxynitrite) was not modified, and Ni-induced intensification of protein tyrosine nitration was less pronounced. Data collectively show that the better Ni tolerance of Brassica juncea may be related to the capability of preventing the induction of redox signalling and consequently to the slighter increase in protein nitration.

Cell wall-associated defence against zinc oxide nanoparticles (ZnO NPs) as well as nitro-oxidative signalling and its consequences in plants are poorly examined. Therefore, this study compares the effect of chemically synthetized ZnO NPs (~45 nm, 25 or 100 mg/L) on Brassica napus and Brassica juncea seedlings. The effects on root biomass and viability suggest that B. napus is more tolerant to ZnO NP exposure relative to B. juncea. This may be due to the lack of Zn ion accumulation in the roots, which is related to the increase in the amount of lignin, suberin, pectin and in peroxidase activity in the roots of B. napus. TEM results indicate that root cell walls of 25 mg/L ZnO NP-treated B. napus may bind Zn ions. Additionally, callose accumulation possibly contribute to root shortening in both Brassica species as the effect of 100 mg/L ZnO NPs. Further results suggest that in the roots of the relatively sensitive B. juncea the levels of superoxide radical, hydrogen peroxide, hydrogen sulfide, nitric oxide, peroxinitrite and S-nitrosoglutathione increased as the effect of high ZnO NP concentration meaning that ZnO NP intensifies nitro-oxidative signalling. In B. napus; however, reactive oxygen species signalling was intensified, but reactive nitrogen species signalling wasn't activated by ZnO NPs. Collectively, these results indicate that ZnO NPs induce cell wall remodeling which may be associated with ZnO NP tolerance. Furthermore, plant tolerance against ZnO NPs is associated rather with nitrosative signalling than oxidative modifications.

Roots have a noteworthy plasticity: due to different stress conditions their architecture can change to favour seedling vigour and yield stability. The development of the root system is regulated by a complex and diverse signalling network, which besides hormonal factors, includes reactive oxygen (ROS) - and nitrogen species (RNS). The delicate balance of the endogenous signal system can be affected by various environmental stimuli, such as the excess of essential heavy metals, like zinc (Zn). Zn at low concentration, is able to induce the morphological and physiological adaptation of the root system, but in excess it exerts toxic effects on plants. In this study the effect of a low, growth-inducing, and a high, growth inhibiting Zn concentrations on the early development of Brassica napus (L.) root architecture and the underlying nitro-oxidative mechanisms were studied in a soil-filled rhizotron system. The growth-inhibiting Zn treatment resulted in elevated protein tyrosine nitration due to the imbalance in ROS and RNS homeostasis, however its pattern was not changed compared to the control. This nitro-oxidative stress was accompanied by serious changes in the cell wall composition and decrease in the cell proliferation and viability, due to the high Zn uptake and disturbed microelement homeostasis in the root tips. During the positive root growth response, a tyrosine nitration-pattern reorganisation was observed; there were no substantial changes in ROS and RNS balance and the viability and proliferation of the root tips' meristematic zone decreased to a lesser extent, as a result of a lower Zn uptake. The obtained results suggest that Zn in different amounts triggers different root growth responses accompanied by distinct changes in the pattern and strength of tyrosine nitration, proposing that nitrosative processes have an important role in the stress-induced root growth responses.

Selenium (Se) results in primary root shortening and the concomitant induction of lateral roots (LRs) (stress-induced morphogenic response, SIMR). Both ethylene (ET) and nitric oxide (NO) are gasotransmitters interacting with each other during plant growth regulation; however, their involvement and interplay in LR growth have not been examined so far. This study investigates the effect of Se on ET and NO levels and interaction in wild-type (WT) and ET insensitive etr1-1 Arabidopsis. In WT, Se at 15 µM concentration triggered LR emergence (LRem) and slight ET level elevation but in etr1-1 Se-induced LR inhibition was accompanied by four-fold ET level increase which can be associated with the increased expression of ACS2 and ACS8. Treatment with ACC + Se decreased LRem and NO level in WT, whereas AVG + Se in the etr1-1 plants resulted in enhanced LRem and increased NO level indicating that ET may inhibit both NO formation and LR emergence in Se-stressed Arabidopsis. The expression of NO-associated genes (NR1, NR2, GSNOR1, GLB1, GLB2), however, did not correlate with NO levels. Application of GSNO together with Se resulted in enhanced LR outgrowth both in WT and in etr1-1, whereas this effect could be reversed by a NO scavenger which indicates the positive regulatory role of NO during LR emergence. Moreover, GSNO has a clear inhibitory and cPTIO has an inducing effect on ET levels. These data indicate for the first time that the antagonistic interplay between ET and NO regulates the emergence of lateral roots in Arabidopsis under Se stress.