Heavy metals in cigarette smoke strongly inhibit pancreatic ductal function and promote development of chronic pancreatitis.

BACKGROUND AND AIMS: Smoking is recognised as an independent risk factor in the development of chronic pancreatitis (CP). Cystic fibrosis transmembrane conductance regulator (CFTR) function and ductal fluid and bicarbonate secretion are also known to be impaired in CP, so it is crucial to understand the relationships between smoking, pancreatic ductal function and the development of CP. METHODS: We measured sweat chloride (Cl-) concentrations in patients with and without CP, both smokers and non-smokers, to assess CFTR activity. Serum heavy metal levels and tissue cadmium concentrations were determined by mass spectrometry in smoking and non-smoking patients. Guinea pigs were exposed to cigarette smoke, and cigarette smoke extract (CSE) was prepared to characterise its effects on pancreatic HCO3 - and fluid secretion and CFTR function. We administered cerulein to both the smoking and non-smoking groups of mice to induce pancreatitis. RESULTS: Sweat samples from smokers, both with and without CP, exhibited elevated Cl- concentrations compared to those from non-smokers, indicating a decrease in CFTR activity due to smoking. Pancreatic tissues from smokers, regardless of CP status, displayed lower CFTR expression than those from non-smokers. Serum levels of cadmium and mercury, as well as pancreatic tissue cadmium, were increased in smokers. Smoking, CSE, cadmium, mercury and nicotine all hindered fluid and HCO3 - secretion and CFTR activity in pancreatic ductal cells. These effects were mediated by sustained increases in intracellular calcium ([Ca2+]i), depletion of intracellular ATP (ATPi) and mitochondrial membrane depolarisation. CONCLUSION: Smoking impairs pancreatic ductal function and contributes to the development of CP. Heavy metals, notably cadmium, play a significant role in the harmful effects of smoking. KEY POINTS: Smoking and cigarette smoke extract diminish pancreatic ductal fluid and HCO3 - secretion as well as the expression and function of CFTR Cd and Hg concentrations are significantly higher in the serum samples of smokers Cd accumulates in the pancreatic tissue of smokers.

Salicylic acid- and ethylene-dependent effects of the ER stress-inducer tunicamycin on the photosynthetic light reactions in tomato plants.

Plant hormones such as ethylene (ET) and salicylic acid (SA) have an elementary role in the regulation of ER stress and unfolded protein response (UPR) in plants via modulating defence responses or inducing oxidative stress. Chloroplasts can be sources and targets of reactive oxygen species (ROS) that affect photosynthetic efficiency, which has not been investigated under tunicamycin (Tm)-induced ER stress. In this study, the direct and indirect effects of Tm on chloroplastic ROS production were first investigated in leaves of wild-type tomato (Solanum lycopersicum L.) plants. Secondly changes in activities of photosystem II and I were analysed under Tm exposure and after application of the chemical chaperone 4-phenylbutyrate (PBA) in different genotypes, focusing on the regulatory role of SA and ET Tm treatments significantly but indirectly induced ROS production in tomato leaves and in parallel it decreased the effective quantum yield of PSII [Y(II)] and PSI [Y(I)], as well as the photochemical quenching coefficient (qP) and the quantum yield of non-photochemical energy dissipation in PSI due to acceptor-side limitation [Y(NA)]. At the same time, Tm increased non-photochemical quenching (NPQ) and cyclic electron flow (CEF) in tomato leaves after 24 h. However, the photosynthetic activity of the SA hydroxylase-overexpressing NahG tomato plants was more severely affected by Tm as compared to wild-type and ET-insensitive Never ripe (Nr) plants. These results suggest the protective role of SA in the regulation of photosynthetic activity contributing to UPR and the survival of plants under ER stress. Interestingly, the activation of photoprotective mechanisms by NPQ was independent of SA but dependent on active ET signalling under ER stress, whereas CEF was reduced by ET due to its higher ratio in Nr plants.

Fusaric acid-evoked oxidative stress affects plant defence system by inducing biochemical changes at subcellular level.

Fusaric acid (FA) is one of the most harmful phytotoxins produced in various plant-pathogen interactions. Fusarium species produce FA as a secondary metabolite, which can infect many agronomic crops at all stages of development from seed to fruit, and FA production can further compromise plant survival because of its phytotoxic effects. FA exposure in plant species adversely affects plant growth, development and crop yield. FA exposure in plants leads to the generation of reactive oxygen species (ROS), which cause cellular damage and ultimately cell death. Therefore, FA-induced ROS accumulation in plants has been a topic of interest for many researchers to understand the plant-pathogen interactions and plant defence responses. In this study, we reviewed the FA-mediated oxidative stress and ROS-induced defence responses of antioxidants, as well as hormonal signalling in plants. The effects of FA phytotoxicity on lipid peroxidation, physiological changes and ultrastructural changes at cellular and subcellular levels were reported. Additionally, DNA damage, cell death and adverse effects on photosynthesis have been explained. Some possible approaches to overcome the harmful effects of FA in plants were also discussed. It is concluded that FA-induced ROS affect the enzymatic and non-enzymatic antioxidant system regulated by phytohormones. The effects of FA are also associated with other photosynthetic, ultrastructural and genotoxic modifications in plants.

The local and systemic accumulation of ethylene determines the rapid defence responses induced by flg22 in tomato (Solanum lycopersicum L.).

Plant defence responses induced by the bacterial elicitor flg22 are highly dependent on phytohormones, including gaseous ethylene (ET). While the regulatory role of ET in local defence responses to flg22 exposure has been demonstrated, its contribution to the induction of systemic responses is not clearly understood. For this consideration, we examined the effects of different ET modulators on the flg22-induced local and systemic defence progression. In our experiments, ET biosynthesis inhibitor aminoethoxyvinyl glycine (AVG) or ET receptor blocker silver thiosulphate (STS) were applied 1 h before flg22 treatments and 1 h later the rapid local and systemic responses were detected in the leaves of intact tomato plants (Solanum lycopersicum L.). Based on our results, AVG not only diminished the flg22-induced ET accumulation locally, but also in the younger leaves confirming the role of ET in the whole-plant expanding defence progression. This increase in ET emission was accompanied by increased local expression of SlACO1, which was reduced by AVG and STS. Local ET biosynthesis upon flg22 treatment was shown to positively regulate local and systemic superoxide (O2.-) and hydrogen peroxide (H2O2) production, which in turn could contribute to ET accumulation in younger leaves. Confirming the role of ET in flg22-induced rapid defence responses, application of AVG reduced local and systemic ET, O2.- and H2O2 production, whereas STS reduced it primarily in the younger leaves. Interestingly, in addition to flg22, AVG and STS induced stomatal closure alone at whole-plant level, however in the case of combined treatments together with flg22 both ET modulators reduced the rate of stomatal closure in the older- and younger leaves as well. These results demonstrate that both local and systemic ET production in sufficient amounts and active ET signalling are essential for the development of flg22-induced rapid local and systemic defence responses.

Ethylene-dependent regulation of oxidative stress in the leaves of fusaric acid-treated tomato plants.

The mycotoxin fusaric acid (FA) induces rapid oxidative burst leading to cell death in plants. At the same time, plant defence reactions are mediated by several phytohormones for instance ethylene (ET). However, previously conducted studies leave research gaps on how ET plays a regulatory role under mycotoxin exposure. Therefore, this study aims to the time-dependent effects of two FA concentrations (0.1 mM and 1 mM) were explored on the regulation of reactive oxygen species (ROS) in leaves of wild-type (WT) and ET receptor mutant Never ripe (Nr) tomatoes. FA induced superoxide and H2O2 accumulation in both genotypes in a mycotoxin dose- and exposure time-dependent pattern. 1 mM FA activated NADPH oxidase (+34% compared to the control) and RBOH1 transcript levels in WT leaves. However, superoxide production was significantly higher in Nr with 62% which could contribute to higher lipid peroxidation in this genotype. In parallel, the antioxidative defence mechanisms were also activated. Both peroxidase and superoxide dismutase activities were lower in Nr but ascorbate peroxidase showed one-fold higher activity under 1 mM FA stress than in WT leaves. Interestingly, catalase (CAT) activity decreased upon FA in a time- and concentration-dependent manner and the encoding CAT genes were also downregulated, especially in Nr leaves at 20%. Ascorbate level was decreased and glutathione remained lower in Nr than WT plants under FA exposure. Conclusively, Nr genotype showed more sensitivity to FA-induced ROS suggesting that ET serves defence reactions of plants by activating several enzymatic and non-enzymatic antioxidants to detoxify excess ROS accumulation.

The combination of Neosartorya (Aspergillus) fischeri antifungal proteins with rationally designed γ-core peptide derivatives is effective for plant and crop protection.

Plant pathogenic fungi are responsible for enormous crop losses worldwide. Overcoming this problem is challenging as these fungi can be highly resistant to approved chemical fungicides. There is thus a need to develop and introduce fundamentally new plant and crop protection strategies for sustainable agricultural production. Highly stable extracellular antifungal proteins (AFPs) and their rationally designed peptide derivatives (PDs) constitute feasible options to meet this challenge. In the present study, their potential for topical application to protect plants and crops as combinatorial biofungicides is supported by the investigation of two Neosartorya (Aspergillus) fischeri AFPs (NFAP and NFAP2) and their γ-core PDs. Previously, the biofungicidal potential of NFAP, its rationally designed γ-core PD (γNFAP-opt), and NFAP2 was reported. Susceptibility tests in the present study extended the in vitro antifungal spectrum of NFAP2 and its γ-core PD (γNFAP2-opt) to Botrytis, Cladosporium, and Fusarium spp. Besides, in vitro additive or indifferent interactions, and synergism were observed when NFAP or NFAP2 was applied in combination with γNFAP-opt. Except for γNFAP2-opt, the investigated proteins and peptides did not show any toxicity to tomato plant leaves. The application of NFAP in combination with γNFAP-opt effectively inhibited conidial germination, biofilm formation, and hyphal extension of the necrotrophic mold Botrytis cinerea on tomato plant leaves. However, the same combination only partially impeded the B. cinerea-mediated decay of tomato fruits, but mitigated the symptoms. Our results highlight the feasibility of using the combination of AFP and PD as biofungicide for the fungal infection control in plants and crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s10526-022-10132-y.

Activation of Local and Systemic Defence Responses by Flg22 Is Dependent on Daytime and Ethylene in Intact Tomato Plants.

The first line of plant defence responses against pathogens can be induced by the bacterial flg22 and can be dependent on various external and internal factors. Here, we firstly studied the effects of daytime and ethylene (ET) using Never ripe (Nr) mutants in the local and systemic defence responses of intact tomato plants after flg22 treatments. Flg22 was applied in the afternoon and at night and rapid reactions were detected. The production of hydrogen peroxide and nitric oxide was induced by flg22 locally, while superoxide was induced systemically, in wild type plants in the light period, but all remained lower at night and in Nr leaves. Flg22 elevated, locally, the ET, jasmonic acid (JA) and salicylic acid (SA) levels in the light period; these levels did not change significantly at night. Expression of Pathogenesis-related 1 (PR1), Ethylene response factor 1 (ERF1) and Defensin (DEF) showed also daytime- and ET-dependent changes. Enhanced ERF1 and DEF expression and stomatal closure were also observable in systemic leaves of wild type plants in the light. These data demonstrate that early biotic signalling in flg22-treated leaves and distal ones is an ET-dependent process and it is also determined by the time of day and inhibited in the early night phase.

Involvement of nitric oxide (NO) in plant responses to metalloids.

Plants respond to the limited or excess supply of metalloids, boron (B), silicon (Si), selenium (Se), arsenic (As), and antimony (Sb) via complex signaling pathways that are mainly regulated by nitric oxide (NO). The absorption of metalloids from the soil is facilitated by pathways that involve aquaporins, aquaglyceroporins, phosphate, and sulfate transporters; however, their regulation by NO is poorly understood. Using in silico software, we predicted the S-nitrosation of known metalloid transporters, proposing NO-dependent regulation of metalloid transport systems at the posttranslational level. NO intensifies the stress-mitigating effect of Si, whereas in the case of Se, As, and Sb, the accumulation of NO or reactive nitrogen species contributes to toxicity. NO promotes the beneficial effect of low Se concentrations and mitigates the damage caused by B deficiency. In addition, the exogenous application of NO donor, sodium nitroprusside, reduces B, Se, and As toxicity. The primary role of NO in metalloid stress response is to mitigate oxidative stress by activating antioxidant defense at the level of protein activity and gene expression. This review discusses the role of NO in plant responses to metalloids and suggests future research directions.

Role of ethylene and light in chitosan-induced local and systemic defence responses of tomato plants.

Plant defence responses can be triggered by the application of elicitors for example chitosan (ß-1,4-linked glucosamine; CHT). It is well-known that CHT induces rapid, local production of reactive oxygen species (ROS) and nitric oxide (NO) resulting in fast stomatal closure. Systemic defence responses are based primarily on phytohormones such as ethylene (ET) and salicylic acid (SA), moreover on the expression of hormone-mediated defence genes and proteins. At the same time, these responses can be dependent also on external factors, such as light but its role was less-investigated. Based on our result in intact tomato plants (Solanum lycopersicum L.), CHT treatment not only induced significant ET emission and stomatal closure locally but also promoted significant production of superoxide which was also detectable in the distal, systemic leaves. However, these changes in ET and superoxide accumulation were detected only in wild type (WT) plants kept in light and were inhibited under darkness as well as in ET receptor Never ripe (Nr) mutants suggesting pivotal importance of ET and light in inducing resistance both locally and systemically upon CHT. Interestingly, CHT-induced NO production was mostly independent of ET or light. At the same time, expression of Pathogenesis-related 3 (PR3) was increased locally in both genotypes in the light and in WT leaves under darkness. This was also observed in distal leaves of WT plants. The CHT-induced endoplasmic reticulum (ER) stress, as well as unfolded protein response (UPR) were examined for the first time, via analysis of the lumenal binding protein (BiP). Whereas local expression of BiP was not dependent on the availability of light or ET, systemically it was mediated by ET.

Plant defence mechanisms against mycotoxin Fumonisin B1.

Fumonisin B1 (FB1) is the most harmful mycotoxin which prevails in several crops and affects the growth and yield as well. Hence, keeping the alarming consequences of FB1 under consideration, there is still a need to seek other more reliable approaches and scientific knowledge for FB1-induced cell death and a comprehensive understanding of the mechanisms of plant defence strategies. FB1-induced disturbance in sphingolipid metabolism initiates programmed cell death (PCD) through various modes such as the elevated generation of reactive oxygen species, lipid peroxidation, cytochrome c release from the mitochondria, and activation of specific proteases and nucleases causing DNA fragmentation. There is a close interaction between sphingolipids and defence phytohormones in response to FB1 exposure regulating PCD and defence. In this review, the model plant Arabidopsis and various crops have been presented with different levels of susceptibility and resistivity exposed to various concentration of FB1. In addition to this, regulation of PCD and defence mechanisms have been also demonstrated at the physiological, biochemical and molecular levels to help the understanding of the role and function of FB1-inducible molecules and genes and their expressions in plants against pathogen attacks which could provide molecular and biochemical markers for the detection of toxin exposure.

The Effect of Foliar Selenium (Se) Treatment on Growth, Photosynthesis, and Oxidative-Nitrosative Signalling of Stevia rebaudiana Leaves.

Selenium (Se) enrichment of Stevia rebaudiana Bertoni can serve a dual purpose, on the one hand to increase plant biomass and stress tolerance and on the other hand to produce Se fortified plant-based food. Foliar Se spraying (0, 6, 8, 10 mg/L selenate, 14 days) of Stevia plantlets resulted in slightly decreased stevioside and rebaudioside A concentrations, and it also caused significant increment in stem elongation, leaf number, and Se content, suggesting that foliar Se supplementation can be used as a biofortifying approach. Furthermore, Se slightly limited photosynthetic CO2 assimilation (AN, gsw, Ci/Ca), but exerted no significant effect on chlorophyll, carotenoid contents and on parameters associated with photosystem II (PSII) activity (FV/FM, F0, Y(NO)), indicating that Se causes no photodamage in PSII. Further results indicate that Se is able to activate PSI-cyclic electron flow independent protection mechanisms of the photosynthetic apparatus of Stevia plants. The applied Se activated superoxide dismutase (SOD) isoenzymes (MnSOD1, FeSOD1, FeSOD2, Cu/ZnSOD1, Cu/ZnSOD2) and down-regulated NADPH oxidase suggesting the Se-induced limitation of superoxide anion levels and consequent oxidative signalling in Stevia leaves. Additionally, the decrease in S-nitrosoglutathione reductase protein abundance and the intensification of protein tyrosine nitration indicate Se-triggered nitrosative signalling. Collectively, these results suggest that Se supplementation alters Stevia shoot morphology without significantly affecting biomass yield and photosynthesis, but increasing Se content and performing antioxidant effects, which indicates that foliar application of Se may be a promising method in Stevia cultivation.

Solution Structure, Dynamics, and New Antifungal Aspects of the Cysteine-Rich Miniprotein PAFC.

The genome of Penicillium chrysogenum Q176 contains a gene coding for the 88-amino-acid (aa)-long glycine- and cysteine-rich P. chrysogenum antifungal protein C (PAFC). After maturation, the secreted antifungal miniprotein (MP) comprises 64 aa and shares 80% aa identity with the bubble protein (BP) from Penicillium brevicompactum, which has a published X-ray structure. Our team expressed isotope (15N, 13C)-labeled, recombinant PAFC in high yields, which allowed us to determine the solution structure and molecular dynamics by nuclear magnetic resonance (NMR) experiments. The primary structure of PAFC is dominated by 14 glycines, and therefore, whether the four disulfide bonds can stabilize the fold is challenging. Indeed, unlike the few published solution structures of other antifungal MPs from filamentous ascomycetes, the NMR data indicate that PAFC has shorter secondary structure elements and lacks the typical ß-barrel structure, though it has a positively charged cavity and a hydrophobic core around the disulfide bonds. Some parts within the two putative γ-core motifs exhibited enhanced dynamics according to a new disorder index presentation of 15N-NMR relaxation data. Furthermore, we also provided a more detailed insight into the antifungal spectrum of PAFC, with specific emphasis on fungal plant pathogens. Our results suggest that PAFC could be an effective candidate for the development of new antifungal strategies in agriculture.

ZnO nanoparticles induce cell wall remodeling and modify ROS/ RNS signalling in roots of Brassica seedlings.

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.

Effects of Jasmonic Acid in ER Stress and Unfolded Protein Response in Tomato Plants.

Endoplasmic reticulum (ER) stress elicits a protective mechanism called unfolded protein response (UPR) to maintain cellular homeostasis, which can be regulated by defence hormones. In this study, the physiological role of jasmonic acid (JA) in ER stress and UPR signalling has been investigated in intact leaves of tomato plants. Exogenous JA treatments not only induced the transcript accumulation of UPR marker gene SlBiP but also elevated transcript levels of SlIRE1 and SlbZIP60. By the application of JA signalling mutant jai1 plants, the role of JA in ER stress sensing and signalling was further investigated. Treatment with tunicamycin (Tm), the inhibitor of N-glycosylation of secreted glycoproteins, increased the transcript levels of SlBiP. Interestingly, SlIRE1a and SlIRE1b were significantly lower in jai1. In contrast, the transcript accumulation of Bax Inhibitor-1 (SlBI1) and SlbZIP60 was higher in jai1. To evaluate how a chemical chaperone modulates Tm-induced ER stress, plants were treated with sodium 4-phenylbutyrate, which also decreased the Tm-induced increase in SlBiP, SlIRE1a, and SlBI1 transcripts. In addition, it was found that changes in hydrogen peroxide content, proteasomal activity, and lipid peroxidation induced by Tm is regulated by JA, while nitric oxide was not involved in ER stress and UPR signalling in leaves of tomato.

Strigolactones Interact With Nitric Oxide in Regulating Root System Architecture of Arabidopsis thaliana.

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.

Nitro-Oleic Acid in Seeds and Differently Developed Seedlings of Brassica napus L.

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.

Nitro-oxidative signalling induced by chemically synthetized zinc oxide nanoparticles (ZnO NPs) in Brassica species.

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.

The potential use of the Penicillium chrysogenum antifungal protein PAF, the designed variant PAFopt and its γ-core peptide Pγopt in plant protection.

The prevention of enormous crop losses caused by pesticide-resistant fungi is a serious challenge in agriculture. Application of alternative fungicides, such as antifungal proteins and peptides, provides a promising basis to overcome this problem; however, their direct use in fields suffers limitations, such as high cost of production, low stability, narrow antifungal spectrum and toxicity on plant or mammalian cells. Recently, we demonstrated that a Penicillium chrysogenum-based expression system provides a feasible tool for economic production of P. chrysogenum antifungal protein (PAF) and a rational designed variant (PAFopt ), in which the evolutionary conserved γ-core motif was modified to increase antifungal activity. In the present study, we report for the first time that γ-core modulation influences the antifungal spectrum and efficacy of PAF against important plant pathogenic ascomycetes, and the synthetic γ-core peptide Pγopt , a derivative of PAFopt , is antifungal active against these pathogens in vitro. Finally, we proved the protective potential of PAF against Botrytis cinerea infection in tomato plant leaves. The lack of any toxic effects on mammalian cells and plant seedlings, as well as the high tolerance to harsh environmental conditions and proteolytic degradation further strengthen our concept for applicability of these proteins and peptide in agriculture.

Effects of Light and Daytime on the Regulation of Chitosan-Induced Stomatal Responses and Defence in Tomato Plants.

Closure of stomata upon pathogenesis is among the earliest plant immune responses. However, our knowledge is very limited about the dependency of plant defence responses to chitosan (CHT) on external factors (e.g., time of the day, presence, or absence of light) in intact plants. CHT induced stomatal closure before dark/light transition in leaves treated at 17:00 hrs and stomata were closed at 09:00 hrs in plants treated at dawn and in the morning. CHT was able to induce generation of reactive oxygen species (ROS) in guard cells in the first part of the light phase, but significant nitric oxide production was observable only at 15:00 hrs. The actual quantum yield of PSII electron transport (ΦPSII) decreased upon CHT treatments at 09:00 hrs in guard cells but it declined only at dawn in mesophyll cells after the treatment at 17:00 hrs. Expression of Pathogenesis-related 1 (PR1) and Ethylene Response Factor 1 were already increased at dawn in the CHT-treated leaves but PR1 expression was inhibited in the dark. CHT-induced systemic response was also observed in the distal leaves of CHT-treated ones. Our results suggest a delayed and daytime-dependent defence response of tomato plants after CHT treatment at night and under darkness.