Salinity (NaCl) and wheat curl mite infestation profoundly alter nitric oxide (NO) metabolism in barley, with NaCl dose-dependent responses indicating that nitrogen metabolism is fine-tuned through NO-signaling pathways. Nitric oxide (NO) serves as a multifaceted regulator in plants' responses to environmental stress, acting as both a signaling molecule and a protective agent, while also exhibiting potential harmful effects. Our study investigated NO metabolism in barley (Hordeum vulgare L.) plants under salinity (50 mM and 100 mM NaCl) and wheat curl mite (WCM) infestation, exposed to either single stressor or both stressors simultaneously. To accomplish our objectives, we adopted an integrated approach that combines biochemical, molecular, and microscopic techniques. We found that these stressors influence the production of NO and its subcellular distribution. Both nitrate reductase (NR) and non-enzymatic processes contribute to the production of NO. Under combined stress (50 mM NaCl + WCM), NO molecules were detected in the cytoplasm, vacuoles, and chloroplasts, along with elevated NR activity. NO fluorescence in cell walls suggests its role in the apoplastic response of barley under dual stress, which induced a probable synergistic effect caused by salinity and WCM effectors. Upregulation of alternative oxidase AOX and phytoglobin Pgb gene expression, along with decreased activity of S-nitrosoglutathione reductase (GSNOR), in all combinations indicates sophisticated regulation of NO metabolism through transcriptional and enzymatic mechanisms. The accumulation of 3-nitrotyrosines under high salinity and dual stress points to increased nitro-oxidative stress. Moreover, an inhibitory effect of a 100 mM NaCl salinity dose was demonstrated on WCM reproduction. Altogether, these findings provide a mechanistic framework for exploiting NO-related pathways as potential targets in breeding or biotechnological strategies aimed at improving barley tolerance to combined abiotic–biotic stress conditions, while simultaneously limiting pest performance under salinity.

Background/Objectives: Glucosinolates (GSLs) from cruciferous vegetables (CVs), sulfur (S)- and nitrogen-containing compounds, are enzymatically hydrolyzed by myrosinase (EC 3.2.1.147) to yield bioactive derivatives such as isothiocyanates (ITCs) and indoles. These metabolites exhibit chemopreventive and anticancer properties. The article compiles evidence regarding the following: (i) the molecular mechanisms regulating the biosynthesis of key derivatives, including sulforaphane (SFN), phenethyl isothiocyanate (PEITC), and indole-3-carbinol (I3C); (ii) epidemiological and clinical findings; and (iii) strategies to link plant science with nutritional interventions for cancer prevention. Methods: An integrative literature review was conducted using Web of Science, Scopus, ScienceDirect, Google Scholar, and PubMed. English-language studies addressing mechanistic insights, nutritional factors, epidemiology, and clinical trials were included. Results: The biosynthesis and metabolism of GSL in plants are regulated by S and several transcription factors that promote or repress GSL production. Additionally, food processing has been shown to influence retention time and the formation of ITCs. In humans, ITCs activate nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated detoxification, induce apoptosis, and modulate epigenetic pathways. Epidemiological data show inverse associations between CV intake and cancer risk, though variability exists. Clinical trials have confirmed the bioavailability and effects of glucoraphanin and SFN on cancer-related biomarkers. Conclusions: The described compounds are bioavailable in humans and modulate the clinically relevant pathways linked to carcinogenesis. Larger, standardized interventions are needed to determine effective intake levels, optimize bioavailability, and define their potential role in evidence-based nutritional strategies for cancer prevention.

Climate changes profoundly influence the level of stress that plants experience. This study was conducted to investigate the redox homeostasis of barley in response to salinity, infestation by Aceria tosichella Keifer. (the wheat curl mite, WCM), and the combined effects of these stress factors. Hordeum vulgare L. plants were treated with sodium chloride at concentrations of 50 mM and 100 mM and were inoculated with WCM. The plants were also exposed to both stressors simultaneously. Biochemical and molecular parameters were measured to evaluate the potential role of reactive oxygen species (ROS) in modulating barley’s response. The analysis of ROS and antioxidant levels revealed distinct patterns based on specific stress factors. Hydrogen peroxide levels increased in all treatments, indicating a plant redox imbalance. In contrast, superoxide anion levels rose only with WCM infestation, suggesting a specific response to this pest. Lipid peroxidation decreased with WCM inoculated combinations. Conversely, carbonyl group content increased with NaCl and combined stresses but declined with WCM. Variations in ascorbic acid to dehydroascorbic acid and reduced to oxidized glutathione ratios suggest complex regulation of antioxidant metabolism. Moreover, significant changes in the activities and gene expression of key enzymes, including catalases, peroxidases, and superoxide dismutases, highlight the critical role of antioxidant mechanisms in plant responses to stress. Our research results indicate that the response of barley to a combination of stress factors, such as salinity and WCM, differs significantly from the responses induced by their single exposure.

Background/Objectives: Leguminous plants (Fabaceae) are essential for global food and nutritional security due to their high protein content, bioactive compounds, and ecological role in nitrogen fixation. However, climate change poses significant threats to their productivity, quality, and safety. This review aims to summarize the nutritional, biochemical, and health-related importance of legumes, while highlighting the effects of climate change-particularly heat stress and pest pressure-on their nutritional value and public health implications. Methods: This review is based on an integrative literature review drawing on scientific databases including Web of Science, Scopus, ScienceDirect, Google Scholar, and PubMed (March-October 2025). The relevant literature on climate change, legume composition, stress physiology, pest-plant interactions, and nutrition- and health-related outcomes was identified using targeted search terms. Evidence from diverse study types was synthesized to provide a broad, interdisciplinary perspective rather than a systematic assessment. Results: Legume seeds are rich in proteins, complex carbohydrates, fibers, and essential fatty acids, and contain valuable phytochemicals, including polyphenols, carotenoids, saponins, and bioactive peptides, with antioxidant, anti-inflammatory, and cardioprotective effects. Nevertheless, elevated CO2 levels and temperature stress can reduce protein, iron, and zinc contents, while altering phenolic and isoflavone profiles. Simultaneously, warming enhances pest proliferation and fungal contamination, increasing mycotoxin exposure and associated health risks. Integrated pest management (IPM) strategies, particularly those emphasizing biological control, show promise in mitigating these risks while ensuring sustainable legume production. Conclusions: Safeguarding the nutritional and ecological value of legumes under changing climatic conditions requires coordinated efforts across plant breeding, agronomy, and food science. Enhancing thermotolerance and pest resistance, reducing pesticide use through IPM, and valorizing legume by-products are key to preserving food safety and human health. Legumes, thus, represent both a challenge and an opportunity in achieving resilient, climate-smart nutrition systems for future generations.

The responses of plants to stress factors are extremely elaborate [...]

Nitric oxide (NO) is an important signaling molecule that is involved in a wide range of physiological processes in plants, including responses to biotic and abiotic stresses. Changes in endogenous NO concentration lead to activation/deactivation of NO signaling and NO-related processes. This paper presents the current state of knowledge on NO biosynthesis and scavenging pathways in plant cells and highlights the role of NO in post-translational modifications of proteins (S-nitrosylation, nitration, and phosphorylation) in plants under optimal and stressful environmental conditions. Particular attention was paid to the interactions of NO with other signaling molecules: reactive oxygen species, abscisic acid, auxins (e.g., indole-3-acetic acid), salicylic acid, and jasmonic acid. In addition, potential common patterns of NO-dependent defense responses against attack and feeding by parasitic and molting Ecdysozoa species such as nematodes, insects, and arachnids were characterized. Our review definitely highlights the need for further research on the involvement of NO in interactions between host plants and Ecdysozoa parasites, especially arachnids.

Abscisic acid (ABA) is a phytohormone that plays a key role in regulating several developmental processes as well as in response to stressful conditions such as drought. Activation of the ABA signaling cascade allows the induction of an appropriate physiological response. The basic components of the ABA signaling pathway have been recognized and characterized in recent years. Pyrabactin resistance, pyrabactin resistance-like, and the regulatory component of ABA receptors (PYR/PYL/RCAR) are the major components responsible for the regulation of the ABA signaling pathway. Here, we review recent findings concerning the PYR/PYL/RCAR receptor structure, function, and interaction with other components of the ABA signaling pathway as well as the termination mechanism of ABA signals in plant cells. Since ABA is one of the basic elements related to abiotic stress, which is increasingly common in the era of climate changes, understanding the perception and transduction of the signal related to this phytohormone is of paramount importance in further increasing crop tolerance to various stress factors.

In response to environmental stress, plants activate complex signalling, including being dependent on reactive oxygen–nitrogen–sulphur species. One of the key abiotic stresses is drought. As a result of drought, changes in the level of hydration of the plant occur, which obviously entails various metabolic alternations. The primary aim of this study was to determine the relationship between the response of barley to drought and the intensity of stress, therefore investigations were performed under various levels of water saturation deficit (WSD) in leaves at 15%, 30%, and 50%. In barley subjected to drought, most significant changes occurred under a slight dehydration level at 15%. It was observed that the gene expression of 9-cis-epoxycarotenoid dioxygenases, enzymes involved in ABA biosynthesis, increased significantly, and led to a higher concentration of ABA. This was most likely the result of an increase in the gene expression and enzyme activity of L-cysteine desulfhydrase, which is responsible for H2S synthesis. Our results suggest that the differential water deficit in leaves underlies the activation of an appropriate defence, with ABA metabolism at the centre of these processes. Furthermore, at 15% WSD, a dominant contribution of H2O2-dependent signalling was noted, but at 30% and 50% WSD, significant NO-dependent signalling occurred.

Cereal plants under abiotic or biotic stressors to survive unfavourable conditions and continue growth and development, rapidly and precisely identify external stimuli and activate complex molecular, biochemical, and physiological responses. To elicit a response to the stress factors, interactions between reactive oxygen and nitrogen species, calcium ions, mitogen-activated protein kinases, calcium-dependent protein kinases, calcineurin B-like interacting protein kinase, phytohormones and transcription factors occur. The integration of all these elements enables the change of gene expression, and the release of the antioxidant defence and protein repair systems. There are still numerous gaps in knowledge on these subjects in the literature caused by the multitude of signalling cascade components, simultaneous activation of multiple pathways and the intersection of their individual elements in response to both single and multiple stresses. Here, signal transduction pathways in cereal plants under drought, salinity, heavy metal stress, pathogen, and pest attack, as well as the crosstalk between the reactions during double stress responses are discussed. This article is a summary of the latest discoveries on signal transduction pathways and it integrates the available information to better outline the whole research problem for future research challenges as well as for the creative breeding of stress-tolerant cultivars of cereals.

Environmental changes are inevitable with time, but their intensification and diversification, occurring in the last several decades due to the combination of both natural and human-made causes, are really a matter of great apprehension. As a consequence, plants are exposed to a variety of abiotic stressors that contribute to their morpho-physiological, biochemical, and molecular alterations, which affects plant growth and development as well as the quality and productivity of crops. Thus, novel strategies are still being developed to meet the challenges of the modern world related to climate changes and natural ecosystem degradation. Innovative methods that have recently received special attention include eco-friendly, easily available, inexpensive, and, very often, plant-based methods. However, such approaches require better cognition and understanding of plant adaptations and acclimation mechanisms in response to adverse conditions. In this succinct review, we have highlighted defense mechanisms against external stimuli (mainly exposure to elevated levels of metal elements) which can be activated through permanent microevolutionary changes in metal-tolerant species or through exogenously applied priming agents that may ensure plant acclimation and thereby elevated stress resistance.

Key message Defence responses of cyst nematode and/or wheat curl mite infested barley engage the altered reactive oxygen species production, antioxidant machinery, carbon dioxide assimilation and photosynthesis efficiency. The primary aim of this study was to determine how barley responds to two pests infesting separately or at once; thus barley was inoculated withHeterodera filipjevi(Madzhidov) Stelter (cereal cyst nematode; CCN) andAceria tosichellaKeifer (wheat curl mite; WCM). To verify hypothesis about the involvement of redox metabolism and photosynthesis in barley defence responses, biochemical, photosynthesis efficiency and chlorophyllafluorescence measurements as well as transmission electron microscopy were implemented. Inoculation with WCM (apart from or with CCN) brought about a significant suppression in the efficiency of electron transport outside photosystem II reaction centres. This limitation was an effect of diminished pool of rapidly reducing plastoquinone and decreased total electron carriers. Infestation with WCM (apart from or with CCN) also significantly restricted the electron transport on the photosystem I acceptor side, therefore produced reactive oxygen species oxidized lipids in cells of WCM and double infested plants and proteins in cells of WCM-infested plants. The level of hydrogen peroxide was significantly decreased in double infested plants because of glutathione-ascorbate cycle involvement. The inhibition of nitrosoglutathione reductase promoted the accumulation ofS-nitrosoglutathione increasing antioxidant capacity in cells of double infested plants. Moreover, enhanced arginase activity in WCM-infested plants could stimulate synthesis of polyamines participating in plant antioxidant response. Infestation with WCM (apart from or with CCN) significantly reduced the efficiency of carbon dioxide assimilation by barley leaves, whereas infection only with CCN expanded photosynthesis efficiency. These were accompanied with the ultrastructural changes in chloroplasts during CCN and WCM infestation.

Key message Defence responses of cyst nematode and/or wheat curl mite infested barley engage the altered reactive oxygen species production, antioxidant machinery, carbon dioxide assimilation and photosynthesis efficiency. Abstract The primary aim of this study was to determine how barley responds to two pests infesting separately or at once; thus barley was inoculated with Heterodera filipjevi (Madzhidov) Stelter (cereal cyst nematode; CCN) and Aceria tosichella Keifer (wheat curl mite; WCM). To verify hypothesis about the involvement of redox metabolism and photosynthesis in barley defence responses, biochemical, photosynthesis efficiency and chlorophyll a fluorescence measurements as well as transmission electron microscopy were implemented. Inoculation with WCM (apart from or with CCN) brought about a significant suppression in the efficiency of electron transport outside photosystem II reaction centres. This limitation was an effect of diminished pool of rapidly reducing plastoquinone and decreased total electron carriers. Infestation with WCM (apart from or with CCN) also significantly restricted the electron transport on the photosystem I acceptor side, therefore produced reactive oxygen species oxidized lipids in cells of WCM and double infested plants and proteins in cells of WCM-infested plants. The level of hydrogen peroxide was significantly decreased in double infested plants because of glutathione–ascorbate cycle involvement. The inhibition of nitrosoglutathione reductase promoted the accumulation of S -nitrosoglutathione increasing antioxidant capacity in cells of double infested plants. Moreover, enhanced arginase activity in WCM-infested plants could stimulate synthesis of polyamines participating in plant antioxidant response. Infestation with WCM (apart from or with CCN) significantly reduced the efficiency of carbon dioxide assimilation by barley leaves, whereas infection only with CCN expanded photosynthesis efficiency. These were accompanied with the ultrastructural changes in chloroplasts during CCN and WCM infestation.

Nitrogen metabolism is important for physiological processes during normal growth and development, as well as plant defence responses. Second-stage juveniles of the cyst nematode Heterodera schachtii penetrate into the root and initiate permanent feeding sites called syncytia, from which they draw all necessary nutrients. To unravel whether the cyst nematode infestation changes nitrogen metabolism, the enzymatic activity and gene expression of nitrate (NIA) and nitrite (NIR) reductases and glutamate dehydrogenase (GDH), as well as levels of the metabolites nitrates, nitrites, and ammonium ions were estimated both locally in inoculated roots and systemically in shoots of inoculated Arabidopsis thaliana plants at 3, 7, and 15 days post-inoculation (dpi). Moreover, gene expression of long hypocotyle 5 (HY5), a basic-region leucine zipper (bZIP) transcription factor, was also determined, to learn more about the potential transcriptional regulation of the analysed enzymes. It was shown that the activity of nitrogen-related enzymes and the content of metabolites fluctuated during the whole period of infection. These results were accompanied by organ-specific and dpi-dependent gene expression patterns. The high gene expression level of HY5 in infected roots at 7 and 15 dpi coexisted with enhanced expression of NIA1 and NIR1 at 7 dpi and NIA2 at 15 dpi. Enhanced HY5 expression level in shoots of infected plants at 15 dpi was followed by up-regulation of NIA1 expression, suggesting that HY5 may regulate the gene expression of NIA and NIR during nematode infection. Our results indicate that changes in plant nitrogen metabolism occur during cyst nematode infection.

This research aimed to establish how Hordeum vulgare responds to abiotic and biotic stress affecting in tandem. Plants were inoculated with Heterodera filipjevi and treated with cadmium (Cd) concentration (5 μM) that can occur in the cultivated soil. To verify the hypothesis about participation of increased antioxidative defence in H. vulgare under stress, biochemical and microscopic methods were implemented. The amount of superoxide anions and hydrogen peroxide was diminished in plants that were both nematode-inoculated and cadmium-treated. Superoxide anions were rendered harmless by increased activity of superoxide dismutase, and H2O2 was scavenged via Foyer-Halliwell-Asada pathway. The unique enhanced antioxidant capacity of double stressed plants was also linked with the accumulation of S-nitrosoglutathione as nitrosoglutathione reductase activity was inhibited. Furthermore, stimulated activity of arginase in these plants could promote polyamine synthesis and indirectly enhance non-enzymatic antioxidant mechanism. Results indicate that different antioxidants operating together significantly restricted oxidation of lipids and proteins, thus the integrity of cell membranes and protein functions were maintained. The ROS deactivation machinery in barley leaves showed an unusual response during stress induced by H. filipjevi infection and cadmium treatment. Plants could induce a multi-component model of stress response, to detoxify Cd ions and efficiently repair stress damage.

Reactive nitrogen species (RNS) are redox molecules important for plant defense against pathogens. The aim of the study was to determine whether the infection by the beet cyst nematodeHeterodera schachtiidisrupts RNS balance inArabidopsis thalianaroots. For this purpose, measurements of nitric oxide (NO), peroxynitrite (ONOO-), proteinS-nitrosylation and nitration, and nitrosoglutathione reductase (GSNOR) inA. thalianaroots from 1 day to 15 days post-inoculation (dpi) were performed. The cyst nematode infection caused generation of NO and ONOO(-)in the infected roots. These changes were accompanied by an expansion ofS-nitrosylated and nitrated proteins. The enzyme activity of GSNOR was decreased at 3 and 15 dpi and increased at 7 dpi in infected roots, whereas theGSNOR1transcript level was enhanced over the entire examination period. The protein content of GSNOR was increased in infected roots at 3 dpi and 7 dpi, but at 15 dpi, did not differ between uninfected and infected roots. The protein of GSNOR was detected in plastids, mitochondria, cytoplasm, as well as endoplasmic reticulum and cytoplasmic membranes. We postulate that RNS metabolism plays an important role in plant defense against the beet cyst nematode and helps the fine-tuning of the infected plants to stress sparked by phytoparasitic nematodes.

Summary Vacuolar processing enzymes (VPEs) play an important role during regular growth and development and defence responses. Despite substantial attempts to understand the molecular basis of plant–cyst nematode interaction, the mechanism of VPEs functioning during this interaction remains unknown. The second‐stage Heterodera filipjevi juvenile penetrates host roots and induces the formation of a permanent feeding site called a syncytium. To investigate whether infection with H. filipjevi alters plant host VPEs, the studies were performed in Hordeum vulgare roots and leaves on the day of inoculation and at 7, 14 and 21 days post‐inoculation (dpi). Implementing molecular, biochemical and microscopic methods we identified reasons for modulation of barley VPE activity during interaction with H. filipjevi. Heterodera filipjevi parasitism caused a general decrease of VPE activity in infected roots, but live imaging of VPEs showed that their activity is up‐regulated in syncytia at 7 and 14 dpi and down‐regulated at 21 dpi. These findings were accompanied by tissue‐specific VPE gene expression patterns. Expression of the barley cystatin HvCPI‐4 gene was stimulated in leaves but diminished in roots upon infestation. External application of cyclotides that can be produced naturally by VPEs elicits in pre‐parasitic juveniles vesiculation of their body, enhanced formation of granules, induction of exploratory behaviour (stylet thrusts and head movements), production of reactive oxygen species (ROS) and final death by methuosis. Taken together, down‐regulation of VPE activity through nematode effectors promotes the nematode invasion rates and leads to avoidance of the induction of the plant proteolytic response and death of the invading juveniles.

Abscisic acid (ABA) is a phytohormone involved in the acquisition of primary dormancy during seeds maturation as well as dormancy maintenance in imbibed seeds. After imbibition, the ABA content decreased to a much lower level in embryos of freshly harvested triticale grains of the Leontino cultivar, which is more susceptible to pre-harvest sprouting (PHS) than embryos of the Fredro cultivar. Lower ABA content in the Leontino cultivar resulted from increased expression of TsABA8'OH1 and TsABA8'OH2, which encode ABA 8'-hydroxylase and are involved in ABA catabolism. Higher ABA content and maintenance of dormancy in Fredro grains were correlated with intensified ABA biosynthesis, which resulted from higher expression of TsNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase. These results suggest that grains of triticale cultivars with different resistance to PHS vary in their ability to metabolize ABA after imbibition. After-ripening did not affect the ABA content in embryos of dry grains of either triticale cultivar. However, after-ripening caused dormancy release in Fredro grains and significantly affected the ABA content and the rate of its metabolism after imbibition. A more rapid decline in ABA content in imbibed Fredro grains was accompanied by decreased transcript levels of TsNCED1 as well as increased expression of TsABA8'OH1 and TsABA8'OH2. Thus, after-ripening may affect dormancy of grains through reduction of the ABA biosynthesis rate and intensified ABA catabolism. Overexpression of TsNCED1 in tobacco increases ABA content and delays germination, while overexpression of TsABA8'OH2 decreases ABA content, accelerates germination, and reduces the sensitivity to ABA of transgenic seeds compared to seeds of wild-type plants. Therefore, these genes might play an important role in the regulation of triticale grain dormancy, thus affecting susceptibility to PHS.

Phytocystatins are a group of proteins with significant potential to regulate activities of cysteine proteinases of native and pest/pathogen origins. The two-domain triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 was characterized in this study. This protein belongs to the second group of phytocystatins and contains all the conserved sequences and motifs as well as both N-terminal (CY) and C-terminal (CY-L) domains that are characteristic of phytocystatins with the C-terminal extension. We demonstrated that TrcC-8 forms stable dimers with a significantly reduced inhibitory activity against papain compared to the activity of monomers, indicating the regulatory nature of the oligomerization. Moreover, according to our research, only the N-terminal domain possesses the ability to form dimers, indicating that this part of TrcC-8 is involved in the dimerization of the full-length protein. Homology modelling of TrcC-8 strongly suggests distinct specificities for the CY and CY-L domains, confirmed in experiments with inhibition of the papain. Our results suggest that the CY domain of TrcC-8 may, although markedly weakly and suboptimally, interact with papain in an analogous mode to tarocystatin, while the CY-L domain of TrcC-8 has distinct specificity than tarocystatin.

Abscisic acid (ABA) is a plant hormone that plays a predominant role in the onset and maintenance of primary dormancy. Peak ABA accumulation in embryos of triticale grains was observed before any significant loss of water and was higher in Fredro, a cultivar less susceptible to pre-harvest sprouting (PHS), than in Leontino, a cultivar more sensitive to PHS. At full maturity, embryonic ABA content in Fredro was twice as high as in Leontino. Two full-length cDNAs of 9-cis-epoxycarotenoid dioxygenase (TsNCED1, TsNCED2), an enzyme involved in ABA biosynthesis, and two full-length cDNAs of ABA 8'-hydroxylase (TsABA8'OH1 and TsABA8'OH2), an enzyme involved in ABA catabolism, were identified in triticale grains and characterized. The maximum transcript level of both TsNCED1 and TsNCED2 preceded the peak of ABA accumulation, suggesting that both TsNCEDs contribute to reach this peak, although the expression of TsNCED1 was significantly higher in Fredro than in Leontino. High expression of TsABA8'OH2 and TsABA8'OH1 was observed long before and at the end of the ABA accumulation peak, respectively, but no differences were observed between cultivars. The obtained results suggest that mainly TsNCED1 might be related to the higher ABA content and higher resistance of Fredro to PHS. However, Fredro embryos not only have higher ABA content, but also exhibit greater sensitivity to ABA, which may also have a significant effect on grain dormancy and lower susceptibility to PHS for grains of this cultivar.

Phytocystatins (PCs) are protein inhibitors of endogenous plant endopeptidases and exogenous pathogen proteinases. We have previously described the protein inhibitor TrcC-4, which is probably involved in the control of protein degradation during triticale seeds germination. The occurrence of the LARFAVXEHN motif supports the TrcC-4 designation as a PC. In this paper TrcC-4 was expressed in Escherichia coli using the pET28 expression vector. TrcC-4(6×His) was purified by affinity chromatography with a single step of purification. Western blot analysis showed the presence of TrcC-4 in both developing and germinating triticale seeds. TrcC-4 protein level was higher both in scutellum of germinating seeds and in developing grains of the triticale cultivar more resistant to pre-harvest sprouting (Zorro) than in a less resistant one (Disco). Furthermore it was demonstrated that the activity of EP8, cysteine endopeptidase responsible for the mass hydrolysis of prolamin during germination, is inhibited by TrcC-4(6×His), as confirmed by native PAGE with gliadin as a substrate. These results suggest that phytocystatin TrcC-4 controls the activity of cysteine endopeptidases involved in germination and, thus, is potentially involved in pre-harvest sprouting.