Introduction

Hydrogen cyanide (HCN) is a linear, triatomic molecule of molecular weight 27.025 g mol−1, a gaseous compound occurring in the environment, as well in plant and animal tissues (Dowd and Ham 1991; Jaszczak et al. 2017; Aranguri-Llerena and Siche 2020). In the literature, the synonyms of HCN are hydrocyanic acid, formonitrile or prussic acid. Compounds with –C≡N group are defined as cyanides (or nitriles) (Jaszczak et al. 2017). For a long time, HCN was recognized as a metabolic poison, at high concentrations leading to death (Zuhra and Szabo 2022). This toxic property was reflected in the use of HCN for suicidal and homicidal purposes, including a chemical warfare agent (Raza and Jaiswal 1994). The physicochemical properties of HCN are: solubility at 20 °C in water (to form an acidic solution), and in organic solvents (alcohol, ether, glycerol, chloroform and benzene), volatility at 20 °C (873,000 mg m−3), boiling point (26.5 °C), vapour density relative to air of 0.948, high vapour pressure (87.6 kPa at 22 °C) (Raza and Jaiswal 1994; Dagaut et al. 2008). Pure, anhydrous HCN has the specific scent of bitter almond and is colourless in both liquid and gaseous states (Raza and Jaiswal 1994). In water solution, at alkaline pH, HCN is partially dissolved and dissociates into H+ and CN−. In the cell, cytosolic pH is close to 7, and cyanides (pKa 9.21) are mostly present in the protonated form of HCN (Dowd and Ham 1991). This compound is able to cross membranes and enter organelles, which may suggest its signalling nature (García et al. 2019).

HCN is produced in all plants, and some plants, known as cyanogenic, accumulate cyanide in the bound forms (cyanogenic glycosides) (Fig. 1A) for various physiological purposes (Siegień and Bogatek 2006; Gupta et al. 2010). Cyanohydrins (hydroxynitriles) are a group of biocompounds with a cyano and a hydroxy group attached to the same carbon atom that release HCN in the reaction catalysed by hydroxynitrile lyases (HNL) (Arnaiz et al. 2022). Moreover, this enzyme is also involved in cyanohydrins formation via condensation of HCN with an aldehyde or a ketone (Zagrobelny et al. 2008; Arnaiz et al. 2022). HNL has been identified in cyanogenic and non-cyanogenic plants, and is considered to play a beneficial role in plant-pest interaction, as was demonstrated for Arabidopsis (Arabidopsis thaliana (L.) Heynh.) (Arnaiz et al. 2022). In tissues of higher plants, HCN is a final product in the ethylene biosynthetic pathway (Fig. 1B). Oxidation of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) results in the hormone formation (from the carbon C-2 and C-3) and HCN (from C-1) in a stoichiometric ratio of 1:1 (Peiser et al. 1984). In the biosynthetic pathway of camalexin (the phytoalexin of Arabidopsis, which synthesis is induced by different plant pathogens), cyanide is released from indole-3-acetonitrile (IAN) as a thiazoline ring is formed (Böttcher et al. 2009) (Fig. 1C). Furthermore, HCN is also produced from glyoxalate in the reaction that requires the presence of hydroxylamine and manganese (Mn2+) (Solomonson 1981; Hucklesby et al. 1982) (Fig. 1D). Hucklesby et al. (1982) demonstrated the formation of HCN in the extracts of spinach (Spinacia oleracea L.), barley (Hordeum vulgare L.) and maize (Zea mays L.), considered to be non-cyanogenic. The authors proposed that in vivo, in higher plants the concentration of HCN liberated via this pathway may attain even 1 µM. The main routes of HCN formation in planta are presented in the Fig. 1.

Fig. 1
figure 1

The main routes of HCN formation in planta. HCN enzymatic liberation from cyanogenic glycoside A. HCN synthesis from ACC during ethylene biosynthetic pathway B.The release of HCN during camalexin biosynthesis C. HCN synthesis from glyoxalate in the presence of hydroxylamine D. Detailed description in the text. SAM—S-Adenosylmethionine; ACC—1-aminocyclopropane-1-carboxylic acid; IAOx—indole-3-acetaldoxime; IAN—indole-3-acetonitrile; DHCA—dihydrocamalexin acid (according to Hucklesby et al. (1982), Siegień and Bogatek (2006) and Böttcher (2009), modified)

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In the environment, HCN is liberated from the natural source (plants and microorganisms) or is of anthropogenic origin. In the soil, HCN may be produced during the cyanogenic plants’ decomposition or as a result of the activity of cyanogenic microorganisms: Chromobacterium, Anacystis, Nostoc, Plectonema or free-living forms of Rhizobium and rhizospheric Pseudomonas (Ebbs et al. 2010 and citations therein). In the rhizosphere of plants that have been colonized by cyanogenic bacteria, cyanide concentration could reach 100 mg kg−1 dry soil. In soil located close to the HCN-releasing industry, cyanide content can be estimated as high as 1000 mg kg−1 dry soil, and in wastewater even an order of magnitude higher (Ebbs et al. 2010 and citations therein). In the soil, where there is no additional source of HCN, its content varies from 0.005 to 0.5 mg kg−1 dry soil (Kjeldsen 1999). In the rhizosphere, the main role of HCN produced by rhizobia seems to be linked to its toxicity to pathogens. However, Rijavec and Lapanje (2016) suggested that the key function of HCN is rather the sequestration of metals and the indirect regulation of nutrient availability (e.g. phosphate), which is beneficial for both the bacteria and plants, especially in the oligotrophic alpine environment.

The increase in the production of metals, nylon, polyurethane foam, polyester wadding, neoprene foam, acrylonitrile etc. accelerates HCN emission (Schnepp 2006). The electroplating industry contributes to pollution by releasing cyanide in the aqueous waste streams. Among cyanide compounds used in industry are HCN, cyanogen chloride, sodium or potassium cyanide salts, gold and silver cyanides. Wastewater containing cyanide are released from the paint, steel and mining industries. Another significant source of HCN in the troposphere is the combustion of fossil fuels and plastic materials (Schnepp 2006; Dagaut et al. 2008). The incomplete combustion of nitrogen-containing materials, such as vinyl, wool silk or paper, liberates HCN (e.g. cotton—130 μg g−1, wool—6300 μg g−1 and paper—1100 μg g−1) (Lawson-Smith et al. 2011 and citations therein). The emission of HCN from urban vehicles has been known and documented for a long time. It is even postulated that vehicular emissions constitute an essential urban source of HCN (Moussa et al. 2016).

HCN emitted into the environment has a negative impact on human health however, its effect on plants is less recognized.

Cyanogenic compounds in plants

It is estimated that more than 3000 different plant species of ferns, gymnosperms, monocotyledonous and dicotyledonous angiosperms produce cyanogenic compounds (Siegień and Bogatek 2006; Zagrobelny and Møller 2011). Recently, the presence of 112 distinct cyanogenic glycosides was described and their chemical diversity has been discussed (Yulvianti and Zidorn 2021).

Cyanogenesis (the process of the HCN liberation from a biological source) is related to the term ‘cyanogenic potential’ (the current content of HCN in a given cyanogenic plant). It has a variable value, depending on the growing conditions of the tested plant, as well as the type of plant tissue/organs used in analyses (Stochmal and Oleszek 1997; Busk and Møller 2002). HCN is formed in plant reproductive organs: seeds, fruits and flowers (Brunt et al. 2006). Young leaves and seedlings are also characterized by a high ability to emit HCN as a molecule of defence activity (Brunt et al. 2006).

Cyanogenic glycosides are derivatives of α-hydroxynitriles (cyanohydrins). Cyanogenic compounds are biosynthesized from amino acids: valine, isoleucine (linamarin, lotaustralin), leucine (proacacipetalin, acacipetalin), phenylalanine (prunasin, amygdalin), and tyrosine (taxiphyllin, dhurrin) (Siegień and Bogatek 2006; Gleadow and Møller 2014). The amino acid precursor determines the chemical nature of the compound, which may be aromatic, aliphatic or cyclopentenoid (Yulvianti and Zidorn 2021). Mostly, cyanogenic compounds are deposited in the vacuoles, but enzymes responsible for their hydrolysis are located in the apoplast or the cytoplasm (Vetter 2000; Siegień and Bogatek 2006). Due to this spatial separation, the release of HCN is possible after the disruption of membrane integrity or during tonoplast rehydration e.g. in cells of imbibed seeds (Siegień and Bogatek 2006). The ability of some plants (especially those of agricultural significance) to HCN release depends on the synthesis and accumulation of cyanogenic compounds in their organs: leaves, stems, fruits, seeds, roots, and tubers. Almond (Prunus dulcis L. (Mill.)), apricot (Armenica vulgaris L.), peach (Persica sp.), cherry (Prunus sp.), flax (Linum usitatissimum L.), sorghum (Sorghum bicolor (L.) Moench), sea arrowgrass (Triglochin maritima L.) and cassava (Manihot esculenta Crantz.) are representatives of cyanogenic species (Geller 2015). In seeds belonging to the Sapindaceae family HCN may be liberated from cyanogenic lipids (cyanogenic compounds derived from leucine with fatty acids esterified to mono- or a dihydroxynitrite moiety) (Tava and Avato 2014 and citations therein). Cyanolipids constitute 3% of the total oil from seeds of Paullinia cupana var. sorbilis (Mart.) Ducke, which are commonly known as guarana (Avato et al. 2003).

The catabolism of cyanogenic glycosides is dependent on the glucoside type. The hydrolysis of the substrate (cyanogen) is catalyzed by one or more β-glycosidases (enzymes able to cleave the β-glycosidic linkage), resulting in a relatively unstable, rapidly degraded cyanohydrin (α-hydroxynitrile) containing hydroxyl groups and sugar. This intermediate may be decomposed spontaneously or via enzymatic activity (α-hydroxynitrile lyase). Cyanohydrin is degraded to HCN and the carbonyl compound—aldehyde or ketone (Poulton 1988). CN− reacts with the ketone group of molecules and intermediate compounds belonging to Schiff’s bases, leading to the formation of cyanohydrins and nitriles (García et al. 2010).

HCN detoxification in plants

The detoxification of cyanides in plants occurs in the presence of two different enzymes: β-cyanoalanine synthase (β-CAS, EC 4.4.1.9) and rhodanese (EC 2.8.1.1) (Fig. 2). The first enzyme is a pyridoxal phosphate-dependent, active in all organs, located in mitochondria (Machingura et al. 2016 and citations therein). The role of β-CAS in higher plants (cyanogenic and non-cyanogenic) was described by Machingura et al. (2016), pointing at its action in the maintenance of cyanide homeostasis, nitrogen and sulphur metabolism. Both ethylene and HCN are modulators of β-CAS activity. The biochemical mechanism of action of this enzyme is linked to the replacement of the cysteine sulfhydryl group with CN− forming β-cyanoalanine. In this reaction, hydrogen sulfide is released (Fig. 2A) (Ebbs et al. 2010; Machingura et al. 2016). Further, β-cyanoalanine is enzymatically converted to asparagine (by an enzyme of nitrilase activity) or aspartate and ammonium (by an enzyme of nitrile hydratase) (Piotrowski et al. 2001; Piotrowski and Volmer 2006). Asparagine may be also converted to aspartate and ammonium via the hydrolytic activity of the two subtypes of asparaginase (Bruneau et al. 2006; Lea et al. 2007). During the response of plants to biotic stress (linking to the higher ethylene production) induction of β-CAS activity may prevent autotoxicity, and minimise carbon and nitrogen loss through HCN emission (Machingura et al. 2016).

Fig. 2
figure 2

The main, enzymatic routes of HCN detoxification in plants. Asparagine formation from β-cyanoalanine, the product of the reaction catalysed by β-cyanoalanine synthase A, thiocyanate formation in the reaction catalysed by rhodanese B. Detailed description in the text (according to Siegień and Bogatek (2006), modified)

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The controversial plant enzyme, rhodanese (thiosulfate:cyanide sulfurtransferase) catalyzes the reaction of cyanide conversion to thiocyanate in the presence of thiosulfate (Fig. 2B). The involvement of this enzyme in HCN detoxification has been speculated for a long time due to its very low activity. Nevertheless, the characterization of two cDNAs encoding rhodanese isoforms in Arabidopsis (AtRDH1 and AtRDH2) strongly confirmed the existence of this enzyme in plants (Hatzfeld and Saito 2000). Studies related to HCN detoxification in humans have indicated that mitochondrial rhodanese mainly uses compounds containing S–S bond (glutathione, thiosulfate, and cystine) as sulfur donors, whereas the thiols, cysteine, and reduced glutathione (GSH) are not utilized (Baskin et al. 1999).

HCN reacts with cystine disulfide bonds in proteins and peptides. The reaction formula is given below:

$${\text{RS}}^{\_} {\text{SR }} + {\text{ CN}}^{ - } \to {\text{ RS}}^{ - } + {\text{ RSCN}}$$

The reaction of cyanide and oxidized glutathione (GSSG) is analogous:

$${\text{GSSG }} + {\text{ CN}}^{ - } \leftrightarrow {\text{ GS}}^{ - } + {\text{ GSCN}}$$

This bimolecular reaction (the cleavage of the disulfide bond) is dependent on pH, temperature, and concentration of cyanide (Catsimpoolas and Wood 1966).

The most abundant non-protein thiol—GSH is a detoxifying molecule, and a non-enzymatic antioxidant in various aerobic organisms (Noctor et al. 2012). As was mentioned, HCN reacts mainly with molecules containing disulfide bonds, e.g. GSSG. However, the report indicating the reaction of cyanide (or cyanamide, NH2CN) with GSH can be found (Gyamfi et al. 2019). Reduced toxicity of cyanide was observed in the glutathione and glutathione-disulfide-pretreated mice (Hatch et al. 1990). Using an LC − MS/MS method, the 2-aminothiazoline-4-oxoaminoethanoic acid (ATOEA) was analysed as a stable product of the interaction of CN− and GSH in rabbit plasma infused with NaCN (Fig. 3). The ATOEA marker for analyses was prepared from the reaction of cyanamide with GSH (Gyamfi et al. 2019). Thus, HCN reaction with glutathione may be associated with non-enzymatic detoxification in animals (ongoing research) and in plants (there are no published data). This also implies the participation of HCN in the regulation of the redox potential.

Fig. 3
figure 3

The proposed, non-enzymatic routes of HCN detoxification in the presence of glutathione A and cystine B. PGA—pyroglutamic acid; ATOEA—2-aminothiazoline-4-oxoaminoethanoic acid; ITOEA—2-iminothiazolidine-4-oxoaminoethanoic acid; ATCA—2-amino-2-thiazoline-4-carboxylic acid; ITCA—2-iminothiazolidine-4-carboxylic acid (according to Gyamfi et al. (2019), modified)

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Molecular mechanism of HCN action

The best known, and described mechanism of HCN toxicity refers to its inhibitory effect on the mitochondrial respiration chain via binding of CN− to the ferric heme a3 of cytochrome c oxidase (COX), the terminal enzyme in Complex IV (Lawson-Smith et al. 2011; Jaszczak et al. 2017). The stable connection of COX-cyanide complex interrupts oxidative metabolism and phosphorylation. The active site of COX, which binds O2, is a binuclear metal centre constituted by a heme a3 and a copper (Cu2+). CN− binds to heme (Fe2+), and the trivalent state of iron (Fe3+) blocks electron transport to O2 leading to metabolic acidosis (Lawson-Smith et al. 2011 and citations therein). The COX oxidation state determines the rate and tightness of cyanide binding. The cyanide affinity for the oxidized form of COX is around two orders of magnitude higher compared to the reaction rate of cyanide and the reduced form of the enzyme. The extremely stable complex of oxidized COX-cyanide decomposes in the presence of reducing equivalents, leading to cyanide dissociation and the formation of active COX (Solomonson 1981). The possibility of the reversibility of COX inhibition by cyanide was observed in in vitro conditions, after washing of mitochondria or pyruvate application (Nůsková et al. 2010). Nevertheless, it may point to the modulatory effect of cyanide in cellular respiration (Zuhra and Szabo 2022). In addition, HCN is an inhibitor of the activity of other cellular electron transporters, e.g. plastocyanin in chloroplasts. The blockage of electron transport to Photosystem I (PSI) is due to the interaction of cyanide with the Cu2+ of the enzyme forming inactive apoplastocyanin (Berg and Krogmann 1975). The inhibition by cyanide of the activity of metalloenzymes, including those containing Fe, Zn or Cu, has been strongly and widely demonstrated (Fig. 4). Cyanide binds to prosthetic groups of Zn/Cu superoxide dismutase (SOD), Fe-containing catalase (CAT) and peroxidases of class III (POX). CN− also acts as an inhibitor of enzymes such as glutamate decarboxylase and nitric oxide synthase (Jaszczak et al. 2017). Zn-containing alkaline phosphatase and carbonic anhydrase are also sensitive to HCN. Cyanides inhibit seleno-enzymes, like glutathione peroxidase (GPX), an antioxidant in animal tissues (Solomonson 1981). It is postulated that HCN does not react directly with free sulfhydryl groups (Fasco et al. 2007). In plants, in GPX (GPX-like), selenocysteine is replaced by cysteine and it makes this protein less sensitive to HCN (Krasuska and Gniazdowska 2012; Bela et al. 2015). Assuming that cyanide inhibits the activity of enzymes related to the maintenance of the homeostasis of reactive oxygen species (ROS) and reactive nitrogen species (RNS), HCN is involved in the nitro-oxidative stress generation in higher organisms. On the other hand, it may impact the redox potential of cells, which is also of signal importance, especially when one considers the results related to the reaction of CN− with GSH (Gyamfi et al. 2019). It has also been revealed that cyanides can inactivate molybdoenzymes e.g. xanthine oxidase (containing non-heme Fe/labile sulfide centres) and sulfite oxidase (with heme in the form of a β-type cytochrome). The inactivation of different molybdoenzymes depends on the redox-state and varies in sensitivity to cyanide (Solomonson 1981). The activity of the key enzyme of the Calvin cycle—carboxylase/oxygenase ribulose-1,5-bisphosphate (RuBisCO), is also lowered in the presence of HCN (Solomonson 1981; Siegień and Bogatek 2006). The toxicity of HCN is associated with the modification of nitrogen metabolism by the inhibition of molybdoenzymes: nitrate reductase (NR) or nitrogenase (Li et al. 1982; Barr et al. 1995).

Fig. 4
figure 4

HCN involvement in post-translational protein modifications. M—metal ions; G—glutathione; PLP—pyridoxal phosphate, NAD+—oxidized form of nicotinamide adenine dinucleotide

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Another action of CN− is linked to the ability to modify cystine-containing proteins (Fig. 4). HCN reacts with disulfide bonds of the cystine peptides or intra- and/or interchain disulfide bonds to produce sulfhydryl group at one cysteine and covalently attached CN to the other cysteine. The sulfur thiocyanate (–SCN) derivatives are generated mostly at pH 7.0 (Fasco et al. 2007; García et al. 2019 and citations therein). At pH above 7.0, and in the presence of other sulfhydryl compound, cyanide may be released from Cys-S-cyanylated derivatives with the formation of a disulfide bond with the exogenous sulfhydryl reagent (Fasco et al. 2007). Moreover, in the absence of a sulfhydryl compound, the intramolecular cyclization of Cys-S-cyanylated derivatives may occur and a stabile metabolite—2-aminothiazoline-4-carboxylic acid (ACTA) is formed (Fasco et al. 2007; Nishio et al. 2022). For a long time, S-cyanylation was not considered to take place under physiological conditions. Nevertheless, exposure to cyanide as a result of poisoning or smoking, increases S-cyanylated-Cys residues in human plasma proteins (e.g. immunoglobulin G and serum albumin) (Fasco et al. 2007), indicating that the reaction takes place in living organisms. In leaves and roots of Arabidopsis wild type ecotype Col-0 and the T-DNA insertion mutant of l β-cyanoalanine synthase (cas-c1), the presence of S-cyanylated-Cys residues in peptides has been demonstrated even during typical, physiological conditions. The increase in the level of such modified residues was detected in the leaves and roots of 2 weeks old cas-c1 Arabidopsis seedlings treated with ACC (García et al. 2019), which were characterized by the enhanced cyanide level (García et al. 2010). In Arabidopsis roots and leaves, among S-cyanylated proteins were identified: proteins of the TCA cycle (e.g. triose-phosphate isomerase, fructose-bisphosphate aldolase, fructose 1,6-bisphosphatase), glycolysis pathway (e.g. glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, enolase), chaperones (e.g. chaperonin-60α, chaperonin-60ß2), chloroplast and mitochondrial heat shock proteins (HSP70-1 and HSP70-2, rotamase FKBP-1, and other HSPs), enzymes of the S-adenosyl-Met cycle (DNA methyltransferase2, MET synthetase1-2, S-adenosyl-L-homocystein hydrolase1) and ribosomal proteins (García et al. 2019). Redox state of the cellular environment is a fundamental modulator of thiol-containing proteins (Corpas et al. 2022). Each modification related to the oxidation of -SH groups or S-cyanylation of cystine acts directly (this particular modification) but also indirectly altering the current possibility of other modifications. The result of S-cyanylation is the formation of the -SH group (Fig. 4), which can undergo further modifications. Whether S-cyanylated proteins are more susceptible to oxidation or S-nitrosation (in the presence of nitric oxide, another gasotransmitter) (Corpas et al. 2022) needs experimental verification.

Furthermore, HCN may be involved in other post-translational protein modifications (Fig. 4). The results of in vitro experiments concerning the incubation of bovine serum albumin (BSA) with a water solution of KCN demonstrated the formation of carbonyl groups, indicating protein carbonylation (Krasuska et al. 2014). However, the exact mechanism of protein carbonylation by CN− requires further investigation. Another non-enzymatic post-translational protein modification is carbamylation, which occurs in the presence of isocyanic acid (HCNO). The irreversible reaction of HCNO with the free amino group of lysine is linked to the modification of protein structure and function and loss of enzyme activity, as was confirmed for SOD (Jaisson et al. 2018). Using COX protein, and cyanide at the nanomolar range it was demonstrated that this gas is engaged in the de-glutathionylation reaction (Randi et al. 2021). The cyanide reacts with cysteine residue covalently bound to glutathione (a nucleophilic displacement). The formation of CN-glutathione (CN-SG) is linked to the restoration of free cysteine residue (Fig. 4) (Randi et al. 2021). Cyanide also binds to pyridoxal 5’-phosphate (PLP) proteins, altering their activity (Bonavita 1960). The involvement of PLP in HCN detoxification has been confirmed using animal study (Keniston 1987). Moreover, cyanide may react directly with the carbon C-4 of the nicotinamide ring of NAD+ forming the nicotinamide-CN complex. Such modification impacts cellular metabolism e.g. ADP-ribosylation of the rat liver mitochondrial fraction (Masmoudi et al. 1988). Most of the research on HCN involvement in post-translational protein modifications concerns animals. In plants, research related to the recognition of a specific modification with the assignment of a physiological function is ongoing.

The mode of action of HCN is also related to the alterations in ions’ cellular homeostasis. The exposure of rat’s cerebellar granule cells to NaCN (100 µM) stimulated Ca2+ influx into the cells (Cai and McCaslin 1992). Moreover, cyanide treatment of animal tissues was linked to the suppression of KATP channel and inwardly rectifying K+ channel activities (Chao et al. 1996; Inoue et al. 1997). Both Ca2+ and K+ play an important role in plant physiology. Thus, it can be expected that HCN also regulates these cations’ homeostasis and physiological processes in plants.

Selected effects of cyanide action on various proteins’ function in animal and plant tissues were summarized in the Table 1.

Table 1 The main effects of cyanide action on protein function (information based on cited literature)
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Physiological functions of HCN in plants

Besides toxicity, HCN is considered a regulatory, even signalling compound. In plants, cyanide function is related to the defence mechanism towards herbivores and pathogenic organisms (Zagrobelny et al. 2008). Cyanogenesis is observed during the response of plants to pathogen attack (Siegień and Bogatek 2006; Zagrobelny and Møller 2011 and citations therein). The hypersensitive response of tobacco (Nicotiana tabacum L.) was stimulated by the treatment with KCN, which promoted the accumulation of alternative oxidase (AOX) transcripts and restored tobacco mosaic virus localization (Chivasa and Carr 1998). HCN formation in ethylene biosynthesis or during camalexin synthesis may be useful in plant-pathogen response (Böttcher et al. 2009; García et al. 2013). In Arabidopsis cys-c1 mutant plants (with loss of mitochondrial β-CAS function), increased cyanide level enhanced AOX-dependent respiration (salicylhydroxamic acid—SHAM resistant respiration) and increased the expression of AOX in leaves and roots (García et al. 2010, 2013). In addition, an expression of PR1 encoding pathogen-related protein induced by salicylic acid was detected in leaves of mutant plants in the absence of the pathogen suggesting cyanide’s impact on plant response (García et al. 2013). The cas-c1 mutation also implicates the salicylic acid-dependent response to pathogens. However, this action is independent of protein ubiquitination or NPR1 (nonexpresser of PR genes, the central regulator of the salicylic acid-mediated pathway) (Arenas-Alfonseca et al. 2021). In roots’ mitochondria, active β-CAS is responsible for sustaining the low level of HCN, for undisturbed root hair development. As was demonstrated for Arabidopsis, plants lacking β-CAS-C1 were characterized by abnormal root hairs, formed as a result of the inhibition of cell elongation (Arenas-Alfonseca et al. 2018). In the cys-c1 mutant, genes encoding cell wall proteins (arabinogalactan-protein (FLA6), arabinogalactan-protein (AGP3), two xyloglucan:xyloglucosyl transferases, xyloglucan endotransglycosylase (XTR9), pectate lyase, two pectinesterases, proline-rich protein (PRP3), proline-rich extensin-like protein, expansin (EXP18)) were down-regulated (García et al. 2010). The transcript profile of the cys-c1 mutant showed lowered levels of transcript related to ethylene signalling and metabolism, e.g. one member of the ACC synthase gene family (ACS6) (García et al. 2010). Moreover, Arenas-Alfonseca et al. (2018) postulated that the role of β-CAS in root hair elongation is independent of ROS production resulting from NADPH oxidase activity.

HCN modulates seed dormancy state. Released cyanides from cyanohydrins present in the smoke after a wildfire stimulate seed germination of many plant species (Flematti et al. 2013). During the imbibition of cyanogenic—e.g. apple (Malus domestica Borkh.) or non-cyanogenic—e.g. sunflower (Helianthus annuus L.) seeds, various pathways of HCN formation were considered. HCN may also exhibit a different biological activity in these seeds (Bogatek et al. 1991; Esashi et al. 1991; Oracz et al. 2008). In cotyledons of apple embryos, pretreated with HCN, the alterations in the activity of enzymes involved in lipid and sugar catabolism were demonstrated (Bogatek and Lewak 1991). HCN regulates the electron transport in the mitochondrial respiratory chain via the inhibition of COX activity and stimulation of the AOX activity (Siegień and Bogatek 2006 and citations therein). Such modulation of the electron transport may trigger signal/signals for seed germination (Siegień and Bogatek 2006). ROS are key regulators of seed dormancy release and germination (Bailly et al. 2008). HCN fumigation of apple or sunflower embryos stimulated ROS production during germination (Oracz et al. 2009; Gniazdowska et al. 2010b,a), and hydrogen peroxide secretion to the germination medium (Gniazdowska et al. 2010b). Moreover, as HCN-shortly-treated apple embryos’ germination proceeded, the increase in the activity of enzymatic antioxidants (SOD, CAT, POX, GPX-like, glutathione reductase) was detected (Krasuska and Gniazdowska 2012). Increased ROS production in sunflower or apple embryos shortly exposed to HCN was accompanied by protein carbonylation. Among carbonylated proteins, storage proteins were identified and stimulation of proteolytic activity was observed (Oracz et al. 2007; Krasuska et al. 2014). In addition, HCN regulates the level of RNS, the nitric oxide (NO) derivatives, which strongly modulate seed physiology (Ciacka et al. 2022 and citations therein). Reduced dormancy of Arabidopsis seeds treated with sodium nitroprusside (SNP, Na2[Fe(CN)5NO]) (a donor of NO and cyanide), potassium ferricyanide (Fe(III)CN) and potassium ferrocyanide (Fe(II)CN) and KCN were observed (Bethke et al. 2006). The authors demonstrated that the effects of cyanide on seed dormancy were prevented by the NO scavenger (c-PTIO). Short-term pretreatment of apple embryos with HCN resulted in the stimulation of NO emission from embryonic axes (Krasuska and Gniazdowska 2012). Thus, there is a network of HCN-ROS-RNS interactions in seeds. HCN released from cyanogenic compounds or ACC can act as a trigger of the ROS and RNS signal. The non-enzymatic ACC decomposition to ethylene was observed in the presence of KCN (in vitro assay), similarly, an increase in ethylene emission from apple embryos shortly pretreated with HCN was demonstrated (Gniazdowska et al. 2010a). HCN fumigation also enhanced the activity of enzymes of ethylene biosynthesis in germinating apple embryos (Gniazdowska et al. 2010a) and increased embryos’ sensitivity to ethylene (Gniazdowska et al. 2010b). However, the treatment of sunflower seeds with HCN was more likely related to modulation of the ethylene signalling pathway, as the stimulation of the expression of the transcription factor Ethylene Response Factor 1 (ERF1) was observed (Oracz et al. 2008). Recently, air-dry Amaranthus spp. seeds gas-priming with HCN has been demonstrated, and such a method was proposed to be useful in horticulture and agriculture for the improvement of seed germination rate (Kępczyński 2021). Under stress conditions, HCN may be toxic and lowers seed germination rate. In tobacco seeds germinated in saline solutions (50 and 100 mM), overexpression of CAS improved the germination rate (Yu et al. 2021).

Cyanides serve as a nitrogen source for plants, fungi and microorganisms (Gupta et al. 2010). The reversibility of the inhibition of NR by cyanide as well as the very high sensibility of the enzyme to this gaseous compound, point at the regulatory function of HCN in nitrogen metabolism (Solomonson 1981). It has been proposed a trophic role of cyanogenic glucosides in young leaves of flax plants grown under nitrogen (N) deficit conditions (Siegień et al. 2021). Such conditions inhibited the plants’ growth, and lowered N and soluble protein contents in leaves. A decreased cyanogenic glycoside (linamarin and lotaustralin) concentration under N-limitation was observed only in young leaves. However, the level of linamarin and lotaustralin was about tenfold higher in young than in mature leaves, in which cyanogenic glycosides play probably a protective role. Despite the N deficit, flax plants accumulated N in cyanoglycosides. The authors proposed that N from cyanoglycosides may be utilized for amino acids synthesis, as high activity of β-CAS, especially in N-deficient leaves was observed (Siegień et al. 2021).

The effects of exogenous cyanide (KCN) on the concentrations and profiles of amino acids were analyzed in rice (Oriza sativa L.) seedlings growing in the presence of NO3−, ammonium (NH4+) or in N-deficiency, as KCN was the only source of N (Li et al. 2023). Stimulated growth of rice seedlings was observed in KCN-treated and NH4+ supplemented plants compared to the plants supplemented with NO3− or N-deficient plants. Moreover, the application of KCN did not affect the total amount of amino acids in rice seedlings at the same N fertilized condition, suggesting that KCN is quickly metabolized by rice seedlings (Li et al. 2023).

Conclusion

Taking into account the cellular multifunctionality, HCN could be included in the group of modulators or gasotransmitters in plant and animal organisms (Siegień and Bogatek 2006; García et al. 2019; Zuhra and Szabo 2022). Wang (2015) formulated the classification and criteria required for a molecule which may be called a “gasotransmitter”: (1) A small, gaseous molecule dissolved or not in the cytosol (biological milieu) (2) Permeability through the membranes, thus intracellular and intercellular movements do not rely on membrane receptors or transporters, (3) Endogenous production depending on specific substrate and enzyme, under metabolic control, the molecule should not be the product of metabolism only, (4) Well-defined specific functions at physiologically relevant concentrations, (5) Endogenously synthesised gas can be mimicked by its exogenously applied counterpart, (6) It is possible to describe its signal transduction pathway and specify cellular and molecular targets.

Although some aspects of cyanide activity and action especially in plants are still not fully recognized, the knowledge of this molecule is in progress. Under physiological conditions of the cells, HCN exists in gaseous form and is diffusible via membranes to reach multiple cellular targets. HCN is produced endogenously in a regulated manner (Zuhra and Szabo 2022). Depending on the concentration, HCN may function as a cytotoxic compound (high, millimolar concentration) or signalling compound (1–10 µM for animal cells; 1–100 µM for plant tissues) (Fig. 5) (Siegień and Bogatek 2006; Zuhra and Szabo 2022). There is observed increased information on the HCN mode of action, e.g. through post-translational modifications of proteins or inhibition of enzymatic activities as a result of binding to transition metal ions (Fig. 5). The specific physiological activity of cyanide in plants can be clarified based on experiments performed using its exogenous sources or after modification of its endogenous emission. According to current knowledge, we propose a dual physiological role of HCN (as a gasotransmitter or/and a toxin), depending on the concentration (Fig. 5).

Fig. 5
figure 5

The model of a dual, physiological/pathophysiological role of HCN in plants. In general, HCN at low concentration (1–100 µM) may act as a signaling molecule. HCN at high concentration—above 100 µM acts as the poison, which is visualized as light or dark cloud, respectively. The shaded background symbolizes concentration dependent impact of HCN on cellular plant metabolism

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