The -cyanoalanine synthase pathway: beyond cyanide detoxification

Plant, Cell and Environment (2016) 39, 2329–2341

doi: 10.1111/pce.12755

Review

The β-cyanoalanine synthase pathway: beyond cyanide detoxification Marylou Machingura1, Eitan Salomon2, Joseph M. Jez2 & Stephen D. Ebbs3 1

Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA, 2Department of Biology, Washington University in St. Louis, St. Louis, MO 63130, USA and 3Department of Plant Biology and Center for Ecology, Southern Illinois University, Carbondale, IL 62901, USA

ABSTRACT Production of cyanide through biological and environmental processes requires the detoxification of this metabolic poison. In the 1960s, discovery of the β-cyanoalanine synthase (βCAS) pathway in cyanogenic plants provided the first insight on cyanide detoxification in nature. Fifty years of investigations firmly established the protective role of the β-CAS pathway in cyanogenic plants and its role in the removal of cyanide produced from ethylene synthesis in plants, but also revealed the importance of this pathway for plant growth and development and the integration of nitrogen and sulfur metabolism. This review describes the β-CAS pathway, its distribution across and within higher plants, and the diverse biological functions of the pathway in cyanide assimilation, plant growth and development, stress tolerance, regulation of cyanide and sulfide signalling, and nitrogen and sulfur metabolism. The collective roles of the β-CAS pathway highlight its potential evolutionary and ecological importance in plants. Key-words: cyanide; cyanide detoxification; nitrilase; nitrile hydratase; β-cyanoalanine; β-cyanoalanine synthase.

INTRODUCTION Cyanide is a common compound in nature, arising from both anthropogenic and natural sources (Fig. 1). Cyanide shows a complex speciation in water and soil. The molecule can exist as free cyanide (CN- or HCN), in complexes with heavy or transition metals (i.e. metal-cyanide complexes), as cyanate or thiocyanide (e.g. CNO- or SCN-, respectively) or as organocyanides (e.g. nitriles). The chief sources of environmental cyanide are anthropogenic, arising from use in or as a by-products from organic chemical, plastic and rubber synthesis, electroplating and steel production, the extraction of precious metals from ore, and the photographic, aluminium and manufactured gas industries (ATSDR 1997). Total cyanide concentrations present in the waste streams can vary from 1– 65 000 mg L 1 (Beck et al. 2005; Dash et al. 2009). Soil bacteria and fungi are among the most prevalent natural sources of free cyanide (Castric 1981; Antoun et al. 1998; Gallagher & Manoil 2001; Blom et al. 2011). For example, some species of Correspondence: S. D. Ebbs, e-mail: [email protected] © 2016 John Wiley & Sons Ltd

Pseudomonas can release more than 100 mg CN- kg 1 soil (Crutzen & Andreae 1990; Kesler-Arnold & O’Hearn 1990; Owen & Zdor 2001; Barber et al. 2003; Rudrappa et al. 2008). Fungal cyanogenesis by Marasmius, Ascomycetes, Basidomycetes and several other genera has been demonstrated (Knowles 1976). There are a limited number of cyanogenic arthropods (Wong-Chong et al. 2005). Once released, atmospheric cyanide has a half-life of 2–4 months (Li et al. 2000), although some references estimate the half-life to be 1–3 years (ATSDR 1995). The half-life in oceans predicted by model simulation is 1–2 months (Li et al. 2000). Forest fires also release cyanide (Andreae & Merlet 2001). Up to 49 μg HCN L 1 have been detected in storm water runoff after a wildfire, and such concentrations are toxic to aquatic life (Crutzen & Andreae 1990). While free cyanide is rapidly degraded by soil microorganisms, formation of complexes between cyanide and transition metals (e.g. Fe and Co) can produce recalcitrant complexes that persist in the soil and are not amenable to biodegradation (Ghosh et al. 2005a). Ethylene biosynthesis is the ubiquitous source of cyanide in plants. Conversion of 1-amino-cyclopropane-1-carboxylic acid (ACC) to ethylene releases cyanoformic acid, which spontaneously decarboxylates to release CN- (Peiser et al. 1984; Manning 1986). Ethylene synthesis occurs throughout plant growth and development, but increases significantly when plants are subjected to either biotic or abiotic stress (Morgan & Drew 1997; Seo et al. 2011), which then leads to increased cyanide production (Woodrow et al. 2002; Liang 2003). Cyanide may also be enzymatically produced using glyoxylate from photorespiration and hydroxylamine from nitrate assimilation (Solomonson & Spehar 1981; Hucklesby et al. 1982). Plants use the toxicity of cyanide for protection. More than 3000 species of plants produce cyanogenic glycosides as storage forms of nitrogen and defence compounds (Koukol et al. 1962; Cutler & Conn 1982; Selmar et al. 1988; Bennet & Wallsgrove 1994; Vetter 2000). While the majority of these species are angiosperms, nearly 300 species of ferns and at least 48 species of gymnosperms synthesize these compounds (Harper et al. 1976). Hydrolysis of cyanogenic glycosides in response to herbivory/tissue damage (Seigler 1991; Kadow et al. 2012) or during decomposition of plant material in soil (Widmer & Abawi 2002) leads to the release of cyanide. Degradation of glucosinolates in the Brassicaceae family (Donkin et al. 1995) 2329

2330 M. Machingura et al. uptake and alternative metabolic pathways for cyanide detoxification, including the rhodanese (i.e. sulfurtransferase) and formamide hydrolyase pathways.

The β-cyanoalanine synthase cyanide assimilation pathway

Figure 1. Endogenous and exogenous sources promote plant exposure to cyanide. Endogenous refers to in vivo sources of cyanide in higher plants (green box). Exogenous sources include the primary natural sources and anthropogenic sources (orange box).

and in camalexin biosynthesis in Arabidopsis thaliana (Böttcher et al. 2009) can also generate cyanide. Moreover, some algae, such as Chlorella vulgaris, produce free cyanide during oxidation of some amino acids such as histidine (Pistorius et al. 1977). Because cyanide is an inhibitor of electron transport and metalloenzymes (Solomonson 1974; Solomonson 1981; Echevarria et al. 1984), organisms rapidly metabolize this poison. In plants, the canonical cyanide metabolism pathway is the β-cyanoalanine synthase (β-CAS) pathway (Tschiersch 1964; Ting & Zschoche 1970; Miller & Conn 1980; Yu et al. 2012a). Here we review more than 50 years of investigations that establish the protective role of the β-CAS pathway, but also reveal the broader importance of this pathway for plant growth and development and the integration of nitrogen and sulfur metabolism. Unless otherwise noted, any reference to cyanide, hereafter, is to free cyanide (i.e. either CN- or HCN). A recent review (Yu 2015) provides detailed information on cyanide

The β-CAS pathway (Fig. 2) is the principal route for cyanide detoxification in plants (Meyers & Ahmad 1991; Ogunlabi & Agboola 2007). The discovery of the pathway dates to early investigations of cyanogenic glycoside metabolism using labelled H14CN to determine the origin of the nitrile group associated with plant cyanogenic glycosides (Blumenthal-Goldschmidt et al. 1963). Surprisingly, cyanogenic and acyanogenic plants showed incorporation of the radiolabel in asparagine instead of cyanogenic glycosides. Subsequent work revealed cyanoalanine as the principle product formed shortly after exposure to cyanide and led to the identification of key enzymes in the pathway (Ressler et al. 1963; Nigam & Ressler 1964; Tschiersch 1964; Floss et al. 1965; Fowden & Bell 1965; Blumenthal et al. 1968). Biochemically, the β-CAS pathway facilitates incorporation of cyanide into cysteine and the allocation of the cyanide nitrogen into amino acids or for ammonia production (Fig. 2). βCAS catalyses the first step in the pathway, which involves substitution of the sulfhydryl moiety of cysteine with cyanide to yield the nitrile β-cyanoalanine and concomitant release of H2S (Hasegawa et al. 1995b; Warrilow & Hawkesford 1998). Structural and biochemical studies of β-CAS from Arabidopsis, spinach (Spinacia oleracea) and soybean (Glycine max) established this enzyme as a member of the pyridoxal phosphate-dependent β-substituted alanine synthase family, which includes the related cysteine biosynthesis enzyme Oacetylserine sulfhydrylase (also known as O-acetylserine thiol-lyase or cysteine synthase) (Hatzfeld et al. 2000; Yamaguchi et al. 2000; Watanabe et al. 2008; Yi et al. 2012, Yi & Jez, 2012). Next, a bi-functional nitrilase/nitrile hydratase

Figure 2. Two pathways for cyanide assimilation, the sulfurtransferase and β-cyanoalanine synthase pathways. Reactants and products of the pathways are shown in normal font and enzymes associated with the pathway are italicized. Portions of molecules derived from cyanide are shown in red. The product of the sulfurtransferase pathway is thiocyanate. In the second pathway, incorporation of cyanide into β-cyanoalanine is the first step. Next, a bifunctional nitrilase/nitrile reductase uses asparagine as an intermediate to formation of aspartate and ammonium. Possible reactions associated with the β-cyanoalanine synthase pathway involving the formation of the dipeptide γ-glutamyl-β-cyanoalanine are shown as dashed lines as the enzymes mediating these reactions are unconfirmed. © 2016 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 2329–2341

The plant β-cyanoalanine synthase pathway 2331 converts β-cyanoalanine into asparagine (nitrilase), which is then metabolized to aspartate and ammonia (nitrile hydratase) (Piotrowski et al. 2001; Piotrowski & Volmer 2006; Jenrich et al. 2007; Kriechbaumer et al. 2007). Early literature suggested A. thaliana and some species of vetches (i.e. Lathyrus and Vicia) incorporate β-cyanoalanine into the dipeptide γ-glutamyl-β-cyanoalanine, which is either an additional intermediate in the β-CAS pathway or an alternate metabolite (Ressler et al. 1963; Ressler et al. 1969; Watanabe et al. 2008). Recent evidence from Arabidopsis suggests that β-CAS produces γ-glutamyl-β-cyanoalanine and that the dipeptide may serve as a storage molecule (Watanabe et al. 2008). Currently, the metabolic relevance of this potential sidereaction and route to β-cyanoalanine is unresolved and requires further investigation. Following the initial identification of the β-CAS pathway, much of the ensuing research focused on cyanogenesis in plants, especially the production of cyanogenic glycosides. This leads to a resurgence in studies of the β-CAS pathway and its role in metabolizing the cyanide released from cyanogenic glycosides (Mizutani et al. 1991; Seigler 1991) and for the synthesis of amino acids in germinating seeds (e.g. Hasegawa et al. 1994; Hasegawa et al. 1995a). This progress prompted additional research into the distribution of β-CAS across plant species and its localization within plant tissues. Through those efforts, a greater understanding of the diverse biological roles of the βCAS pathway emerged.

Distribution of the β-cyanoalanine pathway across plant species and within plant cells Numerous angiosperms show activity of the β-CAS pathway ( Table 1). The list of plants that possess this pathway may be considerably longer if indirect evidence of cyanide assimilation is also considered (Larsen et al. 2004; Yu et al. 2004), with at least 25 additional plant species that could be included. Activity of the pathway does not appear to be restricted to any particular organ. Multiple tissues, including roots, stems, tubers, leaves, buds, cotyledons, fruits and seeds of numerous plants, display both β-CAS and the nitrilase/nitrile hydratase activity (Table 1). Moreover, the activity of each enzyme varies in each tissue across different plants and can also change temporally, increasing as flowers and fruits mature or as flowers or fruits cut from plants age (Manning 1986; Mizutani et al. 1986; Mizutani et al. 1987; Mizutani et al. 1988; Wen et al. 1997). The spatial distribution of the pathway within specific tissues has been deduced by examining the magnitude of β-CAS activity. The activity of this enzyme in leaves is generally greatest in the mesophyll layer. Fractionation studies with sorghum (Sorghum bicolor), maize (Zea mays), pea (Pisum sativum) and leek (Allium ampeloprasum) detected β-CAS activity in the abaxial and adaxial epidermis, the mesophyll and for the monocots in the bundle sheath cells (Wurtele et al. 1984). For leek, pea and sorghum, activity localized to the mesophyll was approximately eightfold greater than activity in the epidermal tissues. A similar difference in activity was observed between the mesophyll © 2016 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 2329–2341

and bundle sheath cells of sorghum (Wurtele et al. 1984). The distribution of activity across the leaf appears to be influenced by developmental factors, at least in some plant species. The spatial distribution of β-CAS activity along 2 cm increments of individual barley (Hordeum vulgare) leaves increased from base to tip with the older apical segments of the leaf containing 70% of the total activity (Wurtele et al. 1985). The increase in activity along the developmental axis of the leaf was attributed in this case to a specific increase in β-CAS activity in the mesophyll cell layer with cell age. These results contrast with those for maize roots in which greater activity was associated with the younger cells of the tip as compared with the older segments of the root (Stulen et al. 1979). Despite spatial differences in the magnitude across and within plant tissues, β-CAS would be required to some degree in all cells because cyanide produced endogenously or taken up from the rhizosphere would exist at physiological pH as HCN (Dzombak et al. 2005; Ghosh et al. 2005b) and be capable of diffusing throughout plant tissues. Within the plant cell, the β-CAS protein localizes to the mitochondria (Hendrickson & Conn 1969; Warrilow & Hawkesford 1998; Hatzfeld et al. 2000; Yamaguchi et al. 2000; Maruyama et al. 2001; Watanabe et al. 2008; Lai et al. 2009). Reports of cytosolic β-CAS activity reflect the low βCAS activity of the cysteine biosynthetic enzyme Oacetylserine sulfhydrylase (Hasegawa et al. 1995a; Maruyama et al. 1998; Liang & Li 2001). Localization of βCAS to the mitochondria is perhaps logical given that cyanide binds irreversibly to the terminal cytochrome c oxidase of the mitochondrial electron transport chain, thereby inhibiting electron flow (Solomonson 1981). In addition to several versions of cytochrome c oxidase (Siedow 1982; McIntosh 1994), plants possess an alternative oxidase, which provides a parallel pathway for mitochondrial electron flow from Complex I and ubiquinone to oxygen by diverting electron flow to oxygen prior to Complex III and IV. The alternative pathway does not make the same contribution to the proton gradient across the inner mitochondrial membrane as flow through cytochrome c oxidase, so there is a concomitant decrease in ATP synthesis (Siedow & Day 2000). Alternative oxidase does not contribute to the removal of cyanide, so the impact of ATP synthesis would presumably increase with the duration and/or concentration of cyanide exposure. The nitrilase/nitrile hydratase that mediates the conversion of cyanoalanine is likely a cytosolic protein, as green fluorescent protein fusions with the Arabidopsis enzyme localize to the cytosol (Piotrowski 2008). This is in agreement with a proposed model in tobacco (Nicotiana tabacum L.) where under normal conditions nitrilase-like proteins interact with ethylene response element–binding proteins (EREBPs) in the cytosol. Upon signalling, the complex disintegrates and EREBPs are released and translocated to the nucleus to activate expression of defence genes such as tomato (Solanum lycopersicum) Pto kinase (Xu et al. 1998). Detection of nitrilase in the nucleus suggests a potential regulatory role for expression of ethylene-associated genes (Piotrowski 2008).

2332 M. Machingura et al. Table 1. Plant species in which the intermediate β-cyanoalanine from the β-cyanoalanine synthase (β-CAS) pathway has been detected or for which activity of pathway enzymes such as β-CAS, nitrilase or nitrile hydratase has been detected in response to the presence of cyanide

Plant species Alfalfa (Medicago sativa) Apple (Pyrus malus) Arabidopsis (Arabidopsis thaliana) Avocado (Persea gratissima) Barley (Hordeum vulgare) Blue lupine (Lupinus angustifolia) Cabbage (Brassica oleracea) California poppy (Eschscholzia californica) Carnation (Dianthus caryophyllus) Cassava (Manihot esculenta) Castor bean (Castor communis) Clover (Trifolium repens) Cocklebur (Xanthium pennysylvanicum) Common flax (Linum usitatissimum) Common vetch (Vicia sativa) Cotton (Gossypium hirsutum) Crabgrass (Digitaria ischaemum) Cucumber (Cucumis satiyus) Flat pea (Lathyrus sylvestris) Grape (Vitus vinifera) Indian mustard (Brassica juncea) Japanese plum (Prunus salicina) Leek (Allium porrum) Lettuce (Lactuca sativa) Lima bean (Phaseolus lunatus) Loquat (Eryobotrya japonica) Lotus (Lotus tenuis) Maize (Zea mays) Mungbean (Phaseolus aureus) Pea (Pisum sativum) Potato (Solanum tuberosum) Rice (Oryza sativa) Rubber tree (Hevea brasiliensis) Satsuma mandarin (Citrus unshiu) Sorghum (Sorghum bicolor) Soybean (Glycine max) Spinach (Spinacea oleracea)

Tissues assayed for β-CAS pathway intermediates or enzyme activity

Reference

Whole seedlings Fruits, seeds Shoot, leaves, roots

Miller & Conn (1980) Han et al. (2007), Mizutani et al. (1988) Hatzfeld et al. (2000); Yamaguchi et al. (2000)

Fruits, seeds Whole seedlings, leaves, seeds

Whole seedlings, leaves Whole seedlings

Yip & Yang (1988) Forslund & Jonsson (1997); Goudey et al. (1989); Tittle et al. (1990); Wurtele et al. (1985) Akopyan et al. (1975), Hendrickson & Conn (1969); Miller & Conn (1980); Piotrowski & Volmer (2006) Miller & Conn (1980) Miller & Conn (1980)

Flowers

Manning (1986)

Shoot, leaves, tubers Shoot

Echeverry-Solarte et al. (2013); Elias et al. (1997); Marrero-Degro et al. (2011) Tschiersch (1964)

Whole seedlings Whole seedlings, seeds

Miller & Conn (1980) Hasegawa et al. (1994); Maruyama et al. (1996)

Shoot, roots

Miller & Conn (1980)

Whole seedlings

Miller & Conn (1980), (Ressler et al. 1963)

Roots

Ting & Zschoche (1970)

Shoot

Abdallah et al. (2006); Grossmann & Kwiatkowski (2000)

Fruits

Hasegawa et al. (1995a)

Seed

Ressler et al. (1963)

Fruits Whole seedlings

Mizutani et al. (1988) Miller & Conn (1980)

Fruits

Mizutani et al. (1991)

Leaves Whole seedlings Leaves

Wurtele et al. (1984) Goudey et al. (1989) Miller & Conn (1980)

Whole seedlings

Miller & Conn (1980)

Whole seedlings Leaves Whole seedlings

Miller & Conn (1980) Kriechbaumer et al. (2007); Wurtele et al. (1984) Goudey et al. (1989); Miller & Conn (1980)

Whole seedlings, leaves, roots Tuber

Goudey et al. (1989); Wurtele et al. (1984) Maruyama et al. (2001); Wen et al. (1997)

Whole seedlings, leaves, seeds Leaves

Lai et al. (2009), Yu et al. (2012) Chrestin et al. (2004)

Fruits

Mizutani et al. (1988)

Shoot, leaves, roots Whole seedlings Shoot, leaves, roots

Jenrich et al. (2007); Miller & Conn (1980); Wurtele et al. (1984) Tittle et al. (1990); Yi et al. (2012) Hatzfeld et al. (2000); Warrilow & Hawkesford (1998)

Whole seedlings, shoot, leaves

(Continues) © 2016 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 2329–2341

The plant β-cyanoalanine synthase pathway 2333 Table 1. (Continued)

Plant species Sweet pea (Lathyrus odoratus) Tobacco (Nicotiana tabacum) Wheat (Triticum aestivum) Wild mustard (Sinapis arvensis)

Tissues assayed for β-CAS pathway intermediates or enzyme activity

Reference

Shoot

Tschiersch (1964)

Leaves, roots

Liang (2003); Liang & Li (2001)

Whole seedlings Whole seedlings

Goudey et al. (1989) Goudey et al. (1989)

Biological roles of the β-cyanoalanine synthase pathway: endogenous cyanide As cyanide is a well-known enzyme inhibitor and uncoupler, the need for a pathway to detoxify cyanide seems clear (for reviews, see Ebbs 2004; Gleadow & Møller 2014; Siegień & Bogatek 2006; Yu 2015). Cyanogenic plants clearly require activity of this pathway to detoxify the cyanide released from cyanogenic glycosides and cyanolipids. Yet, a wide variety of acyanogenic species also contain the β-CAS pathway. The sole function of the β-CAS pathway as a mere cyanide detoxification mechanism for cyanide from cyanogenic glycosides seems inadequate to justify the ubiquity of this pathway in acyanogenic plants. New insights emerging from more recent studies of this pathway suggest that the functional contributions of the pathway and/or its individual enzymes are more diverse (Fig. 3). The pathway likely serves as a metabolic bridge that links cyanide and cyanogenic compounds to primary nitrogen

Figure 3. Functions of the β-cyanoalanine synthase in higher plants. Cyanide homeostasis refers to assimilation of endogenously produced cyanide, such as during production of ethylene under normal conditions or during periods of abiotic or biotic stress. Maintenance of cyanide homeostasis also regulates processes in growth, development and programmed cell death responsive to cyanide. The contribution of the pathway to nitrogen metabolism includes metabolism of cyanide released from cyanogenic glycosides or cyanolipds or as a source of + + NH4 from direct formation of asparatate and NH4 from β-cyanoalanine via nitrile hydratase activity, or from formation of asparagine from cyanoalanine via nitrilase activity. Metabolism of exogenous cyanide includes both anthropogenic and natural sources for the functions indicated. A direct role in cysteine synthesis in the mitochondria has been demonstrated and an indirect role in H2S signalling is likely as a 2result of cyanide assimilation with concomitant release of S . © 2016 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 2329–2341

metabolism in plants (Gleadow & Møller 2014). Relegating the function of this pathway solely to cyanide detoxification may, therefore, be an oversimplification of its occurrence and biological roles.

Assimilation of cyanide from cyanogenic glycosides Cyanogenic glycosides are water-soluble β-hydroxynitriles (cyanohydrins) and β-glycosides found in more than 3000 taxa across the families Fabaceae, Rosaceae, Linaceae, Compositae and others (Koukol et al. 1962). Common examples of cyanogenic glycosides include dhurrin, amygdalin, prunasin and linamarin. The concentration of these compounds varies widely in plants and is largely a result of complex interaction of several factors. These include plasticity within species and populations (Vetter 2000; Hayden & Parker 2002; Ballhorn et al. 2009; Echeverry-Solarte et al. 2013; Gleadow & Møller 2014), biotic factors such as fungal infection (Ballhorn 2011) and abiotic factors such as light, temperature, nutritional status and drought (Siegień et al. 2013; Gleadow & Møller 2014). A principle biological role for cyanogenic glycosides is as a deterrent to herbivory (Bennet & Wallsgrove 1994). During herbivory, stored cyanogenic glycosides or de novo synthesized glycosides, come in contact with the hydrolytic enzymes. βglucosidases cleaves the glycoside to release the sugar and a cyanohydrin molecule. For highly cyanogenic plants, this so called cyanide bomb system (Morant et al. 2008) provides an effective deterrent to those herbivores that cannot detoxify, metabolize or sequester the dose of cyanide received; however, this defence system is a double-edged sword. Release of cyanide during cyanogenic glycoside metabolism risks autotoxicity that the plant must overcome by cyanide detoxification. Although the sulfurtransferase enzyme rhodanese reportedly contributes to detoxification in some cyanogenic plant tissues (Nambisan & Sundaresan 1994; Elias et al. 1997; Papenbrock et al. 2011), the majority of cyanide removal occurs through the β-CAS pathway (Gleadow & Møller 2014). In several cyanogenic plants, including cassava (Manihot esculenta), rubber tree (Hevea brasiliensis), almond (Prunus dulcis), barley and Japanese plum (Prunus salicina), the activities of enzymes in the β-CAS pathway tend to be higher in tissues with the greatest concentrations of cyanogenic glycosides, the highest rate of glycoside synthesis and/or glucosidase activity (Selmar et al. 1988; Mizutani et al. 1991; Nambisan & Sundaresan 1994; Elias et al. 1997; Forslund & Jonsson 1997; Sánchez-Pérez et al. 2008; Kongsawadworakul et al. 2009).

2334 M. Machingura et al. Because the products of cyanide assimilation by this pathway are aspartate, asparagine and ammonium, these relationships also reflect the coupling of detoxification via the β-CAS pathway to nitrogen and amino acid metabolism (Gleadow & Møller 2014; Pičmanová et al. 2015). One suggestion has been that this recycling of cyanogenic glycosides, part of which is the assimilation of liberated cyanide, lowers the cost of herbivory defence (Neilson et al. 2013). Results from almond, cassava and sorghum also suggest that the pool of cyanogenic glycosides represents more than just a mechanism for herbivory defence (Pičmanová et al. 2015). In some cyanogenic plants, for instance, cyanogenic glycosides represent a significant fraction of tissue nitrogen and organic matter. This pool of stored nitrogen provides a substantial resource that can serve as a reserve and to balance the needs of primary metabolism. Cyanide assimilation via the β-CAS pathway contributes to this larger landscape of cyanogenic glycoside metabolism as the bridge to central nitrogen metabolism.

Assimilation of ethylene-associated cyanide The contribution of the β-CAS pathway to the metabolism of ethylene-associated cyanide has been indicated by several studies demonstrating correlations in some plant species (including soybean, barley, wheat (Triticum aestivum) and some climacteric and non-climacteric fruits) between ethylene production, cyanide production and β-CAS activity (Mizutani et al. 1988; Yip & Yang 1988; Goudey et al. 1989; Lurie & Klein 1990; Mizutani et al. 1992; Hasegawa et al. 1995b; Wen et al. 1997; Mizutani et al. 1998). As for cyanogenic glycosides, this likely represents a dual response to preclude cyanide autotoxicity and to recycle carbon and nitrogen that would otherwise be lost from primary metabolism through the release of cyanide (Fujita et al. 2006; Abe et al. 2008; Lin et al. 2009; Duan et al. 2010). There is growing evidence of a signalling role for cyanide in plants associated with growth and development as well as to the response to abiotic or biotic stress (Siegień & Bogatek 2006; Oracz et al. 2008; García et al. 2014). The β-CAS pathway may also contribute to the maintenance of steady-state tissue cyanide concentrations to help regulate cyanide signalling, such as during root development or seed germination (see next sections). Ethylene induces β-CAS activity in a variety of monocot and dicot species (Goudey et al. 1989; Hasegawa et al. 1994; Hasegawa et al. 1995b; Maruyama et al. 1996; Maruyama et al. 1997; Matilla 2000; Liang 2003; Vahala et al. 2003), which illustrates the integration of ethylene (and cyanide) production with cyanide assimilation.

Biological roles of the β-cyanoalanine synthase pathway: regulation of cyanide in growth and development

Root development Control of the steady-state concentration of cyanide by the βCAS pathway contributes to regulation over specific aspects of plant growth and development, including root growth and development. For example, cyanide metabolism is implicated

in root hair formation and elongation in Arabidopsis (García et al. 2010). A T-DNA insertional mutant of the β-CAS gene in Arabidopsis displayed defects in root hair formation and a lack of elongation. When cyanide accumulates, it may become a repressive signal for genes encoding enzymes involved in cell wall rebuilding, root tip development and ethylene signalling (García et al. 2010). As a result, β-CAS maintains the low level of cyanide to maintain proper root hair development. In agreement, Howden et al. (2009) reported that impaired cyanide metabolism in Arabidopsis NIT4 mutants caused root physiological defects. Heterologous expression in Arabidopsis of the bacterial pinA gene, which encodes a nitrilase enzyme that complements the NIT4 mutation in Arabidopsis, improved root development. In addition to endogenous sources of cyanide, cyanogenic bacteria and fungi in the rhizosphere produce cyanide, which can inhibit root growth. Inoculation of chickpea (Cicer arietinum L.) with a cyanogenic strain of Pseudomonas sp. (MRS23) resulted in a >40% reduction in root length in 5 days as compared with the control plants (Goel et al. 2002). A >66% decrease in root length was observed for A. thaliana when grown in the presence of cyanogenic P. fluorescens strain CHAO. The inhibition of root growth in Arabidopsis was reportedly associated with the suppression of expression of auxin-responsive genes at the root tip (Rudrappa et al. 2008).

Seed germination The activity of the β-CAS pathway may fulfil a fundamental biological role during seed germination, at least in some species (Selmar et al. 1988; Esashi et al. 1996; Maruyama et al. 1997). Although the mechanisms by which the pathway is involved in seed germination are unclear, there is a strong correlation between β-CAS activity and changes in corresponding amino acid pools in some seeds. Assimilation of cyanide in seeds provides nitrogen and carbonyl compounds for incorporation into amino acids necessary to initiate seed germination. Seeds of a variety of plant species, such as cocklebur (Xanthium pennsylvanicum), sorghum, barley and almond, store cyanogenic compounds as a source of nitrogen (Selmar et al. 1990; Esashi et al. 1991; Sánchez-Pérez et al. 2008; Yildirim & Askin 2010; Gleadow & Møller 2014; Pičmanová et al. 2015). Immediately prior to and during germination, there is increased evolution of endogenous HCN (Esashi et al. 1996; Siegień & Bogatek 2006; Gianinetti et al. 2007). The cyanide released from these sources may help break seed dormancy and promote germination (Bogatek & Lewak 1991; Esashi et al. 1996; Maruyama et al. 1996; Oracz et al. 2008). Residual β-CAS activity was detected in seeds of rice (Oryza sativa L.) and cocklebur (Hasegawa et al. 1995a); however, β-CAS activity increased considerably during imbibition after induction following an ethylene burst. Similarly, hydrogen sulfide was evolved along with β-cyanoalanine during germination of pigweed (Amaranthus albus) and lettuce (Lactuca sativa) seeds (Taylorson & Hendricks 1973). In this study, addition of βcyanoalanine was the most effective in promoting seed germination as compared with other amino acids, increasing germination from twofold to fourfold. In addition, increased activity of the β-CAS pathway resulted in higher asparagine, © 2016 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 2329–2341

The plant β-cyanoalanine synthase pathway 2335 aspartate and ammonia levels and subsequent incorporation of the amino acids into protein, which promotes rapid seedling development. In addition to providing the embryo with amino acids for protein synthesis, another hypothesis suggests that an elevated amino acid pool lowers the water potential of the embryonic axis, which favours water influx and radical elongation (Grossmann 1996). Another benefit from activity of the βCAS pathway during germination and seedling establishment is an increase in the pool of free thiols (Vahala et al. 2003). Thiols, particularly H2S, promote seed germination in wheat seeds exposed to copper stress indirectly by increasing the pool of free amino acids that can be utilized for protein synthesis during early growth and development (Zhang et al. 2008).

Stress, ethylene and cyanide Abiotic stresses such as water deficit, ozone exposure, salinity and extreme temperatures lead to increases in both ethylene and cyanide concentrations and elevated activity of enzymes in the β-CAS pathway. Changes in the β-CAS pathway parallel increases in ‘stress cyanide’ and indicate a potential role of the pathway in protecting plants from cyanide autotoxicity. For example, deprivation of water in tobacco plants for 1 day results in a 3.5-fold increase in root cyanide concentrations, a 6.3-fold increase in shoots and a 2-fold elevation of β-CAS activity in both tissues (Liang 2003). Similar results were obtained for Arabidopsis (Col 0) plants subjected to acute water deficiency (Machingura et al. 2013). Transcript abundance of β-CAS in Arabidopsis seedlings increased by 2.5-fold. Increased tissue cyanide concentration and increased sensitivity to water deficit were observed in Arabidopsis mutants with a T-DNA insertion in the single gene encoding the nitrilase/nitrile hydratase associated the second step of the pathway (e.g. AtNIT4) (Machingura et al. 2013). Greater salinity tolerance was observed in an octaploid amphiploid genotype derived from a cross between a hexaploid bread wheat and a diploid wheatgrass (Lophopyrum elongatum) endemic to salt marshes (Jacoby et al. 2013). The greater activity of β-CAS displayed by the octaploid relative to the two parental lines may contribute to increased salinity tolerance because of elevated assimilation of ethylene-associated cyanide. Ozone treatment of birch (Betula pendula Roth) and treatment of pea with H2S led to increased β-CAS activity (Vahala et al. 2003; Cheng et al. 2013). Malformation of mango (Mangifera indica) fruits in response to low temperature has been attributed to increased concentrations of ethylene and cyanide and decreased activity of the βCAS pathway (Nailwal et al. 2006; Ansari et al. 2013). The β-CAS pathway also contributes to tolerance against auxinic herbicides. The toxic mode of action for auxinic herbicides such as quinclorac and 2,4-dichlorophenoxyacetic acid (2,4-D) involves stimulation of excess cyanide production via induced ethylene synthesis. Low β-CAS enzyme activity is also observed in some cultivars sensitive to these herbicides (Grossmann & Kwiatkowski 2000; Abdallah et al. 2006). In a study with resistant and susceptible crabgrass (Digitaria ischaemum), quinclorac induced ACC synthase activity in the susceptible biotype leading to ethylene and cyanide accumulation. There was not a significant change in ACC synthase © 2016 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 2329–2341

activity in the resistant biotype (Abdallah et al. 2006). The selective induction of ACC synthase in sensitive cultivars was suggested as the basis of resistance and/or susceptibility in different species (Grossmann & Kwiatkowski 1995). Similarly, quinclorac-tolerant rice plants did not show differences in cyanide and ethylene production or β-CAS activity (Grossmann & Kwiatkowski 1995; Grossmann & Scheltrup 1997). Tolerance to these herbicides in biotypes of smooth crabgrass (D. ischaemum), barnyard grass (Echinochloa crus-galli) and rice is related to innate differences in the induction and activity of β-CAS in response to herbicide treatment (Grossmann & Kwiatkowski 2000; Abdallah et al. 2006). The auxinic herbicide 2,4-D had the same effect (i.e. promotion of ethylene and cyanide synthesis) on susceptible soybean cultivars (Tittle et al. 1990). Other auxinic herbicides promote the same response in susceptible cultivars of some dicots (Grossmann 1996).

Response to pathogen infection Some plant pathogens also promote ethylene and cyanide synthesis in plants. For example, concentrations of ACC and cyanide increased in tobacco leaves in proximity to lesions formed by tobacco mosaic virus (Siefert et al. 1995). Studies with wheat and Fusarium head blight, as well as Arabidopsis and Botrytis cinerea, and rubber tree and bark necrosis syndrome also suggest that susceptibility to these pathogens is related to β-CAS activity (Chrestin et al. 2004; García et al. 2013; Zhang et al. 2013). In wheat, a proteomic analysis showed high expression of β-CAS in lines resistant to head blight compared with its absence in susceptible lines (Zhang et al. 2013). Similarly, proteomic studies of barley following Fusarium exposure found a decrease in β-CAS expression in the pathogen-sensitive genotype, whereas there was no change in expression in resistant genotypes (Geddes et al. 2008). In Arabidopsis, mutant plants lacking the mitochondrial β-CAS (AtCysC1) display greater sensitivity to the pathogen B. cinerea. In contrast, the same mutant line showed enhanced resistance to infection by Pseudomonas syringae and beet curly top virus, which the authors attributed to an enhanced signalling effect of cyanide in the triggering of the hypersensitive response in the infected plants (García et al. 2013). An alteration in the expression of genes or proteins associated with the hypersensitive response was also seen in other studies (Geddes et al. 2008; Zhang et al. 2013) Such results imply that the β-CAS pathway may be induced to metabolize ethylene-associated cyanide resulting from pathogen infection and to maintain a suitable homeostasis during infection. As with the response of the pathway to abiotic stresses, cyanide assimilation may prevent autotoxicity, while minimizing carbon and nitrogen loss through cyanide emission. Specific elicitors produced by biocontrol agents in the rhizosphere, as well as by commercial chemicals used as plant activators for disease control, induce β-CAS activity. In tomato (Takahashi et al. 2006), treatment with cell wall preparations from Pythium oligandrum, as well as the plant activators benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester CGH-245 704 (BTH; Bion) and 3-allyloxy-1,2benzisothiazole-1,1-dioxide (PBZ; Probenazole) induced expression of the β-CAS gene. Increased expression of β-CAS

2336 M. Machingura et al. appeared specific to these chemicals because parallel treatments with abiotic stresses (e.g. heat shock, cold shock, water deficit, salt or osmotic stress and mechanical wounding) did not yield a comparable response. The authors did acknowledge, however, that this might reflect how the treatment conditions were imposed and not a specific response. Nonetheless, the authors speculated that increased β-CAS activity may either be related to the signalling pathway associated with pathogen resistance or be a collateral response associated with ethylene-mediated signalling.

Biological roles of the β-cyanoalanine synthase pathway: assimilation of exogenous cyanide The functional role of the β-CAS pathway in plants extends to the assimilation of exogenous cyanide from either natural or anthropogenic sources. There has been several studies which have examined the uptake and assimilation of exogenous cyanide, either as free cyanide or metal complexed cyanides (e.g. ferrocyanide or ferricyanide), and the contribution of that cyanide to plant metabolism (e.g. Trapp et al. 2001b; Trapp et al. 2001a; Ebbs et al. 2003; Larsen et al. 2004; Samiotakis & Ebbs 2004; Yu et al. 2004; Larsen et al. 2005; Yu et al. 2005; Bushey et al. 2006; Larsen & Trapp 2006; Yu et al. 2007; Yu et al. 2011; Yu et al. 2012a; Yu et al. 2012b). Such studies were prompted by literature describing how plants such as willow (Salix spp) thrive in cyanide contaminated environments (Trapp et al. 2001b). Tracing of 15N-labelled molecules revealed the movement of the cyanogenic nitrogen atom into the plant tissues and supports the central role of the β-CAS pathway in the metabolism of the transported cyanide and for providing tolerance to cyanide contamination (Ebbs et al. 2003; Ebbs et al., 2008; Samiotakis & Ebbs 2004; Machingura & Ebbs 2010; Yu & Gu 2008; Yu et al. 2012a). The capacity of the β-CAS pathway to assimilate exogenous cyanide also prompted considerable focus on the potential phytoremediation of cyanide. In most feasibility studies, hydroponic systems were used to demonstrate that either free cyanide or metal cyanide complexes such as ferrocyanide or ferricyanide could be removed from soil and water by plant species such as water hyacinth (Eichhornia crassipes L.), willow (Salix spp.), poplar (Populus spp.), maize and rice (Trapp et al. 2003; Larsen et al. 2004; Larsen & Trapp 2006; Ebel et al. 2007; Yu & Gu 2007; Yu et al. 2007; O’Leary et al. 2013). Removal of free cyanide was reportedly in excess of 75% of the added cyanide but less for the metal cyanide complexes. Relative few applied and field trials were performed with the majority conducted on defunct sites associated with the manufactured gas industry. In such studies (Trapp et al. 2001b; Trapp et al. 2001a), species of willow, poplar or black elder (Sambucus nigra L.) were planted in soils with elevated concentrations of cyanide and/or iron cyanide compounds. Although extremely high cyanide concentrations led to varying degrees of phytotoxicity, the woody plants species showed strong survival on many of the cyanide-contaminated soils. Importantly, the reduction of cyanide concentration in the soil has been reported for some contaminated sites subjected to

phytoremediation (Trapp et al. 2001b; Trapp & Christiansen 2003) and applications for remediation of cyanide laden wastewaters proposed (Trapp et al. 2003; Ebel et al. 2007; Vedula et al. 2013). For further reading on cyanide uptake and metabolism in plants, readers should see the recent review by Yu (2015).

Biological roles of the β-cyanoalanine synthase pathway: linking nitrogen and sulfur metabolism Results from several studies imply that cyanide assimilation via the β-CAS pathway provides plants with an alternate nitrogen source under appropriate growth conditions. In substitution, studies where plants such as sorghum, wheat or rice were exposed to different combinations of nitrate, ammonium and/or cyanide interactions with ammonium supply were observed. Wheat and sorghum showed no change in total nitrogen when ammonium was replaced with cyanide (Ebbs et al. 2010; Whankaew et al. 2014). Rice plants treated with nitrate and either cyanide or iron cyanides displayed a greater rate of cyanide assimilation than plants treated with ammonium and cyanide (Yu & Zhang 2012; Yu et al. 2012b). Growth of both sorghum and wheat was supported for 8 weeks in hydroponic culture with cyanide as the only source of nitrogen (Whankaew et al. 2014). In rice, free cyanide, ferrocyanide and ferricyanide increased β-CAS activity (Yu et al. 2012a). Plants deprived of nitrogen also display increased accumulation of cyanide (Samiotakis & Ebbs 2004; Yu & Gu 2008; Ebbs et al. 2010; Whankaew et al. 2014), elevated expression of the β-CAS gene (Takahashi & Saito 1996) and higher levels of β-CAS and asparaginase activity (Machingura & Ebbs 2010). In the presence of cyanide, there is also evidence of reduced activity of enzymes associated with nitrogen assimilation, which would be expected concomitantly with increased cyanide assimilation via the β-CAS pathway. For instance, a decrease in activity of enzymes such as nitrate reductase and glutamine synthetase was observed in rice, carrot (Daucus carota) and the alga C. vulgaris in response to treatment with non-toxic concentrations of cyanide, ferricyanide or ferrocyanide (Solomonson 1974; Barr et al. 1995; Yu & Zhang 2012; Yu et al. 2012b). The β-CAS pathway may also contribute to plant sulfur metabolism, as a side-reaction of the enzyme mimics that of Oacetylserine sulfhydrylase from cysteine biosynthesis. As a member of the β-substituted alanine synthase enzyme family (Hatzfeld et al. 2000), β-CAS uses a pyridoxal phosphate cofactor for its reaction chemistry. Biochemical comparisons of Oacetylserine sulfhydrylase and β-CAS show that each enzyme is tailored for their respective reactions and substrates; however, each can catalyse the activity of the other with significantly reduced catalytic efficiency (e.g. Warrilow & Hawkesford 1998; Hatzfeld et al. 2000; Maruyama et al. 2001). The cysteine synthesis side-reaction of β-CAS has been observed for a number of species, including spinach, Arabidopsis, potato (Solanum tuberosum), maize, vetch, cocklebur and cassava (Stulen et al. 1979; Ikegami et al. 1988b; Ikegami et al. 1988a; Ikegami et al. 1989; Maruyama et al. 1998; Warrilow & Hawkesford 1998; Hatzfeld et al. 2000; Jost et al. 2000; © 2016 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 2329–2341

The plant β-cyanoalanine synthase pathway 2337 Yamaguchi et al. 2000; Maruyama et al. 2001; Marrero-Degro et al. 2011). For most plant species studied, O-acetylserine sulfhydrylases are found in the cytosol and chloroplast, and βCAS is localized in the mitochondria (Warrilow & Hawkesford 1998; Hatzfeld et al. 2000; Jost et al. 2000; Maruyama et al. 2000; Maruyama et al. 2001; Heeg et al. 2008; Watanabe et al. 2008; Yi et al. 2012). At one point, there was speculation that cysteine transport between subcellular compartments was limited and that the localization of enzymes with O-acetylserine sulfhydrylase activity in the cytosol, chloroplast and mitochondria was necessary for each compartment to produce its own supply of cysteine (Lunn et al. 1990). Recent evidence from Arabidopsis demonstrating that both cysteine and sulfide move between subcellular compartments questions that assertion (Heeg et al. 2008). However, unlike the plant species earlier where there is subcellular separation of β-CAS from other O-acetylserine sulfhydrylases, A. thaliana is currently the only known species that has a separate mitochondrial β-CAS and O-acetylserine sulfhydrylase for cyanoalanine and cysteine synthesis, respectively (Hesse et al., 1999, Hesse et al., 2004, Watanabe et al. 2008). One aspect of the function of the β-CAS pathway in sulfur metabolism that could perhaps be inferred is as an endogenous source of sulfide (S2-) produced from formation of βcyanoalanine. There is an expanding literature on the role of H2S as a signalling molecule, a regulator of plant growth and development, and as a contributor to abiotic stress tolerance (Hancock et al. 2011; Lisjak et al. 2013; Hancock & Whiteman 2014). These studies raise an intriguing question as to whether the detoxification of cyanide produced in response to stress ethylene and the commensurate production of endogenous H2S by the β-CAS pathway is also an inducer of the subsequent positive effects of H2S in plants under abiotic stress.

CONCLUSIONS Over 50 years, our understanding of the β-CAS pathway in plants and the diversity of its biological functions have evolved considerably. The β-CAS pathway sits at a juncture between carbon, nitrogen and sulfur metabolism and provides an alternate means of nitrogen assimilation and cysteine synthesis. The functions of this pathway include roles in plant growth, development and stress tolerance. Through the connections between ethylene and cyanide and between cyanide and sulfur, the β-CAS pathway is also a point of crosstalk between key signalling and metabolic regulatory pathways in plants. As such, the description of the β-CAS solely in terms of its function in cyanide detoxification is antiquated. Although further studies are necessary to understand the full range of functions dependent on the β-CAS pathway, the known biology suggests opportunities to examine the evolutionary origin and ecological role of this pathway. While ethylene synthesis occurs throughout the plant kingdom, ethylene production in earlier evolving plants (i.e. those derived before the Gnetophyta) is not mediated by ACC oxidase (Osborne et al. 1996) and does not result in the synthesis of cyanide. There is a clear shift to ACC© 2016 John Wiley & Sons Ltd, Plant, Cell and Environment, 39, 2329–2341

mediated ethylene synthesis in plant evolution, so it would be interesting to conduct similar examinations on the evolutionary origin of the β-CAS pathway in relation to the presence of endogenous cyanide as a selection pressure for gene evolution. The natural presence of cyanide in the environment was postulated to be a component of cyanide ‘microcycles’ (Allen & Strobel 1966; Thatcher & Weaver 1976). These microcycles consist of cyanogenic organisms and organisms that either assimilate cyanide as a source of carbon and nitrogen for growth or co-opt the cyanogenic defences of one organism for their own benefit. The role of cyanide in such ecosystems would depend upon the species composition and distribution, but would typically involve plants and insects, both of which utilize the βCAS pathway as their primary mechanism of cyanide assimilation. This may represent a larger ecological role for the pathway in these microcycles. Another already recognized microcycle is the relationship between fungal pathogens, cyanogenic bacteria releasing cyanide as a biocontrol agent, and the plants forming the rhizosphere environment where this interaction occurs. Recall that there are hundreds of plant taxa that produce cyanogenic glycosides in their tissues, often at concentrations that may exceed 1% fresh weight. When those tissues are shed, what happens to the cyanide that is returned to the environment? This return of nitrogen to the soil represents a source of nitrogen not currently considered in terrestrial nitrogen cycles, yet in some ecosystems may represent an unrecognized contributor to labile soil carbon and nitrogen. As a component of the pre-biotic environment, cyanide played a principle role in the evolution of life on earth, contributing to the formation of the first biological macromolecules. Thus, cyanide appears paradoxically to be one of the most important precursors for the generation of life (Oró & LazcanoAraujo 1981). This is summarized eloquently by E. Pfluger (Pflüger 1875), a 19th century scientist who offered some of the first speculations about the origin of biological macromolecules and life itself: If one considers the beginning of organic life, it is not necessary to pay attention first of all to carbon dioxide and ammonia, because both represent the end and not the beginning of life. The beginning is to be found to a very much larger degree in cyanogens (CN). Ironically, despite the central importance of cyanide to the formation of macromolecules and its fundamental role in the origin of life, these concepts have been largely obscured by the narrow emphasis of cyanide as a metabolic poison and inhibitor. While this is a key mode of action for this molecule, cyanide has nonetheless continued to be an important chemical in natural systems with additional in vivo functions that are slowly emerging. The evolution of the β-CAS pathway in plants is undoubtedly a response to the prevalence of cyanide. Continued study of cyanidelinked pathways is likely to provide additional insight into its diverse roles and opportunities to modulate the β-CAS pathway to alter plant growth and development and to improve plant stress tolerance.

2338 M. Machingura et al.

ACKNOWLEDGEMENTS E.S. was supported by a US-Israel Vaadia Binational Agricultural Research Development (BARD) Fund postdoctoral fellowship (FI-504-14).

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Received 12 August 2015; received in revised form 4 April 2016; accepted for publication 6 April 2016