Response to cadmium in higher plants

Environmental and Experimental Botany 41 (1999) 105 – 130

Review

Response to cadmium in higher plants L. Sanita` di Toppi *, R. Gabbrielli Dipartimento di Biologia Vegetale, Laboratorio di Fisiologia, Uni6ersita` di Firenze, 6ia Micheli 1, 50121, Florence, Italy Received 23 March 1998; received in revised form 1 November 1998; accepted 24 November 1998

Abstract The paper summarizes present knowledge in the field of higher plant responses to cadmium, an important environmental pollutant. The principal mechanisms reviewed here include phytochelatin-based sequestration and compartmentalization processes, as well as additional defense mechanisms, based on cell wall immobilization, plasma membrane exclusion, stress proteins, stress ethylene, peroxidases, metallothioneins, etc. An analysis of data taken from the international literature has been carried out, in order to highlight possible ‘qualitative’ and ‘quantitative’ differences in the response of wild-type (non-tolerant) plants to chronic and acute cadmium stress. The dose-response relationships indicate that plant response to low and high cadmium level exposures is a very complex phenomenon indeed: cadmium evokes a number of parallel and/or consecutive events at molecular, physiological and morphological levels. We propose that, above all in response to acute cadmium stress, various mechanisms might operate both in an additive and in a potentiating way. Thus, a holistic and integrated approach seems to be necessary in the study of the response of higher plants to cadmium. This multi-component model, which we would call ‘fan-shaped’ response, may accord with the Selyean ‘general adaptation syndrome’ hypothesis. While cadmium detoxification is a complex phenomenon, probably under polygenic control, cadmium ‘real’ tolerance — found in mine plants or in plant systems artificially grown under long-term selection pressure, exposed to high levels of cadmium — seems to be a simpler phenomenon, possibly involving only monogenic/oligogenic control. We conclude that, following a ‘pyramidal’ model, (adaptive) tolerance is supported by (constitutive) detoxification mechanisms, which in turn rely on (constitutive) homeostatic processes. The shift between homeostasis and ‘fan-shaped’ response can be rapid and involve quick changes in (poly)gene expression. Differently, the slow shift from ‘fan-shaped’ response to ‘real’ cadmium tolerance is caused and affected by long-term selection pressure, which may increase the frequency (and promote the expression) of one or a few tolerance gene(s). © 1999 Elsevier Science B.V. All rights reserved. Keywords: Cadmium; Heavy metal; Higher plants; Phytochelatin; Stress ethylene; Stress protein; Tolerance

* Corresponding author. Tel.: +39-055-275-7392; fax: + 39-055-282-358. E-mail address: [email protected] (L. Sanita` di Toppi)

1. Introduction Heavy metals—a group of metals with density higher than 5.0 g cm − 3 —such as cadmium (Cd), chromium (Cr), mercury (Hg), lead (Pb), aluminium (Al), silver (Ag), tin (Sn) etc., are impor-

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tant environmental pollutants, particularly in areas where there is high anthropogenic pressure. Their presence in the atmosphere, soil and water—even in trace concentrations — can cause serious problems to all organisms, and heavy metal bioaccumulation in the food chain can be highly dangerous. Cd (density = 8.6 g cm − 3) is a widespread heavy metal, released into the environment by power stations, heating systems, metal-working industries, waste incinerators, urban traffic, cement factories and as a by-product of phosphate fertilizers (Table 1). In areas with low anthropogenic pressure, Cd can be released as a result of rock mineralization processes. Baker et al. (1990) reported that Cd never occurs in isolation in natural environments, but mostly as a ‘guest’ metal in Pb/Zn mineralization. As estimated by Wagner (1993), non-polluted soil solutions contain Cd concentrations ranging from 0.04 to 0.32 mM. Soil solutions which have a Cd concentration varying from 0.32 to about 1 mM can be regarded as polluted to a moderate level. Since Cd is a fairly immobile element, its accumulation in soils can become dangerous to all kinds of organisms. Regarding its potential toxicity for soil organisms and soil microbial processes, Cd was classified by Duxbury (1985) as an element of ‘intermediate toxicity’. High to very high Cd concentrations have been found to be carcinogenic, mutagenic and teratogenic for a large number of animal species (Degraeve, 1981). In soil solutions containing Cd concentrations as high as 35 mM, Cd-‘hyperaccumulating’ species are almost the only ones that can grow; for example, amongst higher plants, the Brassicaceae Thlaspi caerulescens (Brown et al., 1994). The degree to which higher plants are able to take up Cd depends on its concentration in the soil and its bioavailability, modulated by the presence of organic matter, pH, redox potential, temperature and concentrations of other elements. In particular, the uptake of Cd ions seems to be in competition for the same transmembrane carrier with nutrients, such as potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), copper (Cu), zinc

(Zn), nickel (Ni) (Clarkson and Lu¨ttge, 1989; Rivetta et al., 1997). Several techniques have been used to estimate fluxes of essential or toxic elements in plant cells: recently, a Cd-selective microelectrode for the measurement of Cd fluxes in the roots of higher plants has been constructed (Pin˜eros et al., 1998). An increased Cd (and Pb) uptake in transgenic yeast expressing a wheat root gene, possibly encoding a putative plasma membrane transporter, has been reported by Antosiewicz et al. (1996). Cd is believed to penetrate the root through the cortical tissue. As soon as Cd enters the roots, it can reach the xylem through an apoplastic and/or a symplastic pathway (Salt et al., 1995a), complexed by several ligands, such as organic acids and/or, perhaps, phytochelatins (Cataldo et al., 1988; Przemeck and Haase, 1991; Senden et al., 1992, 1994; Salt et al., 1995b). Normally Cd ions are mainly retained in the roots, and only small amounts are transported to the shoots (Cataldo et al., 1983). It has recently been hypothesized that Cd accumulation in developing fruits could occur via phloem-mediated transport (Hart et al., 1998). Table 1 Anthropogenic emissions of Cda 103 tonnes year−1 Atmosphere Energy production Smelting and refining Manufacturing process Waste incineration Total

0.79 5.43 0.60 0.75 7.57

Soils Agricultural and animal wastes Logging and wood wastes Urban refuse Municipal sewage and organic waste Solid waste from metal fabrication Coal ashes Fertilizers and peat Discarded manufactured products Atmospheric fallout Total

2.20 1.10 4.20 0.18 0.04 7.20 0.20 1.20 5.30 21.62

O6erall total

29.19

a Modified after Nriagu and Pacyna (1988), in accordance with Mhatre and Pankhurst (1997).

L. Sanita` di Toppi, R. Gabbrielli / En6ironmental and Experimental Botany 41 (1999) 105–130

2. Effects of cadmium toxicity in higher plants In most environmental conditions Cd enters first the roots, and consequently they are likely to experience Cd damage first. In root tip cells of Allium cepa, Cd damaged nucleoli (Liu et al., 1995), and, in rice, it altered the synthesis of RNA and inhibited ribonuclease activity (Shah and Dubey, 1995). Cd also reduced the absorption of nitrate and its transport from roots to shoots, by inhibiting the nitrate reductase activity in the shoots (Hernandez et al., 1996). Appreciable inhibition of the nitrate reductase activity was also found in plants of Silene cucubalus (Mathys, 1975). The inhibition of root Fe(III) reductase induced by Cd led to Fe(II) deficiency, and it seriously affected photosynthesis (Alcantara et al., 1994). In a very general way, Cd in plants causes leaf roll and chlorosis, and reduces growth, both in roots and in stems. This last effect is partly due to the suppression of the elongation growth-rate of cells, especially in the stem, because of an irreversible inhibition exerted by Cd on the proton pump responsible for the process (Aidid and Okamoto, 1992, 1993). Cd interacts with the water balance (Barcelo´ and Poschenrieder, 1990; Costa and Morel, 1994) and damages the photosynthetic apparatus, in particular the light harvesting complex II (Krupa, 1988), and the photosystems II and I (Siedlecka and Baszynsky, 1993; Siedlecka and Krupa, 1996). In Brassica napus plants, Cd lowered total chlorophyll content, carotenoid content, and increased the non-photochemical quenching (Larsson et al., 1998). Furthermore, Cd inhibited the oxidative mitochondrial phosphorylation, probably increasing the passive permeability to H + of the mitochondrial inner membrane (Kessler and Brand, 1995). Cd also actively inhibits the stomatal opening, but how it does so has yet to be established. Probably the stomatal movements are not directly affected by Cd, but rather are due to the strong interference of Cd with movements of K + , Ca2 + and abscisic acid in the guard cells (Barcelo´ et al., 1986; Barcelo´ and Poschenrieder, 1990). Cd significantly reduces the normal H + /K + exchange and the activity of plasma membrane

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ATPase (Obata et al., 1996), and strongly affects (often by inhibiting) the activity of several enzymes, such as glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, malic enzyme, isocitrate dehydrogenase (Van Assche and Clijsters, 1990; Mattioni et al., 1997), Rubisco and carbonic anhydrase (Siedlecka et al., 1997). A marked increase in the phosphoenolpyruvate carboxylase polypeptide, but no further synthesis of glutamate dehydrogenase and glutamate synthase polypeptides was reported in Zea mays seedlings exposed to 20 mM Cd (Ju et al., 1997). In pea plants chromatin alterations were reported (Hadwiger et al., 1973): it is reasonable to hypothesize that excessive amounts of Cd, usually not present in the natural environment, replace Zn ions in the ‘zinc fingers’, and that consequently Cd interferes with the transcription mechanism. Cd was found to produce oxidative stress (Hendry et al., 1992; Somashekaraiah et al., 1992), but, in contrast with other heavy metals such as Cu, it does not seem to act directly on the production of oxygen reactive species (via Fenton and/or Haber Weiss reactions) (Salin, 1988). On the other hand, Cd ions can inhibit (and sometimes stimulate) the activity of several antioxidative enzymes. In Helianthus annuus leaves, Cd enhanced lipid peroxidation, increased lipoxygenase activity and decreased the activity of the following antioxidative enzymes: superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and dehydroascorbate reductase (Gallego et al., 1996). In Phaseolus aureus Cd ions produced lipid peroxidation, decrease of catalase activity and increase of guaiacol peroxidase and ascorbate peroxidase activity (Shaw, 1995). In Phaseolus 6ulgaris roots and leaves, 5 mM Cd enhanced activities of guaiacol and ascorbate peroxidases, and raised lipid peroxidation (Chaoui et al., 1997). Cd treatment notably increased lipid peroxidation in pea plants (Lozano-Rodrı´guez et al., 1997), whereas no peroxidation was noticed in Cd-exposed plants and hairy roots of Daucus carota (Sanita` di Toppi et al., 1998a,b). Varying responses to Cd-induced oxidative stress are probably related both to levels of Cd supplied and to concentration of thiolic groups already present or induced by Cd treat-

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ment. Thiols possess strong antioxidative properties, and they are consequently able to counteract oxidative stress (Pichorner et al., 1993). Potential effects of Cd on host – pathogen interactions were very recently examined: exposure of tobacco plants to Cd, induced the production of cellular proteins (putative anti-viral factors) capable of inhibiting the systemic spread of the ‘turnip vein clearing’ virus (TVCV) (Ghoshroy et al., 1998). Long-term experiments on the potato Phytophtora infestans relations showed that, in the presence of Cd, the development of the disease was inhibited both in leaves and in tubers. The involvement of polyamines in the transduction of P. infestans and Cd signaling has been hypothesized (Stroinski, 1997). 3. Response mechanisms to cadmium stress at cellular level

3.1. General outline In response to Cd stress, the plant cell can resort to a number of defense systems, such as: (1) immobilization; (2) exclusion; (3) synthesis of phytochelatins; (4) compartmentalization; (5) perhaps, synthesis of metallothioneins; (6) synthesis of stress proteins; (7) production of stress ethylene.

3.1.1. Immobilization A first barrier against Cd stress, operating mainly at the root level, can be the immobilization of Cd by means of the cell wall (Nishizono et al., 1989) and extracellular carbohydrates (mucilage, callose) (Verkleij and Schat, 1990; Wagner, 1993). In roots and leaves of bush bean, Cd ions seem to be mostly bound by pectic sites and hystidyl groups of the cell wall (Leita et al., 1996). However, the importance of these mechanisms may vary in accordance with the concentration of Cd supplied, the species involved, etc. For example, a negligible accumulation of Cd in cell walls of tomato suspension cultures (Inouhe et al., 1991) and in root cell walls of S. cucubalus was reported, and no differences in cell wall binding between normal and Cd-tolerant plants were observed (Verkleij et al., 1990).

3.1.2. Exclusion Preventing Cd ions from entering the cytosol through the action of the plasma membrane could theoretically represent the best defense mechanism. As a matter of fact, in early phases of radish seed germination Cd seems to enter the cells through Ca channels in the plasma membrane (Rivetta et al., 1997). Cd stress in Lactuca sati6a and Lupinus albus gave increased levels of asparagine in root exudates. However, this response was probably due to a disfunction of the plant membranes at Cd concentrations above 1 mM, rather than to a specific mechanism of amino acid induction aimed at directly chelating Cd ions (Costa et al., 1997). No doubt, a deeper understanding of these events would be very useful in widening our knowledge of Cd exclusion in higher plants. 3.1.3. Phytochelatins In general, Cd has a high affinity to metabolic processes of the sulphur metabolism, and its first effects are on ATP-sulfurylase (De Knecht et al., 1995) and adenosine 5%-phosphosulfate sulfotransferase (Nussbaum et al., 1988). Once Cd has entered the cytosol, another system strictly related to sulphur metabolism is promptly activated, finally resulting in the production of important complexing agents, termed phytochelatins, which may contribute decisively towards rendering the metal ineffective. Phytochelatins were isolated and purified for the first time by Grill et al. (1985), although since 1973 their presence had been assumed (named ‘organo-mercury complexes’) in Hg-treated tobacco leaves (Anelli et al., 1973; Pelosi and Galoppini, 1973). Heavy metals, and particularly Cd, are able to activate the synthesis of phytochelatins, which have the following general structure: (g-Glu–Cys)n –Gly, where n is the number of repetition of the unit g-Glu–Cys, normally variable from 2 to 11. Phytochelatins form various complexes with Cd (with molecular masses of about 3600 or 2500), due to the presence of the thiolic groups of Cys, which chelate Cd and, as a result, prevent it from circulating as free Cd2 + inside the cytosol (Grill et al., 1985). Using extended X-ray absorption fine structure (EXAFS) spectroscopy (Strasdeit et al., 1991), a

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Fig. 1. Schematic representation of the mechanisms involved in Cd chelation and compartmentalization in the vacuole (modified after Tomsett and Thurman, 1988). For a detailed explanation see the text and the references therein. ‘LMW’, low molecular weight complex; ‘MMW’, medium molecular weight complex (hitherto isolated only from R. serpentina cell cultures); ‘HMW’, high molecular weight complex; GSH, glutathione; PC, phytochelatins; apo-PC, apo-phytochelatins; S2 − , acid-labile sulphur; Org. ac., organic acids.

˚ in the Cd– CdS bond length of 2.5290.02 A phytochelatin complex was reported. Furthermore, some Cd – phytochelatin complexes (see below) include acid – labile sulphur (S2 − ), which give them an improved stability and a higher efficiency in Cd sequestering. Phytochelatins are synthesized from glutathione, by means of the enzyme phytochelatin synthase, a specific g-glutamylcysteine dipeptidyl transpeptidase (Grill et al., 1989). The enzyme is self-regulated, since its reaction products (phytochelatins), which start being synthesized within a few minutes after Cd supply, chelate Cd (which indeed is necessary to activate the enzyme), and the reaction ends unless further Cd is supplied (Loeffler et al., 1989). For the reaction to be concluded it is essential that no free Cd ions are available. The use of Cd-sensitive mutants of Arabidopsis thaliana was extremely useful in the demonstration of the crucial role of phytochelatins in Cd detoxification (Howden and Cobbett, 1992; Howden et al., 1995a,b). In particular, the cad1 mutant, deficient in phytochelatin synthase activity, does not form any Cd–phytochelatin complex (although it has a glutathione

level comparable with the wild-type plants), and is consequently sensitive to Cd. The production of ‘classic’ phytochelatins is a widespread mechanism of Cd detoxification in higher plants (Gekeler et al., 1989). The use of phytochelatins as biomarkers for Cd toxicity was recently proposed by Keltjens and Van Beusichem (1998). Interestingly, there are some noteworthy compounds similar but not identical to phytochelatins, collectively named iso-phytochelatins (Zenk, 1996): (a) a few members of the Fabales (especially the tribe Phaseoleae) produce homophytochelatins, derived from homo-glutathione, typically present in this order. Homo-phytochelatins are characterized by the presence of b-Ala instead of Gly as a terminal amino acid (Grill et al., 1986); (b) among the Monocotyledonae, in some Poaceae hydroxymethyl-phytochelatins were discovered, which contain Ser instead of Gly (Klapheck et al., 1994); (c) in Zea mays there are iso-phytochelatins, with Glu instead of Gly (Meuwly et al., 1993). In plants producing phytochelatins and isophytochelatins, also desGly–phytochelatins, that is to say phytochelatins lacking Gly, can be copresent in varying percentages (Bernhard and

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Ka¨gi, 1987). DesGly – phytochelatins might originate from the degradation of phytochelatins as a result of a carboxypeptidase capable of releasing Gly (Kubota et al., 1995). Interestingly, Imai et al. (1996) reported the production of cadystins (= phytochelatins) in Schizosaccharomyces pombe, in the complete absence of Cd (and other heavy metals) but in the presence of carboxypeptidase and high concentrations of glutathione.

3.1.4. Compartmentalization A very significant role in Cd detoxification and tolerance is played by vacuolar compartmentalization, which prevents the free circulation of Cd ions in the cytosol and forces them into a limited area. The mechanism is shown in Fig. 1. Exposure to Cd stimulates synthesis of phytochelatins, which rapidly form a ‘low molecular weight’ (LMW) complex with Cd (mainly Cd bound to PC3) (Vo¨geli-Lange and Wagner, 1990; Abrahamson et al., 1992; Vo¨geli-Lange and Wagner, 1996), and, in Rau6olfia serpentina cell cultures, a ‘medium molecular weight’ (MMW) complex, characterized by Cd prevalently bound to phytochelatins with a higher polymerization level (Kneer and Zenk, 1992). These complexes acquire acid–labile sulphur (S2 − ), perhaps at the tonoplast level, and form a ‘high molecular weight’ (HMW) complex (Speiser et al., 1992), with a higher affinity towards Cd ions. It is important to bear in mind that Cd detoxification seems to be related to the capacity of phytochelatins to link S2 − groups rapidly and, consequently, to bind free Cd ions more effectively. Thus, particularly the HMW complex, highly stabilized by S2 − groups, seems to be decisive in Cd detoxification. In S. pombe, Ortiz et al. (1992, 1995) found the hmt1 gene, encoding for the protein HMT1, capable of efficiently transporting Cd–phytochelatin complexes into the vacuole. Again, in S. pombe mutant JS237, signal transduction events involving cAMP and Ca ions appeared to be important for vacuolar accumulation of Cd (see Ow, 1996, and references therein). Moreover, it was found that in the presence of MgATP, Cd – phytochelatin complexes (as well as apo-phytochelatins) are transported against the concentration gradient across the tonoplast, by

means of specific carriers, and they accumulate inside tonoplast vesicles up to 38 times more than in the external solution (Salt and Rauser, 1995). Free Cd ions, if present, seem to enter the vacuole by means of a Cd2 + /2H + antiport (Salt and Wagner, 1993; Gries and Wagner, 1998). In the vacuole, because of the acidic pH, the HMW complex dissociates and Cd can be complexed by vacuolar organic acids (citrate, oxalate, malate) (Krotz et al., 1989), and, possibly, by amino acids. Apo-phytochelatins may be degraded by vacuolar hydrolases and/or return to the cytosol, where they can continue to carry out their shuttle role.

3.1.5. Metallothioneins In animals, cyanobacteria and fungi, Cd and other heavy metals can be complexed and detoxified by metallothioneins, a group of gene-encoded Cys-rich (about 30%) peptides, generally lacking aromatic amino acids (Ka¨gi, 1991). In Arabidopsis thaliana, two Cu-induced metallothioneins with a molecular mass of 4500 and 8000 (called MT1 and MT2) were isolated by using polyclonal antibodies, raised against the metallothionein fusion proteins produced in Escherichia coli (Murphy et al., 1997). Moreover, in wheat germ an Ec metallothionein was found to regulate Zn homeostasis during early seed germination (Lane et al., 1987). Cys residues are present in plant metallothioneins as Cys–x– Cys, Cys–x–x–Cys (where x is an amino acid other than Cys), or Cys–Cys clusters. There is no certain indication in the literature of the existence in higher plants of metallothioneins induced by Cd. Thus, their role in Cd detoxification seems to be at present of secondary importance as compared with phytochelatins and stress proteins. By introducing animal metallothionein genes, transgenic plants were obtained by several authors (see Ow, 1996, and references therein). Recently, a preliminary transformation protocol of tobacco plants by means of a chimeric mouse metallothionein gene was set up. 3.1.6. Stress proteins After being subjected to high temperature stress, heavy metal and other chemical treatments, salinity and drought, plant cells often start the

L. Sanita` di Toppi, R. Gabbrielli / En6ironmental and Experimental Botany 41 (1999) 105–130

synthesis of heat shock proteins (hsps), referred to more generally as stress proteins. A possible role of hsps in protecting tomato plants even against injuries caused by cold was recently discovered by Sabehat et al. (1998). Hsps produced by factors other than thermal shock are also called ‘hsp cognates’, ‘hsc’ (Vierling, 1991). Several hsp classes have been found in plants (Vierling, 1991; Waters, 1995; Waters et al., 1996), among which: (a) hsp100, hsp90, hsp70, hsp60; (b) unusually abundant, small hsps (with a molecular mass ranging from 17,000 to 30,000); c) ubiquitin, a highly conserved small hsp with only 76 amino acids (Hershko, 1988). Unfortunately, the exact function and the detailed mechanism of action of small hsps (except ubiquitin), which are more common in plants than in other eucaryota, is still unknown. It has been demonstrated that the DNA of Cd-stressed cells produces specific mRNA transcripts which regulate the synthesis of stress proteins (Scho¨ffl and Key, 1982; Czarnecka et al., 1984; Edelman et al., 1988). In soybean, Cd can regulate stress protein synthesis by inhibiting the splicing of an intron from the pre-mRNA transcribed by a Gmhsp26-A gene (Czarnecka et al., 1988). However, it is not clear if the inhibition of this intron splicing represents a feature of a specific class of transcripts or is a general effect associated with Cd stress. In cell cultures of Lycopersicon peru6ianum, exposed to 1 mM Cd, significant amounts of hsp70 were bound to the plasmalemma, to mitochondrial membranes and to the endoplasmic reticulum (Neumann et al., 1994). This seems to be related to the formation of complexes between Cd and proteins denatured by Cd itself. Hsp70 has a strong affinity for misfolded proteins, and helps them to find their native conformation, by reintegrating them into the proper membrane complex. This behaviour does fit with data by Jungmann et al. (1993), who found that Cd exposure causes protein denaturation also in Saccharomyces cere6isiae. These denatured proteins, in the presence of ATP and various auxiliary factors, provide a substrate for ubiquitin. The ubiquitinated substrate is then degraded by the proteasome, a multisubunit protease complex.

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Mutants deficient in this ubiquitin-dependent system were found to be hypersensitive to Cd. In addition, it has been ascertained that a perturbation of the ubiquitin system can induce a programmed necrotic response in Nicotiana tabacum (Bachmair et al., 1990). On the other hand, the ubiquitin system does not seem to have relevance in coping with Cd stress in Arabidopsis thaliana (Van Nocker et al., 1996). In several species Cd exposure induced the synthesis of a considerable number of stress proteins with a molecular mass ranging from 10,000 to 70,000 Da. In particular, in roots of Phaseolus 6ulgaris, Cd induced the production of proteins with an apparent molecular mass of 42,000 (Leita et al., 1991), 52,000 and 19,000 Da (Marchetti and Leita, 1995). In cell suspensions of Datura innoxia, Cd induced proteins with the following molecular masses: 70,000, 50,000– 65,000, 22,000–24,000 and 20,000 Da (Delhaize et al., 1989b; Urwin et al., 1996). It is noteworthy that the two proteins with a molecular mass of 20,000 Da: (a) were found exclusively in Cdtolerant cells, and not in normal ones; (b) can also be induced by heat shock. In barley plants, as well as in maize, the heat shock-induced H6hsp17 gene was analyzed, in particular in the molecular structure of the promoter: this gene was also expressed in response to Cd stress (Lupotto et al., 1995). In Nicotiana plumbaginifolia callus cultures, the presence of Cd-induced proteins with molecular masses of 42,000, 40,000, 37,000, 21,000 and 19,000 Da was detected (Fenik et al., 1997). In Oryza sati6a, Cd supply induced the following protein pattern: 70,000, 42,000, 40,000, 26,000, 23,000, 15,000 and 11,000 Da (Prasad, 1997; Reddy and Prasad, 1995), and changes in phosphorylation of Cd-induced proteins were also observed. Interestingly, Rivetta et al. (1997) demonstrated that Cd binds to calmodulin and competes with Ca in this binding. Indeed, various Ca/calmodulin-dependent transduction signals are associated with variations in protein phosphorylation (Friedman and Poovaiah, 1991; Behra, 1993; Sherman and Goldberg, 1993).

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3.1.7. Stress ethylene In Phaseolus 6ulgaris, Cd stimulated ethylene biosynthesis-via the MSAE (methionine, S-adenosylmethionine, 1-aminocyclopropane-1-carboxylic acid, ethylene) pathway (Adams and Yang, 1979)—by directly enhancing (during the first 8 h) and then inhibiting the in vivo activity of ACC synthase (Fuhrer, 1982a). Production of stress ethylene increased activity of guaiacol peroxidases, and accumulation of soluble and insoluble (‘lignin-like’) phenolics were detected in Phaseolus 6ulgaris plants exposed to Cd (Fuhrer, 1982b). Furthermore, in the same species (Rodecap et al., 1981), Cd induced a higher production of ethylene in the roots than in the shoots. The stimulation of ethylene production during Cd stress reached peak concentrations within 5 – 10 h after the treatment and then gradually declined to control levels within 1 day. The authors hypothesized that this decrease in stress ethylene production could be attributed to Cd-sequestering, which diminished the Cd stress. In Amaranthus li6idus seedlings, Cd-induced stress ethylene declined with the concomitant rise in Cd concentration (Bhattacharjee, 1997). Because of the scarcity of data available, it is impossible to understand exactly, at a molecular/ cellular level, the relationship between ethylene biosynthesis and Cd stress. It cannot be ruled out that in the plant cell a Cd-generated perturbation of Ca levels could interact with (and stimulate) stress ethylene production. Indeed, it is reported that Ca ions stimulate ethylene production, as an effector of the ethylene-forming-enzyme (EFE) (Burns and Evensen, 1986; Cheverry et al., 1988). Pennazio and Roggero (1992) have indicated that Cd may directly affect EFE activity in soybean. Cd-induced stress ethylene might represent a signal capable of: 1. Accelerating the lignification process (Ievinsh and Romanovskaya, 1991), by increasing the activities of phenylalanine ammonia-lyase and peroxidases (Gaspar et al., 1978; Ecker and Davis, 1987). Fuhrer et al. (1981) reported that Cd-induced stress ethylene restricted water and Cd flux into bean leaves, by the induction of cell wall alterations in the vascular system;

2. Inducing the activity of ascorbate peroxidase, in order to detoxify hydrogen peroxide (Mehlhorn, 1990). Transgenic tobacco plants expressing antisense RNA for ascorbate peroxidase show increased susceptibility to oxidative stress (O8 rvar and Ellis, 1997); 3. Regulating the expression of genes, shown to be ethylene-responsive, encoding metallothioneins (Whitelaw et al., 1997) and/or defense proteins. An important chemical property of ethylene is the p-bond between carbon atoms. Thus, it is also possible that ethylene might directly interact with Cd and with other transition metals (ethylene is thought to bind its receptor through a transition metal cofactor, possibly Zn or Cu) (Ecker, 1995; Bleeker and Schaller, 1996), and/or change the glutathione metabolism and consequently influence phytochelatin synthesis. In carrot in vitro plants and hairy root cultures, stress ethylene production was always highly stimulated by 1 mM Cd (Sanita` di Toppi et al., 1998a,b). Absence of ethylene—after supply of aminoethoxyvinilglycine (AVG), or use of ethylene traps—caused both an inhibition in the phytochelatin synthase activity of carrot cell suspensions and a considerable lowering in the Cd-induced thiol groups in plants (Sanita` di Toppi et al., 1998a).

3.2. Response to chronic and acute cadmium stress In Table 2 a survey of response mechanisms operating in higher plant organs and cell cultures is reported, following various Cd exposure concentrations and exposure times, drawn from about 85 international publications. In the data analysis, we have in most cases included only wild-type (=normal, non-tolerant) plants (except a very few cases in which the inclusion of Cd-tolerant plant systems was in our opinion opportune). In addition, we have never taken into account concentrations of Cd\ 3 mM, which are in any case misleading in the ‘field situation’. From the analysis of 164 values of exposure concentration and 153 values of exposure time (see also Fig. 2), the following data can be inferred:

Table 2 Response of higher plants to Cd stressa Main response mechanism(s)

References

20 1b, 10, 100, 1 mM

Grill et al., 1987 Bhattacharjee, 1997

3 7

Agrostis tenuis cell cultures Amaranthus li6idus seedlings

10, 30, 60, 100 10

3, 7 4

A6ena sati6a roots A. sati6a roots

PCs Decreased production of ethylene (following increase in Cd concentration) Decreased activity of ascorbate peroxidase (following increase in Cd concentration) PCs PCs

100 0.9b, 1.8, 5.3 5.3 5.3 0.5b

7 3, 7 10 h 7 1

Brassica juncea plants B. juncea roots B. juncea xylem sap B. juncea xylem sap Cicer arietinum epicotyls

PCs incorporation of S2− PCs Binding with oxygen or nitrogen ligands Binding with oxygen or nitrogen ligands Expression of a proline-rich protein gene

10, 30 40

3, 7 1

Cucumis sati6us roots Cucurbita moschata fruits

PCs PCs

250

2, 4, 8 h

Datura innoxia cell cultures

Stress proteins

250

up to 2

D. innoxia cell cultures

PCs

D. innoxia cell cultures

PCs

300

1

250

up to 2 h

D. innoxia cell cultures

PCs

125 100, 200, 500

8h up to 2 h, 4 days

D. innoxia cell cultures Daucus carota cell cultures

Stress proteins PCs

100 mM, 1 mM

4

D. carota hairy root cultures Increased production of ethylene, PCs, stress proteins

100 mM, 1 mM

4

D. carota plants

Increased production of ethylene, PCs, stress proteins

Glycine max roots G. max seedlings

PCs Stress proteins

G. max xylem exudate

PCs, organic acids

20 1 mM 10

4 up to 12 h 2h

Inouhe et al., 1994 Salt and Rauser, 1995 Speiser et al., 1992 Salt et al., 1995b Salt et al., 1995b Salt et al., 1995b Mun˜oz et al., 1998 Inouhe et al., 1994 Fujta and Izumi, 1990 Delhaize et al., 1989b Delhaize et al., 1989a Jackson et al., 1987 Robinson et al., 1988 Urwin et al., 1996 Sanita` di Toppi et al., 1998a Sanita` di Toppi et al., 1998b Sanita` di Toppi et al., 1998a Grill et al., 1986 Edelman et al., 1988 Cataldo et al., 1988

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Exposure concenExposure time (days) Treated material tration (mM) (hours, h where indicated)) (mM where indicated)

113

114

Table 2 Response of higher plants to Cd stressa Main response mechanism(s)

500 10, 30, 60, 100 0.01b, 0.1b, 1b 100 10, 30 0.01b, 0.1b, 1b 100 310 100, 300

12 h 3, 7 9 9 3, 7 9 9 2 up to 12

100, 200, 300, 400 mM, 1 mM 50, 100, 150 107 10, 30 40, 80, 400, 870 900 mM, 1 mM 300

ND

Helianthus annuus leaves Hordeum 6ulgare roots Lactuca sati6a roots L. sati6a roots L. sati6a roots Lupinus albus roots L. albus roots Lupinus luteus roots Lycopersicon esculentum cell cultures L. esculentum cell cultures

Decreased activity of ascorbate peroxidase Gallego et al., 1996 PCs Inouhe et al., 1994 Accumulation of asparagine, lysine, hydroxylysine, proline Costa et al., 1997 Accumulation of asparagine, proline, lysine Costa et al., 1997 PCs Inouhe et al., 1994 Accumulation of asparagine, lysine, hydroxylysine Costa et al., 1997 Accumulation of asparagine, lysine, hydroxylysine Costa et al., 1997 Induction of pathogen-related proteins, PCs Gwozdz et al., 1997 PCs Chen and Goldsbrough, 1994 PCs Inouhe et al., 1991

up to 1 1 3, 7 ND ND 10

L. L. L. L. L. L.

1 mM

esculentum cell cultures esculentum cell cultures esculentum roots esculentum xylem cells esculentum xylem cells peru6ianum cell cultures

PCs PCs PCs Citric acid Citric acid PCs

4h

L. peru6ianum cell cultures

PCs, stress proteins

7

Nicotiana rustica leaves

PCs, Vacuolar compartmentalization

up to 7

N. rustica leaves

PCs

200 45 10, 20

4 up to 36 h 4

N. tabacum cell cultures N. tabacum cell cultures Nicotiana tabacum plants

PCs Organic acids, PCs Induction of cellular proteins

10, 30, 60, 100 1, 3 mM

3, 7 3h

Oryza sati6a roots O. sati6a roots

PCs Stress proteins

15

O. sati6a roots and shoots

Accumulation of proline

20 5, 10, 20, 40

100, 500 0.5b, 5, 20 25 1b, 10, 100 mM, 1 mM

6 h, 1, 4 days 1, 3, 4, 8, 10, 12 17 h

Phaseolus aureus roots and Enhanced actvities of guaiacol and ascorbate peroxidases leaves Phaseolus coccineus roots and PCs leaves Phaseolus 6ulgaris leaf discs Increased production of ethylene

References

Mendum et al., 1990 Scheller et al., 1987 Inouhe et al., 1994 Senden et al., 1992 Senden et al., 1994 Bennetzen and Adams, 1984 Neumann et al., 1994 Vo¨geli-Lange and Wagner, 1990 Vo¨geli-Lange and Wagner, 1996 Hirt et al., 1989 Krotz et al., 1989 Ghoshroy et al., 1998 Inouhe et al., 1994 Reddy and Prasad, 1995 Shah and Dubey, 1997 Shaw, 1995 Tukendorf et al., 1997 Fuhrer, 1982a

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Exposure concentra- Exposure time (days) Treated material tion (mM) (hours, h, where indicated) (mM where indicated)

Table 2 Response of higher plants to Cd stressa

5 100 mM, 1 mM 1, 2, 25 mM 50, 125

Main response mechanism(s)

References

4 up to 2

P. 6ulgaris roots and leaves P. 6ulgaris roots and shoots

Enhanced activities of guaiacol and ascorbate peroxidases Increased production of ethylene (above 1 mM Cd)

21

P. 6ulgaris roots, stems and leaves P. 6ulgaris roots, stems and leaves P. 6ulgaris seedlings

PCs, Stress protein(s) only in roots

Chaoui et al., 1997 Rodecap et al., 1981 Leita et al., 1991

PCs, stress proteins

Leita et al., 1993

Increased production of ethylene (above 10 mM Cd), Enhanced activity of guaiacol peroxidases, Increased phenolics (‘ligin like’ material) (above 10 mM Cd) Expression of a proline-rich protein gene

Fuhrer, 1982b

21

1b, 10, 100 mM, 1 mM

18 h

100

up to 2 h

10, 30 100, 500

3, 7 15

P. 6ulgaris stems, leaves and roots Pisum sati6um roots P. sati6um roots and shoots

1, 2, 3

P. sati6um roots and shoots

PCs

21

P. sati6um roots, stems and leaves Polygonum cuspidatum roots

PCs, stress proteins

Raphanus sati6us roots R. sati6us seeds Rau6olfia sepentina cell cultures R. sepentina cell cultures R. sepentina cell cultures

PCs No PCs PCs

0.5b, 5, 20, 50 50, 125 — 10, 30 10, 30, 100, 300 200

From a Cd contaminated area 3, 7 1 4

20 200

3 4

100

1,4,7,14

10, 100 mM, 1 mM 100

8 h, 1, 3, 5, 7 days 3

10, 30 20

3, 7 3

Low cell wall binding

PCs PCs, incorporation of S2− PCs PCs PCs PCs PCs

Inouhe et al., 1994 Lozano-Rodrı´guez et al., 1997 Ru¨egsegger et al., 1990 Leita et al., 1993 Nishizono et al., 1989 Inouhe et al., 1994 Rivetta et al., 1997 Grill et al., 1985 Grill et al., 1987 Kneer and Zenk, 1992 Maitani et al., 1996a Kubota et al., 1995 Maitani et al., 1996b Inouhe et al., 1994 Grill et al., 1987

115

Rubia tinctorum hairy root cultures Rubia tinctorum hairy root cultures R. tinctorum hairy root cultures Sesamum indicum roots S. 6ulgaris (S. cucubalus) cell cultures

PCs PCs

Chai et al., 1998

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Exposure concentra- Exposure time (days) Treated material tion (mM) (hours, h, where indicated) (mM where indicated)

116

Exposure concentration (mM) (mM where indicated) 200 7.5, 60, 120 30, 180 0.3b, 1b, 45, 135, 180, 250, 490 50–750

Exposure time (days) (hours, h, where indicated))

Treated material

Main response mechanism(s)

References

S. 6ulgaris (S. cucubalus) cell cultures S. 6ulgaris (S. cucubalus) leaves S. 6ulgaris (S. cucubalus) plants

PCs, Incorporation of S2−

S. 6ulgaris (S. cucubalus) roots

PCs, incorporation of S2−

up to 2 h

S. 6ulgaris (S. cucubalus) roots

PCs

1, 3, 7

PCs

Kneer and Zenk, 1992 Schat et al., 1997a De Knecht et al., 1995 De Knecht et al., 1994 De Knecht et al., 1995 De Knecht et al., 1992 Inouhe et al., 1994 Bonham-Smith et al., 1987 Inouhe et al., 1994 Lozano-Rodrı´guez et al., 1997 Keltjens and Van Beusichem, 1998 Meuwly and Rauser, 1992 Ju et al., 1997

4 3 up to 10 3

Incorporation of S2−, Accumulation of proline PCs

10 200

3, 7 4

S. 6ulgaris (S. cucubalus) roots and shoots Vigna angularis roots Zea mays roots

10, 30, 60, 100 100, 500

3, 7 15

Z. mays roots Z. mays roots and shoots

PCs Cell wall binding

3.75

2, 5, 8, 12, 15

Z. mays roots and shoots

PCs

3

2, 4, 6, 9, 12 h, up to 7 days 3

Z. mays roots and shoots

PCs

Z. mays seedlings

PCs

30

20 a b

PCs No stress proteins

PCs, phytochelatins (inclusive of homo-PCs, hydroxymethyl-PCs iso-PCs and desGly-PCs). Cd exposure concentrations 51 mM.

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Table 2 Response of higher plants to Cd stressa

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117

Fig. 2. Representation of Cd exposure concentrations plotted against Cd exposure times, drawn from the data reported in Table 2.

1. Mean Cd exposure concentration: 223 mM (n= 164); 2. Mean Cd exposure time: 5 days (n =153). Considering that soil solutions with a moderate level of Cd pollution have a Cd concentration varying from 0.32 to about 1 mM (see above), a threshold-value of 1 mM was arbitrarily chosen to distinguish between low and high exposure concentrations reported in the literature. On this basis, only in about 9% of the reviewed publications Cd was reported to be supplied in concentrations 5 1 mM, whereas about 91% of the experiments examined were performed with much higher concentrations of Cd. Thus, considering also a mean exposure time of 5 days, the response mechanisms reported in Table 2 are generally a consequence of acute Cd stress. So it must be pointed out that there is a lack of direct information regarding the effects of Cd concentrations of environmental relevance (chronic Cd stress), since most of the reported exposure concentrations, while certainly important in physiological studies, are often not realistic in the natural environment. In addition, many publications lack information on the bioavailabil-

ity of Cd, especially those in which whole plants, tissues or cells are grown in artificial substrates (i.e. hydroponic crops, in vitro cultures, etc.), where some complexing agents will probably diminish the concentration of available Cd. Thus, the exposure concentrations of Cd reported in Table 2 do not necessarily indicate levels of Cd certainly available, and therefore they should be considered with caution. However, on the other hand, the complexity of soil metal reactions and transformations makes it very difficult also to predict soil Cd bioavailability, mobility and retention. Thus, no ‘sweeping generalizations’ are possible (for comprehensive reviews on these topics see: Ross, 1994; Ross and Kaye, 1994). Having stated this in advance, it is generally agreed in ecotoxicology of plants, especially in the assessment of air pollutants, that: C1T1 = C2T2 = C3T3 = ...=CnTn = K, where: C1, C2, C3,..., Cn different exposure concentrations;

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T1, T2, T3,..., Tn different exposure times; K is a constant. Let us consider that the life history of an annual plant species may take between 90 and 150 days from germination to seed ripeness (not considering perennial plants with late reproduction, such as trees, in which the life history may take even 100 years or longer). Substituting in the formula the results drawn from Table 2 (an hypothetical ‘average’ experiment with a Cd exposure concentration of 223 mM and an exposure time of 5 days), we will have: C1 × 90= 223 ×5 (life-cycle of 90 days); C1 = 12.4 mM; and 223×5= C3 ×150 (life-cycle of 150 days); C3 = 7.4 mM; that is:

it is necessary to diminish the exposure concentration by a factor of 18 – 30 or more to come up with an environmentally acceptable Cd level (in any case about 10-fold higher than the above mentioned threshold-value of 1 mM), which might ensure the survival of plants in the ‘field situation’ up to reproduction. Again, these calculations show that most of the exposure concentrations and times reported in the literature are characteristic of acute Cd stress, and that the study of chronic Cd stress has been generally neglected. Since there are very few studies in which low Cd levels are considered, at present only preliminary and provisional comparisons between response to acute and ‘natural-field-condition’ Cd stress can be made. The main defense mechanisms present in response to Cd concentrations 5 1 mM (Table 2) do not seem to be ‘qualitatively’ significantly different from response mechanisms present in plants treated with higher Cd concen-

trations. Furthermore, the influence of the exposure time on these responses does not appear to be particularly significant. As a matter of fact, in wild-type plant systems, accelerated lignification, Cd binding to cell walls, to organic acids, to glutathione and phytochelatins, incorporation of S2 − in phytochelatin complexes, Cd induction of stress ethylene and Cd influence on peroxidases, all appear to be response mechanisms ‘qualitatively’ independent of Cd concentrations (and of exposure times). Although we do not have any preliminary ‘hard evidence’ at the moment, it cannot be ruled out that the response of higher plants to low concentrations of Cd might be more connected to the mantainance of cellular homeostatic equilibria than to proper response mechanisms. For instance, it is worth noting that glutathione and ascorbic acid were discovered to regulate the cell division in the apical meristem of Arabidopsis thaliana roots (Sanchez-Fernandez et al., 1997). Redox homeostasis affected by glutahione could modulate root hair tip growth—in response to adverse environmental conditions, such as Cd stress—towards a more advantageous root development, structure and architecture. Interestingly, Vo¨geli-Lange and Wagner (1996) calculated that in Nicotiana rustica leaves exposed up to 7 days to 5 mM Cd (in any case one or two orders of magnitude higher than in moderately polluted or non-polluted environments), constitutively present glutathione might be sufficient to chelate Cd ions present in the cytosol. They conclude: ‘mechanisms for Cd complexation in the cytosol with glutathione and complexation in the vacuole with organic acids may be prominent responses under low-level exposure. Higher level exposures require additional mechanisms like phytochelatins and sulphide’. Of course, further investigations are needed to support this hypothesis. Indeed, cad1-3 Arabidopsis Cd-sensitive mutants, lacking phytochelatins but possessing a glutathione content about 2-fold of that in the wild-type, showed much more sensitivity than the wild-type to low Cd concentrations (0.3 and 0.6 mM) (Howden et al., 1995b). Particularly at low Cd level exposures, a clear distinction between primary response mechanisms

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to Cd stress and those secondary responses that only reflect a Cd-induced decline and senescence of the plant (Noode´n et al., 1997) is extremely hazardous. For example, Schat et al. (1997a) brilliantly demonstrated that proline accumulation under Cd stress, in leaves of tolerant and non-tolerant ecotypes of Silene 6ulgaris, is a consequence of a Cd-imposed water deficit stress (see also Barcelo´ et al., 1986), rather than a consequence of a direct induction of this amino acid caused by Cd. All the same, it cannot be ruled out that proline might have a role: (a) in providing protection to the enzymes against Cd damage (Shah and Dubey, 1997); (b) in protecting macromolecules against denaturation (Schobert and Tschesche, 1978); (c) in conferring rigidity to the cell wall (Mun˜oz et al., 1998), and (d) in scavenging hydroxyl radicals (Smirnoff and Cumbes, 1989). As discussed above, even the production of ethylene, the typical plant senescence phytohormone, induced by low and high Cd concentrations, strictly speaking should be considered neither a primary defense signal nor a foregone consequence of tissue damage, lipid peroxidation and senescence processes caused by Cd. Based on Table 2, perhaps only one (preliminary) distinction might be made: at least as a general trend, stress proteins (hsp70 family chaperones and small hsps) appear to be induced (only?) by high and extremely high exposure concentrations of Cd (range: 50 mM – 3 mM). Stress proteins might act to limit and repair the damage to cell proteins caused by Cd, and, in particular, they could exert protective effects on membranes (Lin et al., 1985; Panaretou and Piper, 1992). Interestingly, Cd-tolerant tobacco cell cultures showed higher tolerance to high temperatures (37.5°C, 2– 8 h) than unselected cell cultures (Huang and Goldsbrough, 1988), and, vice versa, high temperatures induced a 400-fold greater acquired protection against Cd toxicity in wheat leaf segments (Orzech and Burke, 1988). No doubt (see again Table 2), phytochelatins play a decisive role in Cd detoxification at any exposure time and any concentration of Cd (from ‘physiological’ concentrations of 0.3 mM to ‘hyperstressful’ concentrations of 2.5 mM!). The amount of phytochelatin induced by Cd seems to

119

be roughly related to Cd exposure concentrations and times: within species-specific and organ-specific limits of toxicity, the more Cd is supplied (and the longer exposure lasts, within certain limits), it often follows that the more induction of the above mentioned peptides is obtained. In addition, a positive correlation between duration and level of Cd exposure and number of g-Glu–Cys repeat units in phytochelatins is reported (Grill et al., 1987; Vo¨geli-Lange and Wagner, 1996). It has been shown that the energy necessary for phytochelatin production is considerable: since phytochelatins derive from glutathione, Cdstressed cells have to restore the glutathione used to form them, by activating the enzymes catalyzing glutathione biosynthesis (Ru¨egsegger et al., 1990; Ru¨egsegger and Brunold, 1992). Glutathione levels in roots and shoots of at least ten species of monocots and dicots were greatly increased by exposure to 100 mM Cd (Baker et al., 1990). The enhanced synthesis of glutathione reported in poplar transformants, leading to an augmented phytochelatin synthesis, might represent a valuable system in the study of Cd detoxification (Noctor et al., 1996, 1998). However, as discussed by Ow (1996), other factors can be more important than the phytochelatin level for efficient Cd detoxification: for instance, in S. pombe mutants JS563 a FAD/NAD-linked disulphide reductase (perhaps a phytochelatin reductase) was discovered. This enzyme seems to be essential to guarantee sufficient reducing power, otherwise Cd-induced phytochelatins would become oxidized and ineffective for Cd binding. Thus, it is conceivable that also in higher plants high levels of phytochelatins may not be sufficient per se for complete Cd detoxification. Also the rapid formation of HMW complex, highly stabilized by S2 − groups, seems to be particularly decisive in Cd detoxification (Verkleij et al., 1990; Ortiz et al., 1992; Speiser et al., 1992; Kneer and Zenk, 1992). As noted by Vande Weghe and Ow (1997), Cdsensitive mutants of S. pombe are unable to accumulate phytochelatins. These mutants lack the hmt2 gene, which encodes for a HMT2 protein, required for phytochelatin accumulation in the vacuole, and, at the same time, these mutants overproduce S2 − . In fact, Chen and Huerta

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(1997) demonstrated that sulphur is important in alleviating toxic effects of Cd on photosynthesis in barley seedlings. These facts taken together suggest that sulphur metabolism and Cd detoxification could be closely related. The ‘sulphur carrier’ polypeptide has not yet been identified: a fascinating hypothesis is ‘that phytochelatins possess a dual function of sulphate reduction and sequestration of excess metals’ (Steffens et al., 1986). It has been hypothesized that phytochelatins are not only a Cd-detoxification system, but they could primarily represent, above all in the presence of low concentration of metals, key molecules for cellular homeostasis, necessary to transfer metal ions to apoenzymes which need them for their activity (Thumann et al., 1991). The constitutive presence of phytochelatin synthase (Grill et al., 1989) might be a further confirmation of a more general role played by phytochelatins in plant metabolism, not exclusively related to Cd detoxification. Of course, it cannot be ruled out that different systems may govern the transfer of Cd and the homeostasis of other metals within the plant cell: for instance, very specific Cu metallothionein and Cu chaperones (termed Atx1, Lys7 and Cox17) were all involved in the transport of Cu ions in yeast cells (Valentine and Gralla, 1997), and these mechanisms might be of decided importance also in higher plant cells.

4. ‘Fan-shaped’ response and general adaptation syndrome Taken together, all the above indicates that response to Cd stress in wild-type higher plants is a complex phenomenon. Cd evokes a response of parallel and/or consecutive events, rapid physiological and slow morphological processes, in which every mechanism could be at the same time cause and effect of metabolic changes, directly or indirectly related to the ‘management’ of Cd stress. It is possible that the ‘first line’ mechanisms directly coping with Cd stress are provided by phytochelatins and vacuolar compartmentalization—i.e. amount of phytochelatins, rapidity in HMW complex formation, number of g-Glu–Cys

units, high incorporation of S2 − , level of reduction of phytochelatins and, perhaps, signal transduction events involving cAMP and Ca ions. The presence of organic acids (especially in highly vacuolated cells of mature tissues), necessary in the maintenance of osmotic balances, plays also a significant role in Cd compartmentalization in the vacuole. The ‘second line’ mechanisms could be represented by the other above-described systems (essentially stress proteins, stress ethylene and peroxidases), which are possibly less direct, but nevertheless of decided importance. Some of these mechanisms (i.e. synthesis of stress proteins) could mainly exert the essential function of rapid repair of Cd damage, especially in the presence of high Cd concentrations, allowing the correct refolding of those cell proteins damaged by Cd. It is worthy of note that stress proteins are often expressed in the root—the first barrier against Cd stress—several fold more than in other organs. Root development which seeks to avoid contact with Cd ions might represent another defense strategy in Cd-polluted soils. For instance, with regard to Al stress, it was made clear (Parker and Pedler, 1998; Larsen et al., 1998; Degenhardt et al., 1998) that exclusion mechanisms involve: (a) modification in rhizosphere pH; (b) augmented release of chelating-organic acids by the root; (c) abundant mucilage barrier at root tip level. All the above responses might be potentially important also in coping with Cd ions. Moreover, a deeper study of possible protective roles of mycorrhizal symbiosis against Cd stress (especially mycorrhizas of tree and ericaceous species) (see the reviews by: Galli et al., 1994; Turner, 1994) would be required here, since fungi have been demonstrated to bind heavy metals by means of the cell wall, and the production of metallothioneins, phytochelatins, etc. (Howe et al., 1997). In aquatic environments, a stream of Cd-polluted water can pass and affect the aquatic plants for minutes or hours or days. After that, under conditions in which there is a flow of non-polluted water—in a ‘post-stress’ situation—the plants can rapidly repair the damage caused by Cd ions, in particular by means of stress proteins, acting, for instance, as molecular chaperones.

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Fig. 3. ‘Fan-shaped’ response to Cd stress in higher plants. The proposed multi-component model could allow the plant — modulating to various extents the ‘expression’ of each ray of the fan — to cope effectively with Cd stress, by means of mechanisms of avoidance, detoxification and repair.

However, very often it is extremely difficult to set exact borderlines between ‘first line’ and ‘second line’ mechanisms, and a holistic and integrated approach seems to us to be necessary in the study of the response of higher plants to Cd stress. Various mechanisms might operate in re-

sponse to Cd, both in an additive way and in a mutual—probably more effective—potentiating way. Such an integrated response, which we would call a ‘fan-shaped’ response (Fig. 3), is an overall multi-component model. Of course, the ‘fan-shaped’ response does not dogmatically im-

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ply that all the processes involved (the rays of the fan) are always present in all plant systems, always equally important and always acting in co-operation. It only hypothesizes that different plant systems can cope in different ways with Cd stress, by modulating— more or less, directly or indirectly— the ‘expression’ of each ray of the fan, especially in the presence of other stress factors, as usually occurs in the ‘field situation’. Hence, the relative importance of each response mechanism may appreciably differ. It is a matter of debate whether or not heavy metal detoxification mechanisms would accord with the ‘general adaptation syndrome’ (GAS) hypothesis, in which ‘different types of stress evoke similar or even identical stress coping mechanisms’ (Selye, 1936; Leshem and Kuiper, 1996). In our opinion, the proposed ‘fan-shaped’ response to Cd stress would be in accord with the Selyean concept of GAS, in terms of ‘non-specificity’ of the response mechanisms. In fact, metabolisms of glutathione, organic acids, peroxidases, stress proteins, stress ethylene, proline and other amino acids; mechanisms of compartmentalization, lignification, and root development, are all responses common to other heavy metals and to many other stress agents, such as salinity, heat, ozone, drought, etc. Even the production of phytochelatins can be considered a GAS reaction with regard to heavy metal exposure. Perhaps, as in other stresses, so too in Cd stress we can distinguish between Selyean ‘catatoxic’ and ‘syntoxic’ factors. In spite of a significant degree of overlapping between these two groups, phytochelatins, glutathione (and peroxidases) could be considered mainly catatoxic factors; stress proteins, stress ethylene, organic acids, compartmentalization, lignification, proline and other amino acids (and Cd-affected root development), mainly syntoxic factors. As properly reported by Leshem and Kuiper (1996), ‘the relative importance of each factor may differ for plant species, plant organs, or stages of development in the same plant’.

5. Some considerations regarding cadmium tolerance As we have already said, response to Cd in

wild-type (= normal, non-tolerant) higher plants is a complex phenomenon. Indeed, if Cd levels are low (i.e. about 5 1 mM in the soil solution or in the culture medium), but the exposure time is long (years or, in some cell cultures, at least several months), it can be reasonably hypothesized that plant ‘management’ of chronic Cd stress is a whole made up of general cellular homeostatic processes, which may be common also to the ‘management’ of other metals and other stress factors. If Cd levels are high or very high (i.e. \ or  1 mM), and the exposure time is short (hours, days or weeks), plants can ‘manage’ this acute Cd stress by a rapidly induced full ‘fan-shaped’ response, in order to detoxify Cd ions and efficiently repair Cd damage. Most of the experiments reported in the literature fall into this group (Table 2). Here, we are only in the presence of more or less efficient detoxification systems, presumably present in all plants to varying extents, but we are not dealing with a ‘real’ tolerance mechanism. In our opinion, Cd ‘real’ tolerance in higher plants should in general be defined as the natural or artificially given capacity, regulated by interacting genetic and environmental factors, to bear high levels of Cd exposure for a long time, without appreciable detrimental effects on metabolism. Thus, the development of ‘real’ Cd tolerance should be a long-term process (possibly years) and involve modifications in the genetic patrimony of the plant. One of the most critical points could be: while Cd detoxification in wild type plants is a complex phenomenon, probably under polygenic control (shown also by the broad range of different mutations causing hypersensitivity to Cd), ‘real’ tolerance seems to be a simpler mechanism, apparently involving only one or a few specific major genes, capable of conferring a high level of tolerance on their own (Verkleij et al., 1991). For instance, in Cd-tolerant Chlamydomonas reinhardtii, two independent major genes were found (Collard and Mantagne, 1990). There were hardly any intergenic variations between different tolerant lines or populations of S. 6ulgaris (Schat et al., 1996, 1997b). Therefore, Cd tolerance mechanisms seem to ‘overrule’, rather than act in co-operation with the wild-type defense mechanisms, and the ‘fanshaped’ response appears to become less satisfactory here.

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However, the following point should be taken into account: the molecular genetic basis of Cd tolerance have not been incontrovertibly identified. With regard to heavy metals other than Cd, Macnair (1993) reports: ‘in tolerance, while a major gene is apparent, the simple gene model is probably inadequate. It would be more likely to result in the initial spread of a major gene that gives a reasonable degree of tolerance. Subsequently, continued natural selection would lead to more genes spreading that modify and enhance tolerance’. Macnair (1990) had earlier pointed out that some minor genes (modifiers), not conferring tolerance on their own, can modify the major gene(s), perfecting and enhancing its (their) effects on tolerance. Obviously, the above cannot be ruled out either in the case of Cd tolerance, which has not to date been studied sufficiently. Further efforts are urgently required in order to establish definitely whether the genetic control of Cd tolerance is polygenic or oligogenic/monogenic. If only a few specific major genes are involved in tolerance, from the polygenic control of the ‘fanshaped’ response — typical of Cd detoxification mechanisms — the plant system could alter, over time, under the selection pressure: (1) towards an increased frequency of these major gene(s) and (2) towards the over-expression (ethylene-regulated?) of the above mentioned ‘first line’ mechanisms. In our opinion, in an adaptive response as Cd tolerance very probably is, the control is likely to be both genetic and environmental. On the one hand, it is well-known that the induction of high amounts of phytochelatins — not genetically controlled (at least within certain limits, depending on glutathione and Cd levels in the cell) — cannot be considered a mechanism capable of conferring tolerance by itself (Delhaize et al., 1989a; De Knecht et al., 1992, 1994, 1995). On the other hand, however, a ‘minimum’ level of phytochelatins has to be present to allow Cd vacuolar compartmentalization, which is genetically controlled. Cad1 sensitive mutants of Arabidopsis, lacking phytochelatins, were shown to be deficient in their ability to sequester Cd in the vacuole (Howden and Cobbett, 1992). Hmt1 and hmt2 genes (Ortiz et al., 1992, 1995; Vande Weghe and Ow, 1997), which facilitate Cd sequestration in the vacuole and

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overproduce sulphur, were proven to be essential for Cd detoxification and tolerance in S. pombe. Thus, under long-term selection pressure (either in high Cd-contaminated soils or in Cd-containing culture media) the frequency and the expression of such genes, perhaps having also pleiotropic effects, might increase and confer ‘real’ Cd tolerance. Furthermore, the level of reduction of phytochelatins possibly controlled by a FAD/NADlinked disulfide reductase, signal transduction events involving cAMP and Ca ions (Ow, 1996), and an augmented Cd2 + /2H + antiport (Salt and Wagner, 1993; Gries and Wagner, 1998), might represent other decisive factors in conferring or perfecting tolerance. A close relationship between genetic and environmental/physiological control of Cd tolerance was brilliantly noted by Baker et al. (1986), who demonstrated that there was a remarkable loss of tolerance following a 6-year cultivation of Cd-tolerant Holcus lanatus in Cd-uncontaminated soil. As further discussed by Baker et al. (1990), an essential requisite to facilitate the achievement of Cd tolerance is that plant genotypes possess a considerable plasticity, which enables them to adapt to the Cd-imposed pressure. To date, it is not clear whether stress proteins, although certainly involved in the ‘fan-shaped’ response, could be responsible for real long-term Cd tolerance. First, they are all encoded by large multigene families, and this seems to be in contradiction to the above-discussed possible oligogenic/ monogenic control of tolerance. In addition, to our knowledge, only one authoritative experimental investigation dealing with a possible role of stress proteins in Cd ‘real’ tolerance has been published (Urwin et al., 1996): two proteins with a molecular mass of 20,000 Da were found exclusively in Cd-tolerant cells, and not in normal ones. Differently, Neumann et al. (1994) exposed Lycopersicon peru6ianum cell cultures to a high Cd concentration (1 mM) for only 4 h, and therefore the reported hsp70 and hsp17 formation represents a response to acute Cd stress (repair, detoxification), rather than a clear sign of acquired long-term tolerance. Finally, we fully agree with Meharg (1994) when he asserts that (adaptive) tolerance relies on (constitutive) detoxification mechanisms (Table

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Table 3 Possible response patterns to Cd stress in higher plants and their supposed genetic control, in relation to Cd exposure concentration and exposure timea Exposure concentration

Exposure time

Stress

Phenomenon involved Response

Supposed genetic control

Low

Long

Chronic

Homeostasis

High

Short

Polygenic or multigenic Polygenic

High

Long

Homeostatic, constitutive Acute Detoxification and re- ‘Fan-shaped’, constitupair tive Extremely severe Tolerance More specific, adaptive

Oligogenic or monogenic

a

Tolerance is supported by detoxification mechanisms, which in turn rely on homeostatic processes. The shift between homeostatic and ‘fan-shaped’ responses can be rapid and involve quick changes in gene expression. Differently, the slow shift from ‘fan-shaped response to ‘real’ tolerance is caused and affected by the long-term selection pressure, which may increase the frequency (and promote the expression) of one or a few tolerance gene(s).

3), and that ‘the mechanism(s) required to adapt to highly contaminated environments may involve just one of these processes. Identification of individual biochemical pathways such as phytochelatin production, proteolysis, etc., is essential, but without integration into a cellular response, such studies may lose direction and overemphasize the overall importance of a given pathway in achieving tolerance’. The much enhanced expression of vacuolar transporters in Cdtolerant organisms appears to be a pertinent example of the above.

Acknowledgements Particular thanks to Dr A.J.M. Baker, University of Sheffield, and to Dr B. Pawlik-Skowronska, Polish Academy of Sciences, for the stimulating discussions on these topics. We are also grateful to Mr S.F. Walker and to Dr M. Balaban for their linguistic help.

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