Cellular antioxidants including low molecular weight antioxidants and enzymes of the ROS-scavenging system. (Adapted from Pinto et al., 2003)
Metals like zinc, iron and copper are essential micronutrients required for a wide range of physiological processes in all plant organs for the activities of various metal-dependent enzymes and proteins. However, they can also be toxic at elevated levels. Metals like arsenic, mercury, cadmium and lead are nonessential and potentially highly toxic. Once the cytosolic metal concentration in plant turns out of control, phytotoxicity of heavy metal inhibits transpiration and photosynthesis, disturbs carbohydrate metabolism, and drives the secondary stresses like nutrition stress and oxidative stress, which collectively affect the plant development and growth (Krämer & Clemens, 2005).
Plants have developed a complex network of highly effective homeostatic mechanisms that serve to control the uptake, accumulation, trafficking, and detoxification of metals. Components of this network have been identified continuously, including metal transporters in charge of metal uptake and vacuolar transport; chelators involved in metal detoxification via buffering the cytosolic metal concentrations; and chaperones helping delivery and trafficking of metal ions (Clemens, 2001).
This chapter summarizes heavy metal stress and detoxification in plant. Special focus is given to metallothionein, yet vacuolar metal transporters, phytochelatins as well as certain organic acids, amino acids, and chaperones are also addressed with recent advances. Besides, heavy metal-induced oxidative stress and tolerance as an example of abiotic stress cross-talk will be discussed.
1.1. The vacuolar compartmentation mediated by transporter families CDF and Nramp
A balanced cytosolic metal concentration has to be maintained all the time via strict compartmentation and chelation. The plant vacuole is a main storage compartment site for heavy metals present in excess (Ernst et al., 1992). Nickel-hyperaccumulator plant
The CDF (cation diffusion facilitator) transporters, once named as MTP for metal tolerance protein, are involved in mediating the cytoplasmic efflux of transition metal cations such as Zn2+, Cd2+, Co2+, Ni2+ or Mn2+. In
It’s interesting to note the other side, releasing metal ions from the vacuole into the cytosol if required by metabolism. Which transporter takes the challenge? The NRAMPs might be a possible candidate. The plant NRAMP (natural resistance associated macrophage protein) family transport divalent metal cations into the cytoplasm.
Progressive reports implicated that
1.2. Chelation of cadmium ions by phytochelatin
Chelation of metals in the cytosol is a very important mechanism of heavy metal detoxification and tolerance (Fig 1). The principal classes of known metal chelators in plant
are metallothioneins (MTs), phytochelatins (PCs), organic acids and amino acids (Clemens, 2001). Metallothionein and phytochelatin are proteins or peptides with low molecular weight, high cysteine content, and unique metal-binding capacity. In early reports lack of detail amino acid sequence data, metal-binding proteins in plants were generally assumed to be MTs, which in fact covered at least part of PCs. In an old classification system of three-class MT proteins, phytochelatins are somewhat confusingly described as enzymatically-synthesized class III whereas other two are gene-encoded class I and II (Cherian & Chan, 1993). Later the classification system has been improved and now it’s clear plants express both PCs and MTs, which play relatively independent roles in metal detoxification and/or metabolism (Cobbett & Goldsbrough, 2002).
Phytochelatins have been identified in many plants and photosynthetic organisms, ranging from algae, gymnosperms to monocots and dicots. Phytochelatins (PCs) are synthesized from glutathione (GSH) (in some cases, related compounds) by PC synthases (PCS), and play a role in the distribution and accumulation of Cd and some other highly toxic metals like Ag, Hg, As (Cobbett, 2000, Rauser, 1999). Modern techniques including X-ray absorption spectroscopy (XAS), high performance liquid chromatography-mass spectrometry (HPLC-MS), inductively coupled plasma optical emission spectrometer (ICP-OES) help to reveal that cadmium ions are generally bound to phytochelatins in plant. The percentage of Cd bound to PCs in Indian mustard seedlings increased from 34% after 6 hours of Cd exposure to 60% after 72 hours (Salt et al., 1997). In a Cd-hyperaccumulator desert plant tumbleweed (
There are two types of Cd-PC complexes produced during Cd sequesteration: low-molecular-weight (LMW) and high-molecular-weight (HMW). The LMW complex serves as the transient form for transporting Cd2+ from cytosol to vacuole where more Cd and sulfide are incorporated to produce the HMW complex, which turns the main storage form of Cd2+ (Rauser, 1995). The first molecular insight into transporting the PC-Cd complex comes from the
Besides the phytochelatin-Cd2+ complex transported by ABC transporters, cadmium ions can also reach the vacuole via a direct exchange with protons by tonoplast-localized cation/proton exchanger (CAX) transporters. In oat roots, the pH-dependent Cd2+ accumulation in vesicles was accompanied by efflux of protons, which offers the first clue of Cd2+/H+ antiport in plant (Salt & Wagner, 1993). Then several
1.3. Metallothionein: metal-binding protein and more
Metallothioneins (MTs) are ubiquitous low-molecular-weight, cysteine-rich proteins that can bind metals via mercaptide bonds. Since the first MT was characterized from horse kidneys as cadmium-binding proteins in 1957 (Margoshes & Vallee, 1957), plenty of MT genes have been identified in a wide variety of organisms including bacteria, fungi, and all eukaryotic animal and plant species (Robinson et al., 1993).
The spatial structures of MTs have been uncovered as a dumbbell-like shape with two separate domains, α and β, containing in their core clusters built up of several tetrahedral Metal-Cys units (Fig 2). The different metal reactivity and metal affinity of two domains prompt different functional roles of the two metal clusters, that is, N-terminal β domain is involved in the homeostasis of essential metal ions (Kagi & Schaffer, 1988, Willner et al., 1987), and C-terminal α domain, the tight binding sequestration of excess and/or toxic metal ions (Cherian et al., 1994, Wright et al., 1987). As for the spacer region linking the α and β domains, it may contribute to stability or subcellular localization of MT proteins (Domenech et al., 2005), and is necessary for MT metal detoxification function (Domenech et al., 2007, Zhou & Goldsbrough, 1994).
MT proteins are generally classified into mammalian Class I and plant Class II, and plant MTs can be further subdivided into four types based on the number and arrangement of cysteine residues and the length of spacer region (Cobbett & Goldsbrough, 2002). These four-type plant MTs exhibited certain tissue-preferential expression patterns. Type 1 MTs
are expressed much higher in roots than in shoots (Hudspeth et al., 1996), whereas Type 2 MTs are found mainly in leaves (Hsieh et al., 1995, Zhou & Goldsbrough, 1994). Type 3 MTs are expressed abundantly in the ripe fruits (Clendennen & May, 1997, Ledger & Gardner, 1994, Reid & Ross, 1997), and expression of Type 4 MTs, also known as the Ec type, was only found in developing seeds so far (Chyan et al., 2005, Lane et al., 1987).
A vast number of stimuli have been demonstrated capable of inducing MT genes expression in plants, including natural senescence (Bhalerao et al., 2003), hormones like ABA (Reynolds & Crawford, 1996), ethylene (Coupe et al., 1995), wounding and virus infection (Choi et al., 1996), heat shock (Hsieh et al., 1995), sucrose starvation (Hsieh et al., 1996), UV-light (Foley & Singh, 1994), cold and salt stress (Reid & Ross, 1997), etc. Apparently, different types of MTs respond to different factors, which is especially true when treated with heavy metal stress under different concentrations. Copper increased
Ever since the first identification of MTs, its striking metal-binding property has been brought into sharp focus, which suggests MTs play the principal role in metal homeostasis and detoxification. In animals, MTs are well-known metal-binding proteins protecting against cadmium toxicity (Klaassen et al., 1999), while in plant PCs mainly take the charge of Cd detoxification (Zenk, 1996). MTs seem to have a broader spectrum of metal affinity than PCs, which points to more complicated functions. It’s proposed that MTs participate in maintaining the homeostasis of essential copper (Cu) or zinc (Zn) at micronutrient levels, and also in the detoxification of non-essential toxic metals such as cadmium (Cd) and arsenic (As) (Lee et al., 2004, Merrifield et al., 2004, Roosens et al., 2004).
Though modulation of metal concentrations has great impact on cellular redox balance (Bell & Vallee, 2009), MTs may just scavenge reactive oxygen species (ROS) directly. With a large quantity of nucleophilic sulphydryl groups in the structure, MTs provide a fine nucleophilic “sink” to trap electrophiles and free radicals, that is, the multiple cysteine residues can react with superoxide (•O2 –) and hydroxyl radicals (•OH) leading to their degradation (Klaassen & Cagen, 1981, Sato & Bremner, 1993). Moreover, MTs can be recycled via thiolate exchange with GST (Vasak et al., 1985). Now accumulating evidences support hypothesis that MTs function as an antioxidant in plants. In wild watermelon, drought-induced CLMT2 showed an extraordinarily high activity for detoxifying hydroxyl radicals
Thanks to dynamic instability of metal ions in clusters, MTs can exchange metal ions with other metalloproteins universally necessary for a life cycle. There’s zinc transfer between metallothionein and zinc transporter ZnT1 (Palmiter, 2004), chelator EDTA (Leszczyszyn & Blindauer, 2010), SOD (Koh & Kim, 2001, Suzuki & Kuroda, 1995), and other zinc proteins (Jacob et al., 1998). The metal-transfer mechanism should be a cornerstone for MTs' dual role abstracting the toxic metals arsenic (Ngu et al., 2010) or cadmium (Roesijadi, 2000), as well as donating the essential metals like zinc or copper (Liu et al., 2000). In this sense, metal-binding protein MTs are involved not merely in the coordination of metal concentraions, but contribute more to diverse physiological processes like development or senescence. The wheat type 4 MT Ec gene was specially expressed during pollen embryogenesis, and its accumulation correlates well with increase of the plant hormone abscisic acid (ABA). It’s suggested that induced by the ABA signal, this zinc-containing Ec may regulate certain gene expression via zinc trafficking with zinc-dependent DNA/RNA polymerase or zinc-finger proteins (Reynolds & Crawford, 1996). MTs have been implicated during senescence in many plants (Bhalerao et al., 2003, Breeze et al., 2004, Buchanan-Wollaston & Morris, 2000), and hypotheses for MT’s role in senescence primarily reckon on either MTs' chelating and detoxifying abilities which alleviate the senescence-induced metal ion disturbance and oxidative burst, or the release of necessary metal ions to required places for nutrient recycling.
The positive correlation between MT expression in diverse organisms and the environmental metal concentration suggests that MTs can be effective biomarkers of heavy metal pollution. Such monitoring programs have already gained great potential comprehensively in aquatic and terraneous invertebrates (Chu et al., 2006, Dallinger et al., 2004, Navarro et al., 2009). In plants, MTs are favorable candidates for phytoremediation of heavy metal contaminants, a low-cost, effective, and sustainable plant-based approach for environment governance (Eapen & D'Souza, 2005, Memon & Schroder, 2009). On the other side, biofortification of mineral micronutrients in food crops for the benefit of human health, is another application and extension for metal research in plants, and MTs could also be contributive. The
1.4. Organic acids, amino acids and chaperones
The reactive interactions between metal ions and S, N, and O made organic acids and amino acids potential ligands for metal chelation. Citrate has been proposed the major ligand for Cd2+ at low Cd concentration within cell (Wagner, 1993), and can form Nickel-citrate complex in Ni-hyperaccumulating plant
The coordination of nickel with histidine has been confirmed with analyses of Ni-hyperaccumulating and non-accumulating species. Upon Ni exposure, a large and proportional increase of free histidine was detected in xylem sap in Ni-hyperaccumulating
Copper chaperones are a novel class of proteins involved in intracellular trafﬁcking and delivery of copper to copper-containing proteins such as copper-ATPases or copper/zinc superoxide dismutase.
2. Heavy metal-induced oxidative stress and stress tolerance: Cross-talk
Seen from a systemic view, different abiotic stresses may bring general effects on plant growth and development. For example, drought, salt, and cold stresses can all interrupt the cellular water balance leading to osmotic stress, and generate a phytohormone abscisic acid (ABA) for osmotic adjustment (Wang et al., 2003). ABA acts as a key endogenous messenger in stress response, and hence the ABA sigalling pathway is more or less involved during plant cross-adaptive processes (Tuteja, 2007). In addition, all abiotic stresses can accumulate excess ROS (reactive oxygen species) at certain stage of stress exposure leading to oxidative stress. However, ROS are not only toxic compounds, but sometimes play as important regulators for many biological processes in plants such as cell cycle, programmed cell death, hormone signaling, biotic and abiotic cell responses (Laloi et al., 2004). As common consequences of abiotic stresses, osmotic stress and the ubiquitous oxidative stress have been extensively studied and offer more and more evidences for cross-talk at various steps or levels in the complicated network of abiotic stress signalling pathways.
Reactive oxygen species (ROS) such as •O2 –, H2O2 and •OH are unavoidable by-products of aerobic metabolism, and also commonly generated under various stress conditions. The unwelcome result of ROS overproduction is the oxidative stress, which can cause extensive cellular damages (Miller et al., 2008). Therefore, a delicate antioxidant system is indispensably required to supervise the cytotoxic effects of ROS tightly. The plant antioxidant system consists of ROS-scavenging enzymes, such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), as well as low-molecular-weight antioxidants like glutathione, ascorbate, carotenoids, metallothionein, etc (Table 1). Analysis with transgenic plants overexpressing these antioxidant genes revealed that maintenance of a high antioxidant capacity in cells is linked to increased tolerance against various adverse conditions (Guo et al., 2009, Jayaraj & Punja, 2008, Tseng et al., 2007, Wang et al., 2010).
Heavy metal stresses can shift the cellular balance of free radical homeostasis into terrible accumulation of H2O2. For those redox-active transition metals like copper or iron, autoxidation in Fenton reaction and Haber–Weiss reaction will convert H2O2 to the highly reactive •OH molecule in a metal-catalyzed way. Non-redox-active metals like cadmium or mercury can also result in H2O2 accumulation and an oxidative burst via depletion of the antioxidant glutathione (GSH) pool and inhibition of antioxidative enzymes, especially glutathione reductase (GR) (Mithofer et al., 2004, Schutzendubel & Polle, 2002). To cope with heavy metal stress and associated oxidative stress, metallothionein, a well-known metal chelator and also antioxidant would possibly be a good way out.
|Low molecular weight antioxidants|
|Ascorbate||O2 (1△g) , •OH, O2•, HO2•|
|β-Carotene||O2 (1△g) , RO2•|
|Urate||O2 (1△g) , metal|
|Flavonoid||•OH and HOCl|
|Superoxide dismutase||2O2•- + 2H+ → H2O2 + O2|
|Catalase||2H2O2 → 2H2O + O2|
|Glutathione peroxidase||H2O2 or ROOH + 2GSH → 2H2O or ROH + GSSG|
|Ascorbate peroxidase||H2O2 + Ascorbate → H2O + Monodehydroascorbate|
|Thioredoxin||Prot-S2 + Prot’(SH)2 → Prot(SH)2 + Prot’-S2|
|Peroxiredoxin||ROOH + R’(SH)2 → ROH + R’S2 + H2O|
|Glutathione reductase||GSSG + NAD(P)H + H+ → 2GSH + NAD(P)+|
Take OsMT1a for example. Yang et al. reported functional characterization of a rice type 1 metallothionein, OsMT1a. A model has been proposed to elucidate how OsMT1a plays a role in drought tolerance in plant (Yang et al., 2009). On the one hand, OsMT1a can directly scavenge ROS via increasing activities of antioxidant enzymes CAT, POD and APX. On the other hand, OsMT1a lies upstream of some zinc finger transcription factors like Ossiz, and may tune up downstream defense genes in virtue of these transcription factors through Zn2+ trafficking. Additional data reveal that some zinc/cadmium transporter genes including forecasted vacuolar-membrane-localized ABC transporters
Despite their toxicity, ROS have been reevaluated in recent years as key signal molecules for regulating cell function and development (Rhee, 2006). In plants, the elaborate and efficient network of scavenging mechanisms allowed overcoming ROS toxicity and using some of these toxic molecules, mainly the hydrogen peroxide (H2O2) produced by cytosolic membrane-bound NADPH oxidases, as a signal in a wide range of abiotic stress responses (Bailey-Serres & Mittler, 2006, Mittler et al., 2004, Neill et al., 2002). For instance, in response to drought stress, ABA-induced H2O2 regulates the stomatal closing of
3. Outlook and challenges
As the global population and food demand keep increasing fast, and yet the environment has been endangered worse and worse by water deficit and soil salinization, abiotic stress becomes one of the most harmful factors that limit the growth and productivity of crops worldwide. Although we keep moving forward with the understanding of heavy metal stress and detoxification in plant, there are many components of the complex network yet to be identified. Especially much remains unknown about the signalling molecules of the metal-induced signal transduction, including sensing of the cellular metal change and subsequent transcription regulation of metal-responsive genes (DalCorso et al., 2008). In recent years, next-generation sequencing techniques emerge and develop fast, and the microarray-based analyses become available and efficient for transcriptome or proteome high-throughput screenings, which help to identify regulatory factors for the metal homeostasis and still more metal transporters, low-molecular-weight chelators, chaperones as well. In addition, some heavy metal responsive transcription factors can also be induced by other abiotic stresses such as cold, dehydration, Salicylic Acid (SA) and H2O2, suggesting cross-talk exists between heavy metal response and other abiotic stress defense signalling (Fusco et al., 2005, Singh et al., 2002, Suzuki et al., 2001, Weber et al., 2006). Nevertheless, determining the underlying regulatory and cross-talk mechanisms remain a future challenge.
Heavy metal hyperaccumulators are unique plants capable of accumulating high amounts of various toxic elements (Reeves & Baker, 2000), and the active hyperaccumulation is based on mechanisms of internal hypertolerance to cytotoxic metals and a powerful scavenging system compatible for efﬁcient uptake of the pollutants (Salt, 2006). Therefore, comparative studies on hyperaccumulator and non-hyperaccumulator plants will provide us a good view of naturally selected metal hypertolerance and hyperaccumulation. The first core set of candidate genes with high expression in hyperaccumulators has been identified and will be analyzed at biochemical and genetic level (Krämer et al., 2007). Dissecting these genes opens up a wide avenue for understanding the plant metal homeostasis network, and also agricultural genetic engineering for crop tolerance and biofortification, as well as phytoremediation of environmental metal pollution.
This work was supported by grants from Chinese Academy of Sciences (grant numbers: KSCX2-YW-N-010 & KSCX2-YW-N-056), and National Science Foundation of China (grant numbers: 30825029 & 30621001).