JXB Advance Access originally published online on February 10, 2006
Journal of Experimental Botany 2006 57(4):711-726; doi:10.1093/jxb/erj073
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REVIEW ARTICLE |
The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress
1Department of Biosciences, Himachal Pradesh University, Shimla 171 005, India
2Department of Physiology and Biochemistry of Plants, Faculty of Biology, University of Bielefeld, D-33501 Bielefeld, Germany
* To whom correspondence should be addressed. E-mail: karl-josef.dietz{at}uni-bielefeld.de
Received 16 February 2005; Accepted 22 November 2005
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Abstract |
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 Top Abstract IntroductionProlineHistidineOther amino acidsPolyaminesGlutathione and phytochelatinsOther N-containing metabolites...Summary and outlookReferences |
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Plants exposed to heavy metals accumulate an array of metabolites, some to high millimolar concentrations. This review deals with N-containing metabolites frequently preferentially synthesized under heavy metal stress such as Cd, Cu, Ni, and Zn. Special focus is given to proline, but certain other amino acids and oligopeptides, as well as betaine, polyamines, and nicotianamine are also addressed. Particularly for proline a large body of data suggests significant beneficial functions under metal stress. In general, the molecules have three major functions, namely metal binding, antioxidant defence, and signalling. Strong correlative and mechanistic experimental evidence, including work with transgenic plants and algae, has been provided that indicates the involvement of metal-induced proline in metal stress defence. Histidine, other amino acids and particularly phytochelatins and glutathione play a role in metal binding, while polyamines function as signalling molecules and antioxidants. Their accumulation needs to be considered as active response and not as consequence of metabolic dys-regulation.
Key words: Adaptation, amino acids, heavy metal stress, N-containing metabolites, proline, stress defence
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Introduction |
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TopAbstractIntroductionProlineHistidineOther amino acidsPolyaminesGlutathione and phytochelatinsOther N-containing metabolites...Summary and outlookReferences |
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Uptake of excess metal ions is toxic to most plants. The biochemical impact of metal ions on the cells is as diverse as their chemical nature. From the approximately 90 elements present in the earth's crust, about 80% are metals and 60% are heavy metals with specific weights higher than 5 g cm–3. In context of metal toxicity, other elements with only partial metal properties such as As and with a specific weight lower than 5 g cm–3 such as aluminium also need to be considered due to their toxicity to plants. In the literature, the term ‘heavy metal’ is occasionally used with a very broad and misleading meaning. Based on metal co-ordination chemistry, Nieboer and Richardson (1980)
provided a biologically relevant classification of metals into three categories namely, class A with affinity for O-containing ligands, class B with affinity for N- or S-containing ligands, and borderline, i.e. intermediate between the two with affinity for all three groups of ligands with definite preferences. This categorization reflects a general manner in which different metal ions interact with biological systems. Al3+, 
Au+, Cd2+, Cu+, Cu2+, Co2+, Cr3+, 
Fe2+, Fe3+, Hg2+, Mn2+, Ni2+, Pb2+, 
Sn2+, W6+, and Zn2+ are examples of ions with known toxicity to plants. At present, most studies focus on a few metal ions such as Al, Cd, Hg, Ni, and Zn. Typically, root inhibition is observed at micromolar concentrations, i.e. at concentrations more than three to four orders of magnitude lower than is usually encountered in salinity stress.
Phytotoxicity of heavy metals in most parts can be attributed to symplastic accumulation of heavy metals, particularly in the plasmatic compartments of the cells, such as the cytosol and chloroplast stroma (Brune et al., 1995). Metal-induced changes in development are the result of either a direct and immediate impairment of metabolism (Woolhouse, 1983; Van Assche and Clijsters, 1990) or signalling processes that initiate adaptive or toxicity responses that need to be considered as active processes of the organism (Jonak et al., 2004). Transport processes have been recognized as a central mechanism of metal detoxification and tolerance (Hall, 2002; Hall and Williams, 2003). Some metals, for example, Zn and Cu, are essential for normal plant growth and development as they serve as structural and functional components of specific proteins. Other metals, for example, Cd and Pb, have no known function in plants although a Cd requirement for carbonic anhydrase from marine diatoms has been reported (Lane and Morel, 2000).
The generalized dose–response curves for the two kinds of metals differ with regard to their effects on plant growth. Whereas for non-essential metals these curves comprise a no-effect and a toxicity zone, for essential metals the response curves show an additional deficiency zone preceding the no-effect zone of adequate supply. It is implied in both cases that the plants are endowed with an inherent capability of tolerating toxic metals to some extent. Metal ions turn toxic as soon as their concentration exceeds a metal-specific threshold which varies strongly among plant species and ecotypes, and also with metal properties. As an exception to the rule, certain low concentrations of non-essential elements have occasionally been associated with some promotion of plant growth, as for example seen for Alyssum species and Thlaspi goesingense (Küpper et al., 2001).
Populations of certain plant species that chronically experience exposure to elevated metal concentrations, for example, those inhabiting metal-enriched locations, have repeatedly evolved tolerance to the metal(s) in question (Ernst et al., 1990; Schat et al., 1996). Such metal-tolerant ecotypes and genotypes are the examples of accelerated (micro)evolution when the selection pressure is acute. Examples of tolerant species are Arabidopsis halleri, a Zn-hyperaccumulator, Thlaspi species, that are Cd-/Zn- or Ni-hyperaccumulators, Silene vulgaris with Zn-, Cu-, and Cd-resistant ecotypes, and Alyssum bertolonii, a Ni-hyperaccumulator (Ernst and Nelissen, 2000; Küpper et al., 2001; Bert et al., 2003; Freeman et al., 2004). Correspondingly, the dose–response curves of metal-tolerant variants exhibit a wider no-effect or even limited beneficial zone when compared with their non-tolerant counterparts. Apparently, while evolving tolerance, they acquired some specific molecular means of efficiently detoxifying the surplus toxic metal ions through a combination of complexation and safe deposition. Such acquisition of special tolerance traits is not available to non-tolerant genotypes. Metal hyperaccumulation is defined based on certain metal-specific thresholds of metal levels detected in the shoots. Thus, plants with an ability to accumulate Zn, Ni and Cd in excess of 1, 0.1, and 0.01% of dry weight, respectively are considered as hyperaccumulators for these metals (Chaney et al., 1997; Clemens, 2001). The metal hyperaccumulators hold a high potential for being used in clean-up of toxic metal-contaminated soils (phytoremediation). It appears possible to engineer efficient plants for remediation purposes by combining the hyperaccumulation trait with the high biomass production ability of crop species. A clearer understanding of metal tolerance/hyperaccumulation mechanisms will greatly facilitate the realization of this goal. Several transporters for micronutrients have been characterized which exhibit varying degrees of specificity, implying that non-essential metals share some of them for entering the cell (Demidchik et al., 2002; Hall and Williams, 2003). Furthermore, the vacuolar compartmentation of surplus metal concentrations seems to be a strong component of the cellular metal detoxification strategy (Dietz et al., 2001).
Upon exposure to metals, plants often synthesize a set of diverse metabolites that accumulate to concentrations in the millimolar range, particularly specific amino acids, such as proline and histidine, peptides such as glutathione and phytochelatins (PC), and the amines spermine, spermidine, putrescine, nicotianamine, and mugineic acids. Thus, nitrogen metabolism is central to the response of plants to heavy metals. The scheme presented in Fig. 1 gives examples addressed in the paper and the metabolic link. Except for PC with metal-dependent activation of enzyme activity, nicotianamine, and mugineic acid synthesis, the responses may not or not in each case be the primary plant reactions to heavy metals. However, from the data available, it has become clear that changes in the contents of these metabolites bear functional significance in the context of metal stress tolerance. Therefore, this review compiles the information available on selected stress metabolite accumulation under conditions of heavy metal exposure and examines the functional significance of the response. Emphasis has been given to proline that appears to possess a unique function not only under drought but also in heavy metal-stressed plants.
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Proline |
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TopAbstractIntroductionProlineHistidineOther amino acidsPolyaminesGlutathione and phytochelatinsOther N-containing metabolites...Summary and outlookReferences |
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The proteinogenic amino acid proline functions as an osmolyte, radical scavenger, electron sink, stabilizer of macromolecules, and a cell wall component (Matysik et al., 2002
). Under salinity, proline accumulates to whole tissue concentrations up to 1 mol l–1. Increased levels of proline correlate with enhanced salinity tolerance (for a review see Munns, 2005), for example, in tobacco cell culture expressing the regulatory Hal3 gene from Arabidopsis thaliana (Yonamine et al., 2004).
Proline synthesis
Proline is predominantly synthesized from glutamate (Fig. 2). Three enzymatic activities, namely (i) the 
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-glutamyl kinase activity of 1-pyrroline-5-carboxylate synthetase (At2g39800), (ii) the glutamic--semialdehyde dehydrogenase activity of 1-pyrroline-5-carboxylate synthetase (PCS), and (iii) two isogenes of 1-pyrroline-5-carboxylate reductase (PCR) convert glutamate to proline in three exergonic reactions consuming 1 ATP and 2 NADPH per proline. The consumption of two moles of NADPH implies that proline accumulation may serve as an electron sink mechanism. Alternatively, proline is generated from ornithine by ornithine-
-aminotransferase, where 1-pyrroline-5-carboxylate is produced (not shown). Transcript abundances in plant tissues of Arabidopsis thaliana and A. halleri were derived from the NASC-arrays database that contains microarray hybridization results from more than 1000 experiments. Table 1 shows that strong transcript regulation is reported for 1-pyrroline-5-carboxylate synthetase (P5CS1) in Arabidopsis thaliana roots upon treatment with lead, with induction factors (IF) of IF=6 at 25 µM Pb and IF=31 at 50 µM Pb. Other transcripts involved in proline synthesis only revealed weak up- and down-regulation, for instance P5CS2 in leaves from Pb-treated plants with factors of about 1.4. Transcripts of P5CS1 are also up-regulated in response to Cs stress (Sahr et al., 2005).
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Accumulation
Proline is an extensively studied molecule in the context of plant responses to abiotic stresses. Many plants accumulate this compatible solute under water deficit (Aspinall and Paleg, 1981
), salinity (Ashraf and Harris, 2004), low temperature (Naidu et al., 1991), high temperature, and some other environmental stresses. Up-regulation of proline is often encountered in plants, ranging from algae to angiosperms, under heavy metal stress too. Some reports are listed in Table 2. Heavy metal-induced proline accumulation is generally comparable, in magnitude and other characteristics, to that occurring under other abiotic stresses. An increase of up to >20-fold in the free proline content in the leaves of metal non-tolerant Silene vulgaris was observed. Concentrations in the range of 10 to 50 mM are commonly encountered. The proline-inducing ability of different metals varied (Fig. 3). When compared at equimolar concentrations in the growth medium, Cu was most effective in inducing proline accumulation followed by Cd and Zn, respectively. But, when compared at equal toxic strength, proline accumulation decreased in the order Cd > Zn > Cu (Schat et al., 1997). Cu and Cd proved to be strong inducers of proline in other plant species as well (Alia and Saradhi, 1991; Bassi and Sharma, 1993a, b). The magnitude of the metal-induced increase in proline content in other plant species was lower than that in Silene vulgaris, although they were exposed to higher metal concentrations. Often, the increase in shoot proline content is higher than that in roots. Thus, the bulk of metal-induced proline in Silene vulgaris was restricted to the leaves (Schat et al., 1997). Nevertheless, the root proline content in Lactuca sativa increased proportionally with an increase in Cd concentration causing a 13-fold rise at 100 µM Cd (Costa and Morel, 1994). Zn and Cu-induced proline rise in Lemna minor was fairly rapid; it was measurable within 6 h of metal treatment (Bassi and Sharma, 1993a). Similarly, a Cu-dependent increase in the proline level of detached rice leaves was detected within 4 h (Chen et al., 2001). In Scenedesmus, Pro contents increased in response to Cu and Zn (Tripathi and Gaur, 2004). In the case of Cu, Pro contents peaked at 10 µM while it increased steadily with Zn up to 100 µM.
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High constitutive proline in metal-tolerant plants
The metal-tolerant populations of three different species namely, Armeria maritima, Deschampsia cespitosa, and Silene vulgaris have been reported to possess substantially elevated constitutive proline levels in different plant parts even in the absence of excess metal ions when compared with their non-tolerant relatives. A highly Cu-tolerant Armeria maritima population from a copper-containing bog in Wales contained a high proline content in the roots. The trait was heritable since plants derived from the seeds collected from bog plants also exhibited similarly high proline levels. By contrast, the plants from a non-copper site did not show the elevated proline contents (Farago and Mullen, 1979
). Likewise, the Zn-tolerant clones of Deschampsia cespitosa, grown on medium with nitrate as the N source, contained about 4-fold increased levels of proline in roots when compared with the non-tolerant clones (Smirnoff and Stewart, 1987). Unlike Armeria and Deschampsia, the elevated constitutive proline in metal-tolerant Silene vulgaris, 5–6-fold over that in non-tolerant plants, was predominantly restricted to the leaves (Schat et al., 1997). Following the application of excess metals, the responses of D. cespitosa and S. vulgaris populations essentially resembled each other in that the sensitive lines of both species accumulated high proline contents but the tolerant ones did not (Smirnoff and Stewart, 1987; Schat et al., 1997). The proline increase in metal-tolerant S. vulgaris due to very high Cd concentrations was an exception. The decreased relative proline accumulation in the tolerant ecotype could be due to a slower development of water deficit under metal stress (Schat et al., 1997).
Proline in metal tolerance
The occurrence of high constitutive proline contents in metal-tolerant populations of three taxonomically unrelated species is striking. It is tempting to assume a function for proline in increased metal tolerance in these species. The higher proline production has recently been demonstrated to correlate with increased metal tolerance in a transgenic alga (Siripornadulsil et al., 2002). In this study, a gene encoding mothbean 1-pyrroline-5-carboxylate synthetase (P5CS), responsible for catalysis of the first step of proline synthesis, was introduced into the nuclear genome of green microalga Chlamydomonas reinhardtii. The transgenic alga produced 80% higher proline levels than the wild-type cells, grew more rapidly in Cd concentrations as high as 100 µM, and bound 4-fold more Cd than the wild-type cells.
Mechanism of Proline accumulation
Proline accumulation may be realized by increased synthesis, release from macromolecules or decreased degradation. Costa and Morel (1994) suggested that inhibition of proline oxidation is the reason for higher root proline levels. Chen et al. (2001) found excess Cu-induced proline accumulation in detached rice leaves to be related to proteolysis and increased activities of 1-pyrroline-5-carboxylate reductase or ornithine--amino-transferase. One of the consequences of exposure to heavy metals is the deterioration of the plant–water balance (Rauser and Dumbroff, 1981; Poschenrieder et al., 1989; Barcelo and Poschenrieder, 1990). This may trigger the accumulation of proline, for example, in response to Cd in L. sativa (Costa and Morel, 1994). Schat et al. (1997) established the dependence of metal-imposed proline induction on the development of water deficit. They found that suppression of transpiration at raised relative humidity (98%), as achieved by placing the plants under transparent polyethylene covers, inhibited proline accumulation almost completely, even at metal accumulation rates in leaves responsible for up to a 20-fold increase of leaf proline levels at 75% relative humidity in uncovered control plants. In a converse manner, Kastori et al. (1992) concluded that metal-induced proline accumulation in sunflower is a direct consequence of metal uptake excluding a role of water deficit. The conclusion was based on the observation that, in their study, proline accumulation also occurred in isolated ‘fully turgid’ leaf discs floating on solution. However, the metal concentrations used were sufficiently high to damage the permeability functions of membranes causing the loss of osmolytes and hence, turgor (De Vos et al., 1991, 1993). Consequently, the role of water potential in metal-induced proline accumulation was not ruled out. The possibility of ABA mediating Cu-induced proline accumulation in detached rice leaves has been suggested (Chen et al., 2001). Light was identified as another factor that promotes proline accumulation under environmental stresses (Joyce et al., 1992) including Cd exposure (Arora and Saradhi, 1995). The mechanism of light-dependent stimulation is not understood.
Suggested functions of proline
Osmoregulation:
Based on its known properties proline may be involved in plant heavy metal stress by different mechanisms, i.e. osmo- and redox-regulation, metal chelation, and scavenging of free radicals. The role of proline in osmoregulation and, in turn, in conferring plants the ability to withstand water deficit stress is well established (Aspinall and Paleg, 1981; Kavikishor et al., 1995). Transgenic tobacco containing elevated constitutive proline contents was less susceptible to the inhibitory effects of severe osmotic stress (Kavikishor et al., 1995). In addition to contributing to the osmotic adjustment at relatively high concentrations, proline at low concentrations affected the expression of certain genes associated with osmotolerance (Kiyosue et al., 1996). Thus, it is likely that proline offsets the water deficit developed due to the exposure to heavy metals. Proline has been reported to bring about stomatal closure in several plant species (e.g. Rajagopal, 1981). Such an effect would be expected to restrict the metal uptake and translocation via suppression of transpiration. The significance of a resultant reduction in metal toxicity through this mechanism remains to be examined. Osmoregulation appears to be a common element of plant reactions to various abiotic stresses. This implies that the plant populations capable of efficient osmoregulation, such as the drought-tolerant ones, might better cope with the component of metal toxicity that also includes the development of water-deficit, for example, due to root growth inhibition (Barcelo and Poschenrieder, 1990).
Proline-dependent metal chelation:
The possibility of proline involvement in the chelation of metal ions is indicated. Proline was demonstrated to protect glucose-6-phosphate dehydrogenase and nitrate reductase in vitro against Zn- and Cd-induced inhibition. The measurements with a Cd-specific electrode revealed that the proline-dependent enzyme protection was based on a reduction of free metal ion activity in the assay buffer due to formation of a metal–proline complex (Sharma et al., 1998). A role for proline–enzyme or proline–water interactions such as the one suggested in the case of proline-dependent protection of enzyme activity against salinity, heat or dilution (Schobert and Tschesche, 1978; Krall et al., 1989; Solomon et al., 1994) was not evident in this experiment. The preliminary mass spectroscopic analyses made by the authors indicated the formation of proline–Cd complexes of variable masses in an aqueous assay system (SS Sharma, M Georgi, KJ Dietz, unpublished results). Regarding the significance of the metal chelation function of proline in vivo, it is interesting that Cu in the roots of Cu-tolerant A. maritima was thought to exist as a Cu–proline complex (Farago and Mullen, 1979). However, results contrary to this have also been reported. The possibility that cytoplasmic free Pro directly sequesters Cd was examined in the wild type and a Pro-overproducing transgenic Chlamydomonas reinhardtii by determining the chemical identity of the atoms binding Cd using extended X-ray absorption fine structure (EXAFS) (Siripornadulsil et al., 2002). Data suggested that Cd was not complexed by the same conjugates in the wild type and transgenic algae. In P5CS-1-expressing transgenic alga, the EXAFS spectrum was best fit by Cd co-ordination to four S atoms suggesting Cd sequestration by phytochelatins and not Pro. By contrast, Cd was co-ordinated by O and S atoms in the wild-type cells which was consistent with the findings of Adhiya et al. (2002). For metals like Cd, Pro chelation does not seem to be important as Cd strongly induces phytochelatins (PCs) that are likely to chelate most of the root Cd in short-term exposure (DeKnecht et al., 1994). Wagner (1993) suggested citric acid to be a major ligand for Cd at low cellular concentrations. Metal ligands may vary on an organ- and compartment-specific basis even for the same metal. Thus, Salt et al. (1995) found that Cd in Brassica juncea roots was chelated by S-ligand (PCs). But in the xylem sap it was involved with some, hitherto unknown, N- or O-containing ligand. It will indeed be interesting to work out the relative contribution of proline to metal chelation in a cellular scenario, i.e. in the presence of co-occurring ligands. Proline may be of particular importance in binding metal ions that do not form complexes with PC. Currently, emerging metabolomic approaches should be applied to plants exposed to metal ions with diverse properties and that might be rewarding in this context.
Proline as an antioxidant:
Exposure of plants to both redox active, for example, Cu and Hg, and other metals, for example, Cd and Zn, induces the stimulated generation of free radicals that leads to oxidative stress (De Vos et al., 1991; Weckx and Clijsters, 1996, 1997; Dietz et al., 1999). This represents one of the major causes of toxicity particularly due to redox metals. The cells are equipped with an elaborate network of antioxidative enzymes and low molecular weight metabolites which mitigate the oxidative stress (Dietz et al., 1999). Proline has been reported to scavenge different free radicals in certain in vitro generation and detection systems. Smirnoff and Cumbes (1989) demonstrated the hydroxyl radical (OH·) scavenging property of proline. OH· radicals were generated by ascorbate/H2O2 or by xanthine oxidase/hypoxanthine/H2O2 and detected by hydroxylation of salicylate or by denaturation of malate dehydogenase. The reaction product formed between proline and OH· was not determined in this study. Proline might react with OH· under H+-abstraction by forming a stable radical with spin on the C-5 atom (Rustgi et al., 1977). A proline-nitroxyl radical R2N-O· is also known to be formed between Pro and OH· (Floyd and Nagi, 1984). Similarly, Alia et al. (2001) reported the singlet oxygen (1O2) quenching action of proline. They generated 1O2 photochemically by illumination of toluidine blue and detected, through EPR spectroscopy, the 1O2-dependent formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) from 2,2,6,6-tetramethylpiperidine (TEMP). The 1O2 quenching of proline seems to be based on its capability to form a charge-transfer complex due to a low ionization potential. Further evidence for in vitro proline-dependent free radical scavenging has been obtained from graft co-polymerization reactions. The latter are performed in polymer chemistry and involve grafting of some monomer such as methyl acrylate onto a polymeric backbone such as cellulose. The grafting is essentially mediated by free radicals generated via a chemical or a physical initiator and could be taken as a quantitative measure of free radical generation. Inclusion of proline in the reaction system substantially inhibited the extent of grafting initiated either by Ce4+ ions or -radiation (S Kaul, IJK Mehta, SS Sharma, unpublished results). Proline did not interact with superoxide radicals (Smirnoff and Cumbes, 1989). The mechanisms of ROS quenching by proline have been summarized recently by Matysik et al. (2002).
Proline possibly detoxifies the ROS under stress in vivo too (Smirnoff, 1993). The pretreatment of Chlorella vulgaris with exogenous proline was found to counteract lipid peroxidation as well as K+ efflux observed after exposure to the heavy metals Cu, Cr, Ni, and Zn. Proline accumulation in C. vulgaris due to metal treatment was also evident (Mehta and Gaur, 1999). Similarly, proline, added to the nutrient medium, lowered the inhibitory influence of Cu on the growth of cyanobacterium Anacystis nidulans; the effect being more pronounced when proline was added prior to Cu treatment (Wu et al., 1995). The effect was explained in terms of proline-dependent protection to the membranes against Cu-induced K+ leakage. Later, Wu et al. (1998) reported reduced Cu uptake by Chlorella cells containing high proline concentrations. Free proline content correlated positively with Cu tolerance of the lichen photobiont Trebouxia erici (Backor et al., 2004). The toxic metal effects were also alleviated by proline in Scenedesmus armatus (El-Enany and Issa, 2001). Recently, free proline levels have been found to be correlated with the GSH redox state and MDA content in Cd-treated low (wild type) and high (transgenic) proline strains of C. reinhardtii (Siripornadulsil et al., 2002). Thus, the wild-type cells exhibited a 4-fold more oxidized glutathione pool than transgenic P5CS-1 expressing cells when grown in the presence of Cd suggesting a less oxidizing cytoplasmic state in transgenic than in the wild-type cells under Cd stress. Also, the transgenic cells produced much less MDA than the wild-type cells upon exposure to Cd. Based on these findings, free Pro was suggested to act as an antioxidant in Cd-stressed cells; consequently, increased GSH levels favour enhanced phytochelatin synthesis and sequestration of Cd.
In Scenedesmus, Pro pretreatment decreased lipid peroxidation induced by 5 µM Cu by more than 75% similar to methylviologen- and UV-B-induced MDA generation, while Zn-dependent lipid peroxidation was suppressed by 30% only (Tripathi and Gaur, 2004). These data concur with a preferential antioxidant function of Pro by detoxifying hydroxyl radicals making the metal binding unlikely.
Recently, metal-dependent activation of mitogen-activated kinase pathways has been demonstrated in plants following Cd and Cu treatment (Jonak et al., 2004). The data indicate the differential and metal-specific activation of signalling pathways and the significance of ROS in mediating the stress responses. In this context it will also be important to dissect the diverse possible functions of proline in metal-stressed plants.
Proline as regulator:
Specific Pro functions in plant morphogenesis were revealed in antisense transgenic Arabidopsis with decreased levels of pyrroline-5-carboxylate synthetase. In parallel with decreased levels of Pro, the plants revealed morphological alterations in leaves and a defect in inflorescence elongation. Structural cell wall proteins were specifically affected in the antisence transgenics (Nanjo et al., 1999). Based on a parallelism between Cd-induced growth inhibition and a rise in proline, Chen and Kao (1995) suggested proline to mediate the toxic effect of Cd on rice seedlings. The exogenous proline mimicked the Cd effect. In fact, there are a few recent reports indicating the toxicity of exogenous Pro in plants (Hellmann et al., 2000; Deuschle et al., 2001). Nanjo et al. (2003) evaluated the Pro toxicity in Arabidopsis T-DNA tagged mutant pdh that was defective in Pro dehydrogenase (AtProDH), responsible for catalysing the first step of Pro catabolism. The pdh mutant was hypersensitive to exogenous L-Pro at concentrations 
10 mM while wild-type plants grew normally at such concentrations. In a recent study by Yamada et al. (2005) exogenous Pro-dependent growth inhibition and Pro accumulation in Arabidopsis thaliana were lower than that in petunia indicating the species-specificity of the phenomenon. Further research towards integration of the growth inhibiting and protecting properties of Pro is needed.
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Histidine |
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TopAbstractIntroductionProlineHistidineOther amino acidsPolyaminesGlutathione and phytochelatinsOther N-containing metabolites...Summary and outlookReferences |
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Nearly three-quarters of all known metal hyperaccumulator plants are Ni accumulators. The Ni-hyperaccumulation trait in Alyssum species (Brassicaceae) has been demonstrated to be specifically linked to the ability for free histidine production (Krämer et al., 1996
). Whereas the exposure to Ni resulted in a large and proportionate increase in histidine concentration in xylem sap in Ni-hyperaccumulating A. lesbiacum, such a response was lacking in the non-accumulator A. montanum. The significant co-ordination of Ni with histidine was revealed in the extended X-ray absorption fine structure analysis of the xylem sap in A. lesbiacum. Thus, Ni-hyperaccumulation relies on histidine-dependent root-to-shoot translocation of Ni. Furthermore, A. montanum, when supplied with equimolar concentrations of exogenous histidine, transported more Ni to the shoot as a result of enhanced Ni flux into the xylem and showed tolerance to applied Ni concentrations. Similar results have been obtained with Ni non-accumulator Brassica juncea (Kerkeb and Krämer, 2003). The constitutive His contents of A. lesbiacum roots were 4.4-fold higher than that in B. juncea. Kerkeb and Krämer (2003) examined the role of free His in xylem loading in A. lesbiacum and B. juncea. The data are consistent with a model where Ni uptake is independent of His uptake and the enhanced release of Ni into the xylem is associated with a concurrent release of His from an increased root free His pool. Another Ni-hyperaccumulator Thlaspi goesingense exhibited increased root His concentrations when compared with T. arvense. However, none of the three genes of histidine biosynthesis studied was regulated by Ni (Persans et al., 1999). Besides Ni, Salt et al. (1999) identified Zn–His complexes in the roots of Zn-hyperaccumulator Thlaspi caerulescens. Histidine levels have also been demonstrated to correlate with tolerance to Ni and other metals of the yeast Saccharomyces cerevisiae (Pearce and Sherman, 1999). Recently, a cell surface-engineered yeast displaying a histidine oligopeptide (hexa-His) has been shown to adsorb 3–8 times more copper ions than the parent strain and to be more resistant to Cu than the parent (Kuroda et al., 2001). The beneficial role of high histidine levels has been shown in transgenic Arabidopsis thaliana expressing a Salmonella typhimurium ATP phosphoribosyl transferase enzyme (StHisG). The bacterial enzyme is insensitive to feedback inhibition by histidine. The transgenic plants accumulated about 2-fold higher histidine levels than wild-type plants and showed more than 10-fold increased biomass production in the presence of toxic Ni in the growth medium (Wycisk et al., 2004) convincingly demonstrating a mechanistic link between His and moderate Ni tolerance. |
Other amino acids |
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TopAbstractIntroductionProlineHistidineOther amino acidsPolyaminesGlutathione and phytochelatinsOther N-containing metabolites...Summary and outlookReferences |
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Zinc toxicity, in terms of root growth inhibition, in Deschampsia cespitosa (metal tolerant and non-tolerant clones) varied with the source of N in the growth medium. Zn was invariably less toxic when N was supplied as ammonium rather than nitrate. The ammonium-grown non-tolerant clone was found to accumulate asparagine in the roots; subsequently formed Zn-asparagine complex may reduce Zn toxicity (Smirnoff and Stewart, 1987
). Properties of asparagine as ligand towards Cd, Pb, and Zn have been reported from in vitro studies (Bottari and Festa, 1996). The accumulation and employment of specific amino acids in metal tolerance and hyperaccumulation is a species-specific trait. Homer et al. (1997) determined the amino acids in three species of Philippine Ni-hyperaccumulators namely, Walsura monophylla, Phyllanthus palwanensis, and Dechampetalum geloniodes. The Ni content in the leaf extracts of these plants was 23.7, 34.3, and 134 µmol g–1, respectively. Among all amino acids Pro was highly abundant in all cases. Thus, the above species contained 29, 14.3, and 3 µmol g–1 proline, respectively. As an exception, glutamine was also present in high concentration (29.8 µmol g–1) in W. monophylla. These findings indicate that association between amino acids and metals could be important particularly because Ni complexes with amino acids are considerably more stable than those with carboxylic acids (Homer et al., 1997). The Ni-hyperaccumulator Stackhousia tryonii was analysed for changes in amino acid contents of xylem sap when grown at low or high Ni (Bhatia et al., 2005). Total amino acid concentrations slightly decreased from 22 to 18 mM and the proportion of Gln declined from 48% to 22 mol% while Ala, Asp, and Glu increased. Asp and Ala may contribute to complexation of Ni in xylem (Bhatia et al., 2005).
Zn administration to tomato and soybean strongly affected amino acid concentrations (White et al., 1981a). A study of metal complex equilibria in xylem fluids suggests that the major portion of Cu and Ni was bound to asparagine and histidine (White et al., 1981b). Also present in exudates from high Zn-treated tomato were Cu–aspartate and Cu–threonine complexes. Amino acids do not bind Zn efficiently at acidic pH. Thus, organic acids are the predominant ligands at low pH, but amino acid–Zn complexes are increasingly formed as the pH increases (White et al., 1981b). Cysteine contents increased by factors of 4.5 and 3.8 in tolerant and sensitive ecotypes of Silene vulgaris, respectively, in response to metal stress that caused similar inhibition of root growth (Harmens et al., 1993). Cysteine is required for methionine and glutathione/phytochelatin synthesis, and, therefore, is a central metabolite in antioxidant defence and metal sequestration. Genetically increased capacity for metal-induced Cys synthesis was shown to support survival of Arabidopsis under acute Cd stress (Dominguez-Solis et al., 2004). The significance of these alterations in amino acid composition for metal binding in vivo is difficult to assess. However, it should be kept in mind that amino acids are asymmetrically distributed between plasmatic and secretory compartments. In barley mesophyll cells, the concentrations of total amino acids in the extravacuolar compartment were about twice that of the vacuole and may be in the range of 100 mmol l–1; similar values were likewise observed in chloroplasts (Krause et al., 1984; Dietz et al., 1990). Such high concentrations further increase under stress and are likely to contribute to heavy metal binding.
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Polyamines |
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TopAbstractIntroductionProlineHistidineOther amino acidsPolyaminesGlutathione and phytochelatinsOther N-containing metabolites...Summary and outlookReferences |
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Polyamines are ubiquitous in all organisms. They influence a variety of growth and development processes in plants and have been suggested to be a class of plant growth regulators and to act as second messengers (Evans and Malmberg, 1989
; Slocum and Flores, 1991; Kakkar and Sawhney, 2002). The polyamines are cations due to protonation at cytoplasmic pH, i.e. putrescine2+, spermidine3+, and spermine4+. This accounts for their binding ability to nucleic acids (Flink and Pettijohn, 1975) and affinities for phospholipids in plasmamembranes (Grimes et al., 1986). The levels of polyamines and the activities of their biosynthetic enzymes in plants increase under environmental stresses (Evans and Malmberg, 1989). Polyamines are synthesized from ornithine, citrulline or arginine following decarboxylation to yield putrescine and the addition of one or two aminopropyl groups from S-adenosyl methionine to generate spermine and spermidine (Kakkar and Sawhney, 2002). Polyamine functions in plants are only slowly unravelled. Transgenic and mutant plants with altered polyamine levels reveal strong developmental aberrations (reviewed in Kakkar and Sawhney, 2002). In addition, polyamines have an antioxidative property by quenching the accumulation of 
probably through inhibition of NADPH oxidase (Papadakis and Roubelakis-Angelakis, 2005).
Polyamine contents are altered in response to the exposure to heavy metals. Weinstein et al. (1986) showed an up to 10-fold increment in putrescine content in Cd-treated oat seedlings and detached oat leaves with a marginal rise in spermidine and spermine content. The Cd-dependent increase in putrescine was blocked by difluoromethylarginine (DFMA), an inhibitor of arginine decarboxylase (ARGdc), linking this enzyme with Cd-induced putrescine biosynthesis. The response of different polyamines to Cd treatment strongly varied in Phaseolus vulgaris in an organ-specific manner. Putrescine increased in root, hypocotyl, and epicotyl whereas spermidine increased in hypocotyl, decreased in leaves, and did not change in roots. By contrast, spermine level decreased in all seedling parts. Al-induced changes in putrescine of Catharanthus roseus were influenced by the cell age. Al addition to the suspension cultures induced an increase in putrescine level within 24 h in freshly transferred cells but reduced the same at later stages (2–7 d) (Zhou et al., 1995). Elevated putrescine and 1,3 diaminopropane (DAP) due to Hg treatment were also observed in the green alga Chlorogonium elongatum (Agrawal et al., 1992).
Transcripts encoding polyamine synthetic genes were non-responsive or slightly up- and down-regulated in response to Zn-, Pb-, and Cs-exposure (Table 1). For example, spermine/spermidine synthase mRNA was increased with IF=2.6 in leaves of Arabidopsis halleri exposed to 25 µM Zn, and with IF=2.9 in roots of Cs-stressed Arabidopsis thaliana. However, there was no uniform response of the tissues to metal stressors.
A specific role of polyamines in plants under metal stress is not yet known. However, there is a strong possibility that they can effectively stabilize and protect the membrane systems against the toxic effects of metal ions particularly the redox active metals. Data supporting this are available both from in vivo and in vitro studies. Thus, NADH- and ascorbic acid-dependent lipid peroxidation in rat liver microsomes was inhibited by polyamines; spermine was most effective. Spermine binding to membrane phospholipids explained the observed inhibition (Kitada et al., 1979). Using membrane vesicles prepared from mixed soybean phospholipids, Tadolini et al. (1984) showed that polyamines inhibit lipid peroxidation when bound to the negative charges on the vesicle surface. A pretreatment of sunflower leaf discs with exogenous spermine was recently found to reverse almost completely the Cd- or Cu-induced lipid peroxidation (thiobarbituric acid reactive material) (Groppa et al., 2001). It is important here that the isolated plasmamembranes from root and hypocotyl of Cd-treated mungbean seedlings contained greater polyamine amounts than those from controls (Geuns et al., 1992). In addition, polyamines namely, spermine, spermidine, putrescine, and cadaverine have been demonstrated to scavenge free radicals in vitro (Drolet et al., 1986). Furthermore, polyamines block one of the major vacuolar channels, the fast vacuolar cation channel, and their accumulation could decrease ion conductance at the vacuolar membrane to facilitate metal ion compartmentation (Brüggemann et al., 1998).
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Glutathione and phytochelatins |
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TopAbstractIntroductionProlineHistidineOther amino acidsPolyaminesGlutathione and phytochelatinsOther N-containing metabolites...Summary and outlookReferences |
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The significance of glutathione and the metal-induced phytochelatins (PCs) in heavy metal tolerance has been summarized intensely in excellent reviews (Rauser, 1995
, 1999; Hall, 2002). Depletion of glutathione appears to be a major mechanism in short-term heavy metal toxicity (Schützendübel and Polle, 2002). In accordance with this hypothesis, a good correlation between glutathione contents and tolerance index was observed with 10 pea genotypes differing in Cd sensitivity (Metwally et al., 2005). Freeman et al. (2004) reported the concentrations of glutathione and also Cys and O-acetyl-L-serine (OAS) in shoot tissue to correlate strongly with the Ni-hyperaccumulation ability of various Thlaspi hyperaccumulator species. High GSH concentrations in hyperaccumulator T. goesingense coincided with high constitutive activity of serine acetyl transferase (SAT); SAT catalyses the acetylation of L-Ser to OAS which in turn provides the carbon skeleton for Cys biosynthesis. Elevated GSH levels in T. goesingense also coincided with the ability both to hyperaccumulate Ni and to resist its damaging oxidation effects. Further, the arsenate hypertolerance in Aspergillus sp. P37 has recently been linked to an enhanced capacity to maintain a large intracellular glutathione pool under conditions of As exposure and to sequester As(GS)3 in vacuoles. As-induced formation of vacuoles with accumulated thiol species was observed (Canovas et al., 2004). In addition to its function as an antioxidant, metal-dependent activation of PC synthase puts strain on the glutathione pool. PCs are the best known example of metal-induced metabolite accumulation and are implicated in cellular metal detoxification (for review see Steffens, 1990; Rauser, 1995). They are thiol-rich low molecular weight peptides synthesized by plants in response to exposure to heavy metals; synthesis from GSH is catalysed by phytochelatin synthase. The PC–metal complex is often sequestered in the vacuoles. From a variety of experimental approaches the involvement of PCs in the detoxification of Cd and As (Cobbett, 2000,; Schmöger et al., 2000) appears definite as they could also be linked to the differential heritable tolerance to Cd in Arabidopsis thaliana (Howden et al., 1995) and As in Holcus lanatus (Hartley-Whitaker et al., 2001). However, PCs are important for detoxification of only a limited set of metals such as Cd2+, Cu2+ and 
while Zn2+ and Ni2+ are poor inducers of PCs and exhibit low binding affinity. Most other metals lack significant binding at all. Although overexpression of PC synthase (AtPCS1) in S. cerevisiae did not alter the sensitivity to Zn and Ni, it induced an enhanced tolerance to Cd, As, and Hg (Vatamaniuk et al., 1999). There seems to be no kingdom barrier as to the role of PCs in metal detoxification. Thus, Caenorhabditis elegans was recently demonstrated to express a gene coding for a functional phytochelatin synthase (PCS). Further CePCS complemented the Cd sensitivity of a S. pombe PCS knock-out strain by conferring the PCS activity (Clemens et al., 2001; Vatamanuik et al., 2001). PCS is post-translationally activated by metal binding to specific activating sites (Maier et al., 2003). In addition to this mechanism, recent work has shown that transcriptional activation of PCS is another level of regulation that is dependent on isoform, developmental stage, and growth condition (Lee and Korban, 2002; Finkemeier et al., 2003). In barley, mRNA levels of PCS were stimulated in response to Cd when the plants were low in nitrogen, pointing to an N-saving mechanism at low N (Finkemeier et al., 2003). PCS protein accumulation in leaves was also seen in Brassica juncea treated for 6 d with 25 µM Cd, but the response was absent from roots (Heiss et al., 2003). On the other hand, not all plants respond to Cd with PC synthesis. In Salix treated with Cd, phytochelatins (-(Glu-Cys)nGly; n=2–3) were not detectable, suggesting that heavy metal stress defence in Salix is independent of the phytochelatins PC2 and PC3 (Landberg and Greger, 2004). Despite this surprising result, a central but not exclusive role of phytochelatins in specific metal tolerance is apparent. |
Other N-containing metabolites in relation to metal stress |
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TopAbstractIntroductionProlineHistidineOther amino acidsPolyaminesGlutathione and phytochelatinsOther N-containing metabolites...Summary and outlookReferences |
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Higher plants also synthesize other N-containing amino acid-derived metabolites upon metal stress such as betaines, mugineic acid, and nicotianamine. While betaines represent general stress metabolites, the other two have a specific function in metal homeostasis and metal stress defence. Glycinebetaine, the trimethyl glycine, is a commonly encountered osmolyte that accumulates in plants under salinity and drought stress. Its synthesis predominantly depends on decarboxylation of serine to ethanolamine that is subsequently methylated with S-adenosylmethionine as the methyl donor. Armeria maritima ecotypes were collected from sandy grassland, Zn-contaminated sites or salt marshes (Köhl, 1996
). Upon exposure to drought, the plants from the Zn-mines accumulated more proline, but also more betaine, than plants from sites without metal contamination.
Nicotianamine (NA) is ubiquitously present in the plants and is synthesized from three molecules of methionine by nicotianamine synthase (NAS) (Fig. 4). The Arabidopsis thaliana genome contains four nas genes. Initially, nicotianamine was identified in the context of Fe nutrition. Three carboxylic acid groups within each molecule enable high efficiency binding of Fe and other transition metals. NA is a precursor for secreted mugineic acids, chelates Fe, and participates in Fe transport (Schmidt, 2003). Ectopic overexpression of nicotianamine aminotransferase (NAAT) in tobacco, which synthesizes NA but not phytosiderophores, consumed endogenous NA and caused a severe decrease in Cu>Fe>Zn>Mn contents in leaves to levels of element deficiency and reproductive sterility (Takahashi et al., 2003). Grafting of naat-tobacco shoots on wild-type roots substantially restored Fe, Cu, and Zn concentrations in naat-tobacco and sterility was overcome. From the data the authors proposed multiple functions of nicotianamine in the delivery of metals, particularly at the reproductive stage (Takahashi et al., 2003). Recent work indicates that NA is also important in heavy metal detoxification. The Zn-hyperaccumulator plant Arabidopsis halleri expresses the nas-2 and -3 genes at a very high constitutive level, for example, nas-2 with a 73-fold higher transcript level compared with A. thaliana (Weber et al., 2004). The response of nas 1–4 transcripts to elevated Zn supply was not uniform, neither in the sensitive A. thaliana and A. halleri, nor between roots and leaves (Becher et al., 2004). Heterologous expression of Atnas2 in the yeast Schizosaccharomyces pombe conferred increased Zn tolerance with a shift of IC50 from 50 µM in the wild type to 300 µM in the mutant yeast strain (Weber et al., 2004). The data suggest an important role of NA in metal homeostasis especially of hyperaccumulating species and possibly in hyperaccumulation. NA could be involved in the transfer of excess metals from the roots to the shoots. Plants with decreased and increased NA contents should be tested for altered metal tolerance.
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Mugineic acids are synthesized in graminaceous species from NA by transamination, oxidation, and hydroxylation reactions (Kobayashi et al., 2005
) and, thus, are based on methionine building blocks as well (Fig. 4). They belong to the group of phytosiderophores that are secreted under iron deficiency to promote iron acquisition from the rhizosphere. Nicotianamine aminotransferase (NAAT) catalyses the transfer of amino group from NA for the biosynthesis of phytosiderophores. In addition to mobilizing Fe, phytosiderophores appear to affect heavy metal solubility, leading to an increased bioavailability, for instance, of Cd (Collins et al., 2003). Recently, the H+-symporter ZmYS1 has been identified in maize that transports metal complexes of NA and phytosiderophores. The KM-values were 19 µM for deoxymugineic acid–Ni-complex and 165 µM for NA–Ni-complex (Schaaf et al., 2004). The transporter also carried Cu-, Fe-, and Zn-complexes following its expression in yeast. Since eight homologues of ZmYS1 have been identified based on sequence similarity in the genome of Arabidopsis thaliana, a plant that lacks phytosiderophore production, it is hypothesized that these transporters may be involved in metal ion distribution in general (Curie et al., 2001; Schaaf et al., 2004). Mugineic acid certainly needs more attention towards its role in metal delivery and distribution under conditions of excess metals particularly on Fe-limited soils. |
Summary and outlook |
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TopAbstractIntroductionProlineHistidineOther amino acidsPolyaminesGlutathione and phytochelatinsOther N-containing metabolites...Summary and outlookReferences |
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The compiled data demonstrate the significance of nitrogen-containing metabolites beyond phytochelatins and glutathione in plant response and acclimation to heavy metals. Since the various metal ions have specific chemical properties and induce distinct responses of adaptation and damage development, it is not surprising that accumulating N-metabolites display a variety of functions, i.e. metal ion chelation, antioxidant defence, protection of macromolecules, and possibly signalling. The case of proline exemplifies the functional diversification of a compound initially addressed as a compatible solute in the context of osmotic and salinity stress: Proline is suggested to quench ROS and reactive nitrogen species (RNS) and to relieve the oxidative burden from the glutathione system. This may facilitate phytochelatin synthesis and enhance metal tolerance (Siripornadulsil et al., 2002
). Direct metal binding by Pro is suggested although a thorough evaluation of this function in vivo is required. It is difficult to dissect the distinct contributions of individual functions of a compound from the overall tolerance, particularly if the compound is essential for basic metabolism. In a more straightforward manner, current research may focus on the identification of novel players in metal tolerance by employing post-genomic approaches such as metabolite profiling and cDNA array analyses (Becher et al., 2004; Weber et al., 2004; Urbanczyk-Wochniak and Fernie, 2005). The Fe deficiency-induced up-regulation of all genes of the methionine cycle and nicotianamine synthesis represents an intriguing and convincing example of the power of this kind of approach (Kobayashi et al., 2005). This Fe-related metabolism is directly linked to Zn stress and tolerance as judged by the up-regulation of nicotianamine synthase transcripts in cDNA array analyses with Arabidopsis thaliana and Arabidopsis halleri (Becher et al., 2004; Weber et al., 2004). On the other hand, mutants need to be checked routinely for altered metal sensitivity. Such reverse genetic approaches will certainly elucidate novel regulatory and metabolic relationships, such as, for example, the role of a mitochondrial Prx IIF (Finkemeier et al., 2005). Insertional inactivation of Prx IIF gene had no effect on Arabidopsis under optimum conditions, but growth was severely impaired under stress such as Cd. It would not be surprising if the list of N-metabolites involved in metal homeostasis and heavy metal defence expands in the near future. |
Acknowledgements |
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Expert help from Miriam Hanitzsch in the NASC array analysis is gratefully acknowledged. This review and parts of the own cited work originate from a collaboration funded by the Indian National Science Academy (INSA, India) and the DFG as well as DAAD (Germany).