The Society for Integrative and Comparative Biology
A Modulating Role for Antioxidants in Desiccation Tolerance1
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1 Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, West Sussex RH17 6TN, United Kingdom
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Most organisms depend on the availability of water. However, some life-forms, among them plants and fungi, but very few animals, can survive in the desiccated state. Here we discuss biochemical mechanisms that confer tolerance to desiccation in photosynthetic and non-photosynthetic organisms. We first consider damage caused by water removal and point out that free radicals are a major cause of death in intolerant tissue. Free radicals impair metabolism and necessitate protection and repair during desiccation and rehydration, respectively. As a consequence, desiccation tolerance and prolonged longevity in the desiccated state depend on the ability to scavenge free radicals, using antioxidants such as glutathione, ascorbate, tocopherols and free radical-processing enzymes. Some ‘classic’ antioxidants may be absent in lower plants and fungi. On the other hand, lichens and seeds often contain secondary phenolic products with antioxidant properties. The major intracellular antioxidant consistently found in all life forms is glutathione, making it essential to survive desiccation. We finally discuss the role of glutathione to act as a signal that initiates programmed cell death. The failure of the antioxidant system during long-term desiccation appears to trigger programmed cell death, causing ageing and eventual death of the organism. In turn, this suggests that a potent antioxidant machinery is one of the underlying mechanisms of desiccation tolerance.
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Organisms consist mostly of liquid water and losing it is deleterious. Hardly any animals and higher plants can survive desiccation, but the majority of the latter have desiccation tolerant "orthodox" seeds. Very few higher "resurrection" plants, but numerous cryptogams such as bryophytes, lichen-forming fungi and algae have desiccation tolerant vegetative tissues. In the desiccated state, they can survive for very long periods of time. When water is available, these plants revive from the desiccated state, and seeds imbibe and eventually germinate. Vegetative desiccation tolerant tissues undergo frequent desiccation and rehydration cycles throughout their entire life, but seed desiccation tolerance is lost after germination. Other organisms that can survive desiccation include (cyano)bacteria, yeast, insects, rotifers, nematodes, and the brine shrimp Artemia. These are dealt with elsewhere (Alpert, 2005
; Clegg, 2005; Potts, 1994; Ricci, 1998; Ricci and Pagani, 1996), while we concentrate on plants. Among the various desiccation tolerant life forms, seeds appear to have the most remarkable longevity in the dry state. The oldest seed that was germinated with credibility was a 
1,000 years old seed of sacred lotus from an ancient lake bed in China (Shen-Miller et al., 1995). The following sections discuss free radical formation and harmful effects of oxidative stress during desiccation, and how antioxidants counteract it. We then consider the responses to desiccation and rehydration of three models of desiccation tolerant organisms, a resurrection plant, a lichen and seeds. When the antioxidant system of an organism fails during long-term desiccation, oxidative processes prevail and induce programmed cell death, thus limiting longevity, even in desiccation tolerant organisms. |
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Oxygen—a two-edged sword
Oxygen, the basis of all aerobic life, is a highly oxidizing molecule, which is necessary for its role in respiration. However, this characteristic allows it to form free radicals and to participate in other oxidative chemical reactions (Finkel and Holbrook, 2000
; Abele, 2002) that are directly correlated with degenerative processes and illnesses (Marx, 1985; Skulachev, 2000, 2002). Free radicals are atoms or molecules with an unpaired electron. This unpaired electron is readily donated, and as a result, most free radicals are highly reactive. Oxygen radicals (Halliwell and Gutteridge, 1999; Elstner and Osswald, 1994; Finkel and Holbrook, 2000) include singlet oxygen (1O2), superoxide (
), the hydroxyl radical (·OH) and nitric oxide (NO·). Together with hydrogen peroxide (H2O2), which is not a free radical but is never-the-less highly reactive, these molecular species are termed reactive oxygen species (ROS).
In this section, we outline a few major pathways of ROS production in cells and the potential of ROS to damage macro-molecules (for detailed reviews see Halliwell, 1987; Halliwell and Gutteridge, 1999; Elstner and Osswald, 1994; Finkel and Holbrook, 2000; Abele, 2002). ROS also play positive roles in signalling pathways and plant disease resistance, which include rapid extracellular ROS production, the "oxidative burst" (Bolwell, 1999), but these beneficial roles of ROS lie outside the framework of this paper.
Pathways that produce reactive oxygen species
Most triplet oxygen (ground state oxygen; 3O2) is reduced to water in the respiratory electron transport chains. When the terminal oxidases, cytochrome c oxidase and the alternative oxidase, react with oxygen, four electrons are transferred and water is the product (equation 1).
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However, the four-electron reduction of oxygen to water is always accompanied by transfer of one, two, or three electrons onto 3O2. For example, the superoxide anion () is formed by the capture of one electron by oxygen (equation 2), and its formation is an unavoidable consequence of aerobic respiration (Møller, 2001). In addition, some enzymes (e.g., nitropropane dioxygenase, galactose oxidase, xanthine oxidase) catalyse reactions in which a single electron is transferred from the substrate onto 3O2. Enzymes in the cell wall and plasma membrane (NAD[P]H oxidases, peroxidases, poly- and di-amino-oxidases) can, in this way, also form extracellular , that may then play a role in signalling and inter-cellular cross-talk (Bolwell 1999; Martinez et al., 2000; Guillén et al., 2000; Delannoy et al., 2003).
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Auto-oxidation of some reduced compounds (e.g., flavins, pteridines, diphenols, and ferredoxin; equation 3) can also transfer a single electron to 3O2 (Halliwell, 1987; Halliwell and Gutteridge, 1999).
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Compared to other oxygen radicals, is less reactive, having a half-life of 2–4 µs, and a low cellular concentration (<10–11 M). It cannot react directly with membrane lipids to cause peroxidation, and cannot cross biological membranes (Vronova et al., 2002). Most formed in biochemical systems reacts with itself non-enzymatically or enzymatically (catalysed by superoxide dismutase) to form H2O2 (equation 4).
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Hydrogen peroxide formed by the previous or other reactions, and can react together to form the hydroxyl radical (·OH) (equation 5) in the iron-catalysed Haber-Weiss reaction. The hydroxyl radical is the most reactive and aggressive species known to chemistry having a half-life of 1 ns.
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In the cell wall, a Fenton-type reaction (equation 6) may be catalysed by apoplastic peroxidases (Chen and Schopfer, 1999).
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Hydrogen peroxide is formed by dismutation, but in addition, oxidases such as glycolate oxidase, glucose oxidase, urate oxidase, oxalate oxidase (the so-called "germin-like" oxidase), and amino acid oxidases can transfer two electrons onto 3O2 to form H2O2 (Halliwell, 1987; Halliwell and Gutteridge, 1999; Rea et al., 2002) (equation 7).
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In photosynthetic tissues, light energy can be transferred from photo-excited pigments onto 3O2, raising it to a more reactive, excited state known as singlet oxygen, 1O2 (equations 8 and 9) (Halliwell, 1984; Frank et al., 1999). Excess excitation or inhibition of photophosphorylation increase 1O2 production.
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In non-photosynthetic tissue, 1O2 can also be produced by electron transfer from 
OH to (equation 10). 1O2 can react directly with polyunsaturated fatty acid side chains and initiate lipid peroxidation (Halliwell, 1987).
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Enhanced formation of reactive oxygen species during desiccation
Desiccation, as other biotic and abiotic stresses, ultimately cause loss of control mechanisms that maintain low ROS concentrations (Kranner, 2002; Kranner et al., 2002). The resulting increase in ROS will lead to deteriorative processes such as ageing and eventually death (Harman, 1956, 1987; Beckmann and Ames 1998). In non-photosynthetic tissue, ROS formation during desiccation will inevitably be enhanced through imbalances in the respiratory electron transport chains. Vegetative photosynthetic tissues suffer additional damage when desiccation occurs in the light and water deficit restricts photosynthesis (equations 8 and 9). In the desiccated state, free radical production is more likely to occur through the non-enzymatic reactions and auto-oxidations outlined above than through enzyme-catalyzed routes. However, the possibility of enzyme catalyzed reactions in the dry state cannot be entirely excluded. If tissues are desiccated until their cytoplasm enters the glassy state (Sun and Leopold, 1997), this will significantly decrease molecule mobility and thus the speed of chemical reactions. However, a glassy cytoplasm is still in the liquid phase, which will allow chemical reactions to take place, presumably at very slow rates. Highly reactive molecules such as ROS are the most likely source of damage to nucleic acids, proteins and lipids. In particular, the. OH radical can attack and damage almost every molecule found in living cells. It can, for example, hydroxylate purine and pyrimidine bases in DNA (Halliwell, 1987), thus enhancing mutation rates. Oxidative damage to proteins changes their configuration, mostly by oxidizing the free thiol residues of cysteine to produce thiyl radicals. These can form disulphide bonds with other thiyl radicals, causing intra- or inter-molecular cross-links. ROS such as 1O2 and. OH can also abstract hydrogen radicals from lipids thus initiating peroxidation. Lipid peroxides decompose to give volatile hydrocarbons and aldehydes (Esterbauer et al., 1991; Valenzuela, 1991). The latter can act as second toxic messengers that disseminate initial free radical events (Esterbauer et al., 1991).
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ANTIOXIDANTS |
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Aerobic metabolism generally depends on a stringent control of ROS by antioxidants (Finkel and Holbrook, 2000
; Abele, 2002). However, as desiccation greatly enhances ROS formation, an effective antioxidant machinery is bound to be an essential trait of desiccation tolerant organisms. Protection mechanisms that include sugars, glass formation and expression of LEA proteins are discussed elsewhere (Alpert, 2005 Crowe, 1998).
"Classic" antioxidants
The major water-soluble antioxidants found in biological systems are glutathione (
-glutamyl-cysteinyl-glycine; GSH) and ascorbic acid (Asc) (Noctor and Foyer, 1998). Tocopherols and ß-carotene (Munne-Bosch and Alegre, 2002) are the main lipid-soluble antioxidants. An example of how antioxidants scavenge free radicals directly is given for GSH and ·OH in equation 11. At physiological pH, GSH is present as the glutathiolate anion (GS–). Glutathione disulfide (GSSG), formed as shown in equation 12, is re-reduced by the enzyme glutathione reductase (equation 13). This way, ·OH is scavenged and the antioxidant is recycled.
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Ascorbate (Asc) also reacts rapidly with , ·OH and 1O2 (Halliwell and Gutteridge, 1999), forming the rather unstable monodehydroascorbate (MDHA) and then dehydroascorbate (DHA). Regeneration of Asc may occur via a Mehler-peroxidase reaction sequence or through the Asc-GSH cycle (see below) at the expense of NADPH (Foyer and Halliwell, 1976). Asc also supports the regeneration of membrane-bound antioxidants such as carotenoids and 
-tocopherol.
Plants also use pathways that dissipate light energy as heat. In particular in the xanthophyll cycle (Frank et al., 1999; Demmig-Adams and Adams, 2000) solar radiation is dissipated as heat while violaxanthin undergoes de-epoxidation to antheraxanthin and then zeaxanthin. Thus formation of 1O2 is prevented.
ROS scavenging enzymes
ROS scavenging enzymes include superoxide dismutase (SOD), ascorbate peroxidase (AP) and other peroxidases, mono- and dehydroascorbate reductases, glutathione reductase (GR) and catalase (for an overview see Elstner and Osswald [1994]).
Superoxide dismutases catalyze the dismutation of to H2O2. Thus, is removed and further conversion into ·OH is prevented (equation 14). Peroxidases catalyse H2O2-dependent oxidation of substrates (S) (equation 15). Catalases break down high concentrations of H2O2 very rapidly (equation 16), but are much less effective than peroxidases at removing H2O2 present in low concentrations because of their lower affinity (high Km) to H2O2.
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An interplay between antioxidants and enzymes to scavenge ROS in plants was first suggested by Foyer and Halliwell (1976). They postulated an ascorbate-glutathione cycle for the scavenging of H2O2 produced from by SOD in which ascorbate and glutathione play an important role as reductants. This cycle involves reactions of GSH, Asc, GR, AP, mono- and dehydroascorbate reductases. Moreover, it may also be linked to -tocopherol (Finckh and Kunert, 1985).
Other antioxidants
A wide range of other compounds such as flavonoids, sugars, polyols, proline and polyamines also have antioxidant properties (Smirnoff, 1993). Particularly phenols (Rice-Evans et al., 1996) have remarkable antioxidant properties in vitro. However, seed phenols occur mainly in the seed coat, phenolic lichen products are mainly extracellular deposits, and resurrection plants accumulate their phenols in vacuoles. The role of (poly)phenols as cytoplasmic antioxidants is therefore uncertain. However, together with flavonoids, they can act as ‘sun screen’ pigments, shading the desiccated photosynthetic apparatus, and this will help avoiding 1O2 formation. This appears to be particularly important in resurrection plants (Farrant et al., 2003) and lichens (Kranner et al., 2005).
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ANTIOXIDANTS IN DESICCATION TOLERANT ORGANISMS—THREE CASE STUDIES |
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The following section reviews our work on mechanisms that protect from desiccation-induced oxidative stress in three model organisms.
The resurrection plant Myrothamnus flabellifolia
Myrothamnus flabellifolia, a short woody shrub from Southern Africa, survives severe desiccation of its vegetative organs. By investigating mildly to sub-lethally desiccated plants, Kranner et al. (2002) demonstrated that recovery from desiccation correlates with a capacity to maintain, or rapidly re-establish, a number of antioxidant systems on rehydration. After desiccation for up to 4 months, the plants recovered normal metabolism and growth upon desiccation. Leaves retained high concentrations of chlorophyll during desiccation, a source of potentially harmful 1O2 production. Desiccation triggered conversion from violaxanthin to zeaxanthin, suggesting that energy was dissipated as heat through the xanthophyll cycle. Glutathione and Asc were converted to GSSG and DHA, respectively, indicative of free-radical scavenging activity and the failure to re-reduce the active forms of these antioxidants. After 4 months of desiccation, re-watering induced formation of Asc and GSH, simultaneous reduction of GSSG and DHA, and rapid production of -tocopherol and of various carotenoids.
However, 8 months of desiccation caused sub-lethal damage to the plants. Upon rehydration, they abscised all their leaves. They grew new ones thereafter, but this required 3 months of recovery. This lethal damage to the leaves coincided with a complete breakdown of antioxidants; Asc, GSH, -carotene and -tocopherol were totally depleted and did not recover upon rehydration. Taken together, these results suggest that (i) a resurrection plant can survive desiccation without damage as long as its antioxidant and photoprotective machinery is functional, (ii) that the longevity in the desiccated state is limited and (iii) that lethal damage to leaves correlates with the breakdown of antioxidants. In turn, this suggests, that antioxidants confer desiccation tolerance.
The lichen symbiosis—Cladonia vulcani
Lichens are also extremely tolerant of desiccation. These organisms consist of a fungal "mycobiont" and a green algal (and/or cyanobacterial) "photobiont." For free-living fungi and algae, lichenization involved the evolution of a complex above-ground structure that neither of them could form alone. Lichenization requires that the fungus gives up its saprophytic life-style below ground; the photobiont may also cease its hidden life in bark, soil (Mukhtar et al., 1994) or small crevices in rocks (Ascaso et al., 1995). So far, it was unclear whether biochemical interaction between fungal and algal partners is involved in conferring tolerance to the main stresses associated with the new above-ground life-style: desiccation and irradiation. Recent results show that antioxidant and photoprotective mechanisms in the lichen Cladonia vulcani are more effective by two orders of magnitude than those of its isolated partners (Kranner et al., 2005). The only "classic" antioxidant present in the fungus is GSH. Neither the Cladonia vulcani mycobiont nor the photobiont contain Asc; tocopherol is only found in the photobiont. When isolated, both isolated alga and fungus suffer oxidative damage during desiccation. On its own, the alga only tolerates very dim light and its photoprotective system is only partially effective. Without the alga, the fungus's GSH-based antioxidant system is slow and ineffective. However, in the lichen, the two symbionts appear to induce up-regulation of protective systems in each other. In the lichen, where it is exposed to higher light intensities, the alga has lower chlorophyll concentrations, which helps avoiding 1O2 formation. In addition, it has higher concentrations of photoprotective pigments involved in non-photosynthetic quenching of light energy, and of the antioxidant -tocopherol. In addition, total glutathione (GSH plus GSSG) is present in the lichen at a level 30% greater than the sum of the contents in isolated alga and fungus. The lichen therefore appears to be better adjusted to cope with oxidative stress than its isolated partners. This mutually enhanced resistance to oxidative stress, and in particular its desiccation tolerance, are required for life above ground. This new life-style increases the chance of dispersal of reproductive propagules. To summarize, for a lichen, protection from oxidative stress is essential for survival in the desiccated state. Moreover, the enhanced antioxidant capacity contributes to the evolutionary success of the lichen symbiosis (Kranner et al., 2005).
Orthodox seeds of Pisum sativum
Orthodox seeds appear to depend on antioxidant protection in the desiccated state in a similar way to that described above. They may not use Asc, which is only present in very small amounts in dry seeds (Tommasi et al., 1999; de Gara et al., 1997). However, the patterns of GSSG and GSH formation during desiccation and rehydration, respectively, as described for vegetative tissues, are very similar in orthodox seeds during maturation (that coincides with desiccation) and germination (that starts with the uptake of water). Maturation drying will enhance ROS formation, and, coinciding with putative ROS attack, GSH is converted to GSSG which accumulates (Kranner and Grill, 1993) because desiccation limits its re-reduction to GSH. However, GSSG may also have a protective role. By binding to protein SH-groups, GSSG protects these from irreversible oxidation (e.g., to sulfonic acids) and irreversible formation of intra-molecular disulfide bonds that would cause denaturation of proteins during desiccation (Kranner and Grill, 1996). Mature, dry pea seeds, for example, contain about 25% of their total glutathione as GSSG. When imbibition allows the seed to germinate, GSSG is reduced to GSH and when reduction is complete, the activity of GR declines (Kranner and Grill, 1993). Fast removal of GSSG and re-establishment of a reducing intra-cellular redox environment (Schafer and Buettner, 2001) are essential because high GSSG concentrations (Ernst et al., 1978; Fahey et al., 1980; Dhindsa, 1987) and oxidative conditions (Shenton and Grant, 2003), respectively, inhibit protein synthesis and induce programmed cell death (Schafer and Buettner, 2001).
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LONGEVITY IN THE DESICCATED STATE—EVEN DESICCATION TOLERANT ORGANISMS DO NOT LIVE FOREVER |
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Breakdown of antioxidants changes the intra-cellular redox environment and initiates programmed cell death
As pointed out in the last section, the longevity of desiccation tolerant organisms in the dry state is limited, and viability loss correlates with the breakdown of antioxidants. If not scavenged, ROS can damage macro-molecules either directly, as explained elsewhere in this manuscript, or they may act as signals that induce programmed cell death (PCD), the mechanisms by which cells eliminate redundant cells (Raff, 1998
; Hengartner, 2000). Our recent work (I.K., unpublished data), suggests that a substantial increase in glutathione half-cell reduction potential (EGSSG/2GSH), caused by continuous oxidative stress during ageing in the desiccated state, is one of the "death triggers" that initiate PCD. In the final, or execution phase of PCD, DNA is cleaved into inter-nucleosomal fragments (Raff, 1998), and this restrains any recovery. EGSSG/ 2GSH, being one of the early PCD triggers, can be used as a universal marker of viability. In the following, we show how EGSSG/2GSH can be applied to assess viability in the three desiccation tolerant model systems, Pisum sativum seeds, the resurrection plant M. flabellifolia and in the lichen C. vulcani.
Glutathione redox potential in three model systems as a marker of viability
We have recently shown that plant and fungal cells lose viability when EGSSG/2GSH attains values of –180 to –160 mV, using data containing >250 values from 20 species in 11 plant orders and 3 species in 2 orders of lichenized fungi (I.K., unpublished data). This corresponds to the apoptotic zone in human cells (Schafer and Buettner, 2001). In this zone, repair mechanisms may still allow recovery, but plant material with EGSSG/2GSH more positive than –160 mV is dead or dying. Figure 1 demonstrates how the increase in EGSSG/2GSH is typically correlated with viability loss in Pisum sativum seeds. In these seeds, DNA fragmentation increased with decreasing viability, indicative of the executioners of programmed cell death, (meta)caspase-activated DNAses (I.K., unpublished data).
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Similarly, EGSSG/2GSH increases towards more positive values during desiccation of M. flabellifolia. As for seeds (Fig. 1), viability loss can already be predicted in the dry state (Fig. 2a), but the differences in EGSSG/2GSH between viable and non-viable leaf materials become even more pronounced during rehydration (Fig. 2b).
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EGSSG/2GSH is also a useful marker to assess viability in C. vulcani and its isolated photo- and mycobiont (Fig. 3). While lichen and alga recovered after 2 months of desiccation, the isolated fungus suffered detrimental damage after 2 months of desiccation. On rehydration, EGSSG/2GSH in the isolated fungus increased to values that are more positive than –50 mV, clearly indicating detrimental damage.
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To summarize, desiccation tolerant organisms have a most remarkable ability to ‘shut down’ their metabolism and to survive long periods in the desiccated state, and a potent antioxidant system is one of the underlying mechanisms of desiccation tolerance. However, when oxidative processes prevail during long-term desiccation, or ageing in the desiccated state, antioxidants break down. When major parts of the total glutathione pool are converted into GSSG, EGSSG/2GSH increases and becomes a signal that initiates PCD. On the cellular level, this will help the organism to eliminate damaged cells, but if this process occurs repeatedly, the whole organism will lose viability.
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ACKNOWLEDGMENTS |
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We thank H. W. Pritchard for discussions and helpful comments on this manuscript. The Millennium Seed Bank Project is supported by the Millennium Commission, The Wellcome Trust, Orange Plc. and Defra. The Royal Botanic Gardens, Kew, receive grant-in-aid from Defra.
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FOOTNOTES |
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1 From the Symposium on Drying without Dying: The Comparative Mechanisms and Evolution of Desiccation Tolerance in Animals, Microbes, and Plants presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2005, at San Diego, California.
2 E-mail: i.kranner{at}rbgkew.org.uk
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