The Regulation of Iron Uptake and Possible Functions of Nicotianamine in Higher Plants

Biochem. Physiol. Pflanzen 183, 257 - 269 (1988) VEB Gustav Fischer Verlag lena

BPP Review

The Regulation of Iron Uptake and Possible Functions of Nicotianamine Higher Plants GUNTER SCHOLZ, ROSWITHA BECKER, UDO

In

W. STEPHAN, ARMIN RUDOLPH and AXEL PICH

Forschungsbereich Biowissenschaften und Medizin der Akademie der Wissenschaften der DDR Zentralinstitut fUr Genetik und Kulturpflanzenforschung Gatersleben , German Democratic Republic Key Term Index: iron, model, nicotianamine, regulation, uptake, Lycopersicon esculentum MILL., cv. Bonner Beste, mu!. chloronerva

Summary At present two different strategies of iron uptake under conditions of iron shortage are known for higher plants. In most cases, with the exception of grasses, an increased release of protons and reductants by root tips is accompanied by an increase ofthe reduction potential at the surface of root cells, leading to a higher availability of iron present in the rhizosphere. Anatomically, the most striking response is the formation of numerous transfer cells within the root epidermis which very likely are the sites of increased metabolic activity (Strategy I). Roots of graminaceous plants respond to iron limitation in the environment by the release of phytosiderophores of the mugineic acid type , potent chelators of ferric iron. Ferri-phytosiderophores are re-absorbed by the roots, thus improving the iron nutrition of the plant (Strategy II). Both strategies imply the presence of a trigger mechanism, perhaps an iron-sensitive sensor, that responds to the iron status of the cell and delivers a signal for switching on and off specific inductive iron uptake mechanisms. Nicotianamine, a compound structurally related to mugineic acid, is of general occurrence among plants. Its biological activity is very likely linked with its ability to form stable complexes with iron (II) and other divalent heavy metal ions. Experiments with the nicotianamine-auxotroph tomato mutant chloronerva revealed its regulatory function in the uptake of Fe, Cu, Mn, and Zn. It is assumed that nicotianamine could act within both strategies as an iron (II) carrier between the site(s) of iron (III) reduction and the 'sensor' which perhaps is located in the mitochondria. The regulation of other divalent cations can be explained by the antagonism between iron and heavy metals.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Two strategies of inducible iron acquisition at low external supply Some facts about nicotianamine and phytosiderophores . The primary function of nicotianamine: A hypothesis. References. . . . . . . . . . . . .

258 258 260 263 267

Abbreviations: ESR, electron spin resonance spectroscopy; NA, nicotianamine = (2S: 3'S: 3"S)-N-[N-(3amino-3-carboxypropyl)-3-amino-3-carboxypropyl]-azetidine-2-carboxylic acid; ORD, optical rotatory dispersion spectroscopy.

17

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Introduction In the field of mineral nutrition of higher plants investigations on the regulation of iron uptake were among the fastest advancing branches of scientific endeavour during the past years. The current views about iron acquisition by plants, especially under conditions of limited supply, have been reviewed by several authors, among others by LANDSBERG (1984) ; BROWN and JOLLEY (1986); MARSCHNER et al. (1986); ROMHELD and MARSCHNER 1986a) and BIENFAIT (1987). According to ROMHELD and MARSCHNER (1986a) two different strategies were observed within distinct taxonomic groups which are shortly summarized below:

Two strategies of inducible iron acquisition at low external supply Strategy I (Fig. I A) is operating in dicots and was also detected in monocots with the exception of grasses (ROMHELD and KRAMER 1983). The response of dicots to low levels of iron in the rhizosphere consists mainly of an extrusion of protons by the roots into the environment (BROWN 1963; ROMHELD and MARSCHNER 1984) together with a decrease of the reduction potential at the surface (plasmalemma) of epidermal root cells (SUMONS et al. 1984 a) and, more or less, also with a release of reductants (BROWN and AMBLER 1973, OLSEN et al. 1981). Since the solubility of Fe (III), the prevailing iron ion in aerated soils, is positively correlated with the third power of the proton concentration, a pH decrease in the rhizosphere by one unit theoretically leads to a 1,OOO-foid increase in Fe (III) solubility. The soluble Fe (III) species can be reduced by low-molecular weight root-born reductants in the rhizosphere, or perhaps more efficiently by an inducible reductase (,Turbo' -reductase) at the plasma membrane with cytosolic NADPH as intracellular electron donor (SIJMONS et al. 1984b). These inductive biochemical processes go along with several anatomical changes of apical root zones. The most prominent one at the cellular level is the development of transfer cells with a highly invaginated cell surface and an accumulation of mitochondria within a dense cytoplasm in the vicinity of the plasmalemma (KRAMER et al. 1980). According to our present knowledge these transfer cells are the sites of the biochemical iron deficiency response mechanisms of non-grasses (LANDSBERG 1986). These inductive response mechanisms are perhaps triggered by a signal originating from the root cells themselves where a hypothetical 'sensor' in the cytoplasm or within organelles may initiate the whole complex of adaptive responses if the concentration of iron entering the cell (or organell) decreases below a certain level (BIENFAIT et al. 1987). It is further thought that this whole complex could be controlled by a single gene, T3820 FER of Lycopersicon esculentum (BROWN et al. 1971). The primary product of this gene is still obscure. But there is some reason to assume, at least hypothetically, that it could be identic with, or closely related to, the 'iron level feeler' i.e. the molecular 'sensor' mentioned above (BIENFAIT 1987), perhaps an iron-dependent enzyme or an iron-dependent repressor protein that controls genes responsible for the inductive iron deficiency response mechanisms, as has been deduced from investigations with bacteria by BRAUN and BURKHARDT (1982) and by SCHAFFER et al. (1985). After a sufficient level of iron in the cells is attained the inductive processes are slowed down or switched off until a new demand emerges and the machinery is induced again. Thus, an oscillating behavior can be observed under certain conditions, as was demonstrated with sunflowers by VENKATRAJU and MARSCHNER (1972), ROMHELD and MARSCHNER (1981). 258

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rhizosphere / soil

free space

plasma membrane

cytoplasma

I I

I

:

phytosidel'OJfiJre

phy1o·

nS~roJfure----+"';";;;';;""~-----""'------de novo synthesis

Fe(m ) hydrox~

: :

L'd

: phytosiderop/lore

phytos~~ recycling?

Fe(m)Phytos~rophore

I I I I

B

:

Fig. I . Schematic presentation of two different strategies of iron uptake by higher plants, induced b y iron shortage in the rhitosphere (RbMHELD and MARSCHNER , 1986 a, modified). A. Strategy I (dicots and non-grass monocots): Solubili zation ofFe(III) by extrusion of protons and phenolics with chelating (and perhaps also reducing) properties, reduction by membrane-bound reductases and uptake of Fe2+ at specific membrane sites . B. Strategy II (grasses): Excretion of Fe(lIl)-specific phytosiderophores and uptake o f the ferri-phytosiderophores with subsequent intracellular iron reduction and chelate splitting. The intracellular r eductase(s) and cooperating electron d onors are still unknown . R' ase , ferric reductase; Fe 2 +US, Fe2+ uptake system; PES, phytosiderophore excreting system; FPTS, ferri -phytos iderophore transport system ; ICR , intracellular reductase; PN, pyridine nucleotides .

This is only a rough survey to demonstrate some important aspects of this rapidly developing field of research . In fact, some authors (e.g . LANDSBERG 1984) prefer whole-plant activities as iron deficiency responses with auxin as a signal, the synthesis of which is increased upon iron deficiency in the shoot apices and within young, most actively growing leaves with a high iron demand. Although BIENFAIT et al. (1987) were able to demonstrate that iron-efficiency reactions can be developed by roots on their own , without the need for a signal , they do not exclude the existence o f amodulating influence from the leaves. 17*

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At concentrations of available iron above the inductive level the 'Turbo' -reductase exhibits only low activity which seems to be constitutive for rhizodermal cells and sufficient to cover the needs of the plant. The biochemical mechanisms of the 'Turbo' and the non-inducible 'Standard' reductase (which uses ferricyanide as electron acceptor) are still unknown. It is even doubtful whether the 'Standard' reductase is really part of the iron uptake mechanism (BIENFAIT 1987). Strategy II (Fig. 1 B) is an alternative adaptation of plants to limited iron supply in the environment which until now has been observed in grasses exclusively. It resembles the wellknown inducible iron uptake system of microorganisms where low-molecular weight siderophores of the hydroxamate or catechol type are synthesized and released upon iron deficiency. These natural chelators are very specific for ferric iron and are highly effective in sequestering Fe (III) in the environment due to their extremely high stability constants. After chelation the ferri-siderophores are re-absorbed by specific receptors at the outer cell membrane (NEILANDS 1981). Contrary to microbial siderophores where abundant and still growing information is available about detailed structures, genetic regulation etc. little is known about equivalent mechanisms in higher plants. TAKAGI (1976) observed the release of low-molecular weight amphoteric substances by grass roots under limited iron supply. Subsequent investigations revealed the presence, among others, ofmugineic acid and 2' -deoxymugineic acid, derivatives of azetidine-2-carboxylic acid (Fig. 2) which are also potent chelators for Fe (III) (SUGIURA et al. 1981; MINO et al. 1983; SUGIURA and NOMOTO 1984). Since they possess biological properties very similar to the hydroxamate and catechol type siderophores they fulfill the definition given for siderophores by NEILANDS and LEONG (1986) as ". . . low-molecular weight, virtually Fe (IIJ)-specific ligands produced ... as scavenging agents in order to combat low iron stress" and are therefore regarded as phytosiderophores. As described by ROMHELD and MARSCHNER (1986 b), phytosiderophores which are very effective in solubilization of ferric hydroxide are released from iron deficient barley (Hordeum vulgare) but not from cucumber (Cucumis sativus) roots. After formation of Fe (III) complexes these naturally iron chelates are absorbed 100 to 1,000 times faster than synthetic chelates, as e.g. Fe (III) EDTA, and reduced within the plant. These results indicate the existence of a specific inductive iron deficiency response mechanism in barley and perhaps in all graminaceous plants that, contrary to strategy I, involves exudation of phytosiderophores, chelation of Fe (III) in the rhizosphere, and re-absorption of the chelates by the roots, without release of protons, increase of the reduction potential and development of transfer cells. Although the details of this alternative scheme still await investigation, there is only little doubt about the ecological efficiency of strategy II as is easily demonstrated e.g. by the vigorous growth of grasses even on soils with high pH and bicarbonate where many dicots suffer severe iron chlorosis (ROM HELD and MARSCHNER 1986b). Some Jacts about nicotianamine and phytosiderophores From a structural point of view the phytosiderophores mentioned above are closely related to nicotianamine (Fig. 2). Some authors have expressed the view that NA may be synthesized in vivo from three molecules of azetidine-2-carboxylic acid (KRISTENSEN and LARSEN 1974) and that in monocots NA may perhaps play an indirect role as a precursor of 2' -deoxymugineic 260

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AZETIDINE -2-CARBOXYLIC ACID

+

R1=H; R2= NH3: NICOTIANAMINE R1=

R2=OH: MUGINEIC ACID

R1=H;R2=OH : 2'-DEOXYMUGINEIC ACID

Fig. 2. The nicotianamine family: Physiologically significant derivatives of azetidine-2-carboxylic acid.

acid after transamination and reduction (FUSHIY A et al. 1982; ROMHELD and MARSCHNER 1986b; BIEN FAIT 1987). According to MORI and NISHIZAWA (1987) methionine is incorporated into phytosiderophores in the order avenic acid - deoxymugineic acid - mugineic acid - epihydroxymugineic acid and/or hydroxymugineic acid . This implies that NA could well be an intermediate, orginating from methionine, and after desamination leading to avenic acid . But experimental evidence is still lacking. Moreover, nicotianamine (NA) is not restricted to strategy II plants but is generally distributed among multicellular plants (RUDOLPH et al. 1985; for references see also PROCHAZKA and SCHOLZ 1984). But FUSHIYA et al. (1982) failed to detect NA in root exudates of iron deficient monocots. Furthermore , NA does not significantly chelate ferric iron at physiological pH (BENES et al. 1983). NA is present in all parts of the plant with preference to young, rapidly developing organs , as e.g. young leaves, margins of expanding leaves, shoot apices and root tips (STEPHAN and RUDOLPH, unpublished results). It is easily transported within plants both from roots to the shoot and vice versa as demonstrated by grafting experiments (RUDOLPH and SCHOLZ 1972). Apart from some Iiliaceous plants which are still to be investigated more closely because in some species their NA content seems to be low, the only higher plant lacking detectable amounts ofNA is the NA auxotroph tomato (Lycopersicon esculentum) mutant chloronerva (RUDOLPH and SCHOLZ 1972), for which NA supplied via the roots or the leaves functions as a 'phenotypically normalizing factor' . It subdues all mutant characters and causes growth and development that under optimal conditions makes the mutant indistinguishable from its wild-type (for references see SCHOLZ and BOHME 1980; RIPPERGER and SCHREIBER 1982; PROCHAZKA and SCHOLZ 1984 ; SCHREIBER 1986) . Although it is known for long that chloronerva, despite its interveinal chlorosis of young leaves, accumulates large amounts of iron, even in the veins of chlorotic leaflets and that the mutation did not only affect the regulation of iron uptake but more generally the uptake also of other divalent heavy metals (SCHOLZ et al. 1985 a, 1987), the primary role of NA in these processes is still obscure. First speculations about the possible function(s) of the 'normalizing factor' were stimulated by observations on complex formation with iron and copper by ORD measurements . Whereas a positive Cotton effect was achieved immediately after addition of CUS04 to a solution ofthe 'normalizing factor' in acetate buffer atpH 5.0, the addition of freshly prepared FeS04 solution exerted a significant effect only after 24h standing in the laboratory atpH 4.5 . This effect was inhibited by the simultaneous addition of citrate. It was therefore concluded that complex formation did occur only after oxidation, i.e. that Fe3+ rather than Fe 2 + was involved (SCHOLZ 1970). The latter result was later on confirmed by RIPPERGER and SCHREIBER (1982), although in the unphysiological range of pH 2. The idea of complex formation was supported by identification of the 'normalizing factor' with NA by BUDMfNSKY BPP 183 (1988) 4

261

et al. in 1980. At that time the close structural relationship between NA and mugineic acid was already known from investigations of TAKEMOTO et al. (1978). Furthermore, it was deduced from a Dreiding model that "NA has an optimal molecular structure for complex formation with iron. Not only are six functional groups present, necessary for octahedral coordination, but the distances between the groups are also optimal for the formation of chelate rings: Three 5-membered rings formed by the ex-amino acid residues and two 6-membered rings formed by the 1,3-diaminopropane moities. The spatial location of the oxygen atoms on one side of the complex and the methylene groups and/or the azetidine ring system on the other might playa decisive role in the biological function of the complex" (BUDESfNSKY et al. 1980). On these grounds the authors concluded that NA might possibly function as a phytosiderophore. This approach was, however, challenged by a growing number of other observations. NA, unlike mugineic acid and 2' -deoxymugineic acid, failed to increase the 59Fe (III) uptake and chlorophyll synthesis of chlorotic, iron-deficient rice plants in nutrient solution (MINO et al. 1983). Furthermore, until now there is no evidence for the release of NA from roots of iron deficient grasses (FUSHIYA et al. 1982). The existence of a Fe(III)NA complex has been claimed from ESR data at 77 K by SUGIURA et al. (1981) but without any detailed information about stability constants etc. As already mentioned above, BENES et al. (1983) were unable to detect complex formation at physiological pH by potentiometric titration of NNFe(CI0 4 h solution. However, they observed a series of NA complexes with Fe2+ and other divalent transition metal ions instead. The highest stability constant was with Cu2+ (Tab. 1). This is a Table I. Stability constants log K of nicotianamine and mugineic acid complexes with various heavy metal ions.

Nicotianamine Mugineic acid I) BENE~

Fe(1l1)

Fe(Il)

18.1

12.1 8.1

Mn(lI) 8.8

Zn(II) 14.7 10.7

Co(II) 14.8

Ni(ll) 16.1

Cu(lI) 18.6 18.3

I)

2)

et al. (1983) et al. (1981)

2) SUGIURA

property that NA seems to share with mugineic acid, the chelating activity of which with Fe3+ is strongly inhibited by Cu 2 + and Zn 2 + , obviously due to competition for binding ligand atoms (SUGIURA and NOMA TO 1984). But in spite of the apparent structural similarity between NA and mugineic acid (Fig. 2), the presence of a terminal alpha amino group instead of hydroxyl and the lack of a hydroxyl function at the 2'position of the NA molecule results in a dramatic shift in specificity as compared to mugineic acid (NEILANDS and LEONG 1986), and thus to a prevalence for Fe 2 + rather than Fe 3 + as complexing ion. The assumption that the function ofNA is intimately linked with complex formation rather than with functions as a growth factor or as a cofactor of macromolecules is supported by its lack of stereospecifity: According to RIPPERGER et al. (1982) the optical antipode, (+)nicotianamine, exerts the same biological activity as the naturally occuring isomer in restoring mutant characters of chloronerva. Both substances are identic with respect to their complexing capacities but not in the spatial arrangements of their carbon chains and functional groups which is prerequisite for specific binding to macromolecules. Furthermore, experiments with a series of derivatives of NA and of model substances led to the conclusion that any changes of ligand atom groups involved in the formation of octahedral metal chelates lead to inactivation 262

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of biological complementation in the mutant chloronerva (see. e.g. RIPPERGER 1986). The ability of NA to form complexes with heavy metal ions is, however, only one precondition for its presumable carrier function. Others are the small molecular size and the relative hydrophobic nature of these complexes, as revealed by observations from our laboratory. Although a final decision on the valency state of iron that accepts NA as ligands at physiological pH cannot be made at present, the majority of results indicate complex formation with ferrous rather than ferric iron as a decisive property. This is not to say that chelation with Fe 3 + does not occur at all and that positive Cotton effects reported above are artifacts, but the stability constant of Fe (III) N A under the experimental conditions seems to be very low, so that this complex escapes detection by titration (BENES et al. 1983). The primary function of nicotianamine : A hypothesis We suggest that the primary function of NA is inside rather than outside the plant. This assumption is supported by the facts reported above, according to which NA excretion has not been observed. In addition, overcoming of mutant characters of chloronerva is achieved by external supply of NA, both to the roots and to the leaves. A role in short distance (iron) transport through membranes from cell to cell or between cell compartments has been envisaged by SCHOLZ and BOHME (1980). As outlined on page 258 the inducible iron uptake processes are perhaps triggered by the iron status of the cells (or of some specific cells). This affords a kind of 'iron level feeler' or 'sensor' inside the cell. It is generally accepted that iron after reduction at the plasmalemma (strategy I) or after trans-membrane transport of Fe (III)phytosiderophores and subsequent reduction (strategy II) is transported within the cytoplasm of root cells as ferrous iron. In order to protect the cell from deleterious effects ofFe 2 +, at least above a certain concentration, and to prevent re-oxidation (BIENFAIT and V AN DER MARK 1983) the ferrous iron should be transported within the cell by means of a hypothetical ferrous iron carrier (MARSCHNER 1986). We want to forward the proposal that NA is a candidate for this carrier function. This view is supported by the following observations: 1. The tomato mutant chloronerva shows an excessive iron uptake if supplied with FeEDT A or FeEDDHA at 5 to 20/-tM or 20 to 50 /-tM concentration, respectively (STEPHAN et aI., in preparation). Large amounts are transported into the shoot, leading to high concentrations, especially in older leaves. But only a small portion seems to reach the intercostal areas of fast growing leaflets, thus causing chlorosis (Fig. 3) whereas relatively large amounts remain concentrated along their veins, as was demonstrated by autoradiography (SCHOLZ 1965). After supply of NA either to the nutrient solution or to the leaves vigorous growth commences and chlorosis is subdued, although iron absorption by the roots decreases almost to the normal wild-type level (SCHOLZ et al. 1985 a). These observations might be explained by the assumption that due to lack ofNA, Fe 2 + is not transported efficiently enough on its route from the reduction site(s) at the plasmalemma through the cytoplasm and the membranes of cell organelles to the iron 'sensor'. In the case of root cells the 'sensor' remains unsaturated and the inductive high-efficiency iron uptake system continues working. Proton extrusion into the medium is observed up to lO/-tM FeEDTA in the medium, causing acidification down to pH 4. At the same time 'Turbo' -reductase activity shows only a peak at 2/-tM FeEDT A. Contrary to the mutant, wild-type roots reveal acidification only at very low FeEDT A concentrations in the medium and inductive iron reductase activity decreases at about 20/-tM FeEDTA. These BPP 183 (1988) 4

263

Fig. 3. A young leaflet o/the mutam chloronerva. showing typical chlorosis o/ intercostal areas.

biochemical responses in the mutant are accompanied by stunted roots with thickened tips and abundant root hairs as symptoms of apparent iron deficiency, even at iron concentrations in the nutrient solution up to more than 10 flM FeEDT A (Fig. 4) (STEPHAN et aI., in preparation). In fast growing mutant leaves the high iron demand of expanding cells cannot be covered for similar reasons : Although iron is imported from the roots probably in the form of Fe (III) citrate (TiFFIN 1966) and reduction capacity is available as photochemical and enzymatical activity (BROWN et al. 1979 , REDINBAUGH and CAMPBELL 1983) , transport from the xylem of the leaves to the most remote parts of intercostal areas is inhibited , causing local chlorosis. Plastids from chlorotic cells are poorly developed and exhibit a distorted lamellar substructure (ADLER and SCHOLZ 1986) that could be interprete
264

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Fig. 4. Roots of20-days-old tomato seedlings grown ill Ilutrient solution with 10 iJM FeEDTA. A. Roots of the mutant chloronerva with thickened root tips and areas of root hairs as symptoms of apparent iron deficiency. B. Wild-type roots with very thin main and expanded lateral roots. Inserts show a respective root at higher magnification.

the wild-type 'Bonner Beste' or sunflowers grow fairly well at concentrations as low as I [lM, although significantly less vigorous than at lO[lM concentration and with more or less chlorotic leaves. Under this iron regime addition of NA to the nutrient solution led to a substantial recovery of growth as well as of chlorophyll content with a concomittant increase of iron concentration in the wild-type plants (SCHOLZ et al. 1985 b). This observation could be explained by an enhanced Fe 2+ transport from the reductive sites of the roots to the sites of demand under the influence of exogenously applied NA, even in NA containing genotypes. 3. In chloronerva high iron uptake is accompanied by a high uptake of other divalent ions such as Mn2+, Zn 2 + and Cu2+, whereas the absorption of Rb+ and P0 4 3 - seems to be unaffected. This high heavy metal ion uptake decreases together with the decreasing iron uptake after addition of NA (SCHOLZ et al. 1987). This effect can easily be explained by the antagonism between iron and heavy metals which is operating in many plant species and has been observed under several condition (DEKoCK 1956; SCHERER and HOFNER 1980; ROMHELD et al. 1982; JOLLEY et al. 1986). Despite high iron concentrations within roots the 'sensor' in chloronerva remains unsaturated in the absence of NA and the plants 'feel' an apparent iron deficiency. Therefore an additional uptake of heavy metal ions is induced that declines upon external addition of NA.

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enzyme synthesis

~ I,'. / /'"-'"

plasma

membrane

NA

cytoplasma

de novo synthesis?

-~

? I

apopIasmic

t transpor:t via plasniodesmata __ o Fe +NA - F e ( ] I ) - N A - - - - o - to adjacent cells t I

uptake

Fe () ][ -~--reduction

2+

I I I

I

NL """,,(:... '~'-------- "'",,,--"""""" r; ~ I

I

I

I~I-~-

Syn\heSiS

synthesiS

t

))

iron deficiency message ?

Fig. 5. Scheme of hypothetical nicotianamine (NA) functions in plant cells: Complexation of Fe 2 + and symplast transport to the sites of demand within the cytoplasm or organelles, among others also to the 'sensor' of inductive iron uptake processes (see Fig. I.) which could perhaps be located within the mitochondria.

The possible action of NA is modelled by Fig. 5. NA functions as a scavenger for Fe2+ after reduction at the cell membrane (strategy I) or after trans-membrane transport by ferriphytosiderophores and reduction within the cell (strategy II), thus protecting the cytoplasm from the deleterious effects of ferrous ions and channelling it to the sites of demand, among others also to the 'sensor' which could be located within the cytoplasm or the organelles, among which mitochondria are favourite candidates. NA could therefore fulfill its function within both strategies. Other phenomena may also be explained on the basis of complex formation between NA and divalent heavy metal ions. For instance, according to unpublished results from our laboratory , heavy metal toxicity in tomato and sunflower is prevented by the simultaneous addition of equimolar amounts ofNA or Na2EDTA to the nutrient solution. In the case ofCu2+ this result was not achieved by a NA supply to the leaves . Since roots of all plants hitherto tested contain endogenous NA (RUDOLPH and SCHOLZ 1972), this effect seems to be located in the nutrient solution rather than within the plant and most likely involves unspecific complex formation (ERNST 1974). The high stability ofthe Cu 2 +NA comlex (BENES et a1. 1983) renders the heavy metal less available to the plants , as is indicated by a decrease of copper uptake (SCHOLZ et aI., in preparation). It is interesting to speculate whether external copper detoxification by root exudates from copper-tolerant Silene cucubalus (LOLKEMA et al. 1986) could be related to the de-toxifying effect ofNA. This would afford release ofNA by the roots, at least into the apperent free space, under conditions of copper toxicity. Summarizing the facts and arguments reported above, we want to conclude that NA may keep a key position as intracellular Fe2+ carrier in the regulation of iron and heavy metal uptake and transport as a link between the iron status of the cell and the triggering of iron shortage response mechanisms. However, a large portion of work is still necessary to convert ideas and speculations into sound experimental evidence. 266

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Acknowl{'dgement We are indebted to Dr. H. F. BIENFAIT and to Dr. P. C. SIJMONS, Amsterdam, for extensive discussions of this field of research.

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Received May 22, 1987; revised/arm accepted September 25, 1987 Authors' address: Dr. habil. GUNTER SCHOLZ, Dr. ROSWITHA BECKER, Dr. UDO W. STEPHAN, Dr. ARMIN RUDOLPH and Diplom-Biochem. AXEL PICH, Zentralinstitut fUr Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR, CorrensstraBe 3, Gatersleben, DDR - 4325.

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