Strategy II Plants

APOPLASM

C. Iron

2. Strategy II Plants

In contrast to strategy I plants, grasses are characterized by a different mechanism for Fe acquisition, with Fe-mobilizing root exudates as main feature. In response to Fe deficiency, graminaceous plants (strategy I1 plants) (39) are able to release considerable amounts of non-proteinaceous amino acids (Fig. SB), so called phytosiderophores (PS), which are highly effective chelators for Fen1 (Fig. 8)

PS blosynthesls Fe (Me) mobllhatlon

(in vesicles ?)

anion channel 7)

' I

FOPS + SID t PS + FeSlD

6

Figure 8 Model for root-induced mobilization of iron and other micronutrients (Zn,

Mn, Cu) in the rhizosphere of graminaceous (strategy 11) plants. (Modified from Ref. 1 .) Enhanced biosynthesis of mugineic acids (phytosiderophores, PS) in the root tissue. (A) Biosynthesis of PS. (B) Exudation of PS anions by vesicle transport or via anion channels, charge-balanced by concomitant release of K'. (C) PS-induced mobilization of Fe111 (MnII, ZnII, CuII) in the rhizosphere by ligand exchange. (D) Uptake of metal-PS com- plexes by specific transporters in the plasma membrane. (E) Ligand exchange between microbial (M) siderophores (SID) with PS in the rhizosphere. (F) Alternative uptake of microelements mobilized by PS after chelate splitting.

(46,176). This release takes place predominantly in subapical root zones (30).

Unlike FeIII-citrate, FeIII-PS chelates are stable even at high soil pH levels

>

7 ( 162,177,178). Due to the formation of high-affinity FeIII-PS complexes (Fig.

8C), there is only minimal competition by chelation with Ca2+, Mg?+ and AI3+, which are usually present in high concentrations in many soils (15 1). However, recent studies indicate that sulfate, and especially phosphate, applied as fertilizers at high rates, may inhibit PS-promoted FeIII dissolution, mainly by displacement of PS from the surface of Fe (hydr)oxides (179). Unlike strategy I plants, where Fe111 reduction is a prerequisite for iron uptake, strategy I1 involves a specific transport system for FelII-PS complexes, located at the plasma membrane in roots of graminaceous plants (Fig 8D) (1 80). The uptake system requires metabolic energy (46) and is highly specific with respect to FeIII as metal ligand and to PS as organic chelators as well ( 1 S l ) . FeIII chelated by synthetic or microbial siderophores is not recognized by this FeIII-PS transporter (39). However, Fe111 complexes with certain microbial siderophores, such as rhizoferrin, can improve Fe uptake i n graminaceous plant species via exchange chelation with phytosidero- phores (Fig. 8E; 186).

From the ecological point of view, strategy I1 has advantages over strategy I, especially in well-buffered calcareous soils with high pH, since Fe mobilization and Fe uptake by strategy I1 is less dependent on the external (soil) pH than strategy I ( I 80).

After entering the cytosol, the behavior of FeIII-PS complexes is still un- known, but the reduction potential of - 102 mV suggests that Fe liberation is possible via reduction by common physiologically available reductants such as NAD(P)H (-320 mV) and glutathione (-230 mV)(15 I ) .

Tolerance to Fe deficiency in different graminaceous plant species is roughly related to the amount of PS exuded under Fe-deficient conditions (bar- ley

>

wheat > oat

>

rye

>

maize

>

sorghum

>

rice), although considerable genotypical variation exists within each single plant species ( 1 50,18 I , 182). The ability to accumulate high amounts of PS in the rhizosphere (up to 1 mM in Fe- deficient barley) (39) seems to be associated also with a distinct diurnal rhythm of exudation, which is restricted to several hours after onset of the light period (46), and with restriction of PS release to subapical root zones (30). Temporal and spatial concentration of PS exudation may be a strategy to counteract microbial degradation (39,40,183), dilution by diffusion into the bulk soil (184), and immo- bilization by sorption of Fe to phospholipids during FePS uptake ( 1 85). In maize and sorghum, with a comparatively high susceptibility to Fe deficiency chlorosis, PS are continously released at a relatively low rate (1 86) in the subapical root zones (Neumann, unpublished).

The molecular mechanism of PS exudation is still not clear. Biosynthesis of PS seems to be regulated by the intracellular iron level ( I 87). Synthesis in the root tissue increases when Fe supply is limited even before iron deficiency c h h -

rosis appears and rapidly declines after reapplication of iron ( 188- 192). PS are derived from nicotianamine, which is ubiquitous in higher plants with putative functions in regulating the physiological availability of Fe and/or transport of copper and other micronutrients in the xylem (13,194). Nicotianamine is synthe- sized from L-methionine via trimerization of S-adenosyl-methionine (Fig. 8A) in a reaction sequence similar to ethylene biosynthesis (Yang cycle), with continu- ous recycling of L-methionine (195). I n graminaceous plants, PS formation pro- ceeds by transamination and hydroxylation of nicotianamine to deoxymugineic acid (DMA). DMA is either released as PS into the rhizosphere (e.g., maize) or converted to higher hydroxylated PS derivatives such as mugineic acid (MA), hydroxymugineic acid (HMA), epi-hydroxy mugineic acid (epi-HMA), disticho- nic acid A and avenic acid A, which were identified as PS in barley, rye, and oat (1 8 I , 189,190,196,197). Several genes involved in the biosynthetic pathway have been cloned (191,198-200). In roots of Fe-deficient barley, biosynthesis proceeds throughout the whole day with a decline of the internal PS levels during the period of release ( 1 5 1,201 ). Inhibitory effects of KCN and DCDD and of low root-zone temperatures suggest that PS biosynthesis and the exudation process are highly dependent on metabolic energy (67,178,202). Low root carbohydrate concentrations under low light conditions and a stimulation of PS release at high light intensities may indicate the importance of a continuous supply of photosyn- thates from the shoot to the roots for the biosynthesis of PS in the roots (203).

Ultrastructural investigations in Fe-deficient barley roots revealed, particu- larly in epidermal cells, the formation of large ER vesicles with attached ribo- somes and filled with fibrous materials prior to the release of PS. When PS exuda- tion was terminated, these large vesicles disappeared. Therefore, a function of vesicles for storage and/or transport of PS (Fig. 8) has been implicated in the mechanism of diurnal PS exudation in barley (93). Accordingly, PS release was almost completely inhibited during the period of root exudate collection by short- term (2-h) application of the vesicle transport inhibitor brefeldin A in barley but not in maize (Table 5 ) , where diurnal variation in PS exudation is not detectable ( 1 86). However, in both plant species, PS release was also inhibited by short- term application of various anion-channel antagonists (Table 5 ) (87), suggesting involvement of anion channels in PS exudation, which is associated with a con- comitant equimolar release of Kt as counterion (Fig. 8) (87,178). Since vesicle transport in higher plants is usually implicated in the secretion of polysaccharides and proteins ( M ) , another putative function of the vesicles in barley roots may be synthesis and transport of proteins to the plasmamembrane, which regulate the diurnal exudation of PS or of the channel proteins themselves. Mori (202) suggested that biosynthesis and release of PS in barely might be triggered by diurnal changes in temperature and not by light signals, but this could not be confirmed by the findings of Kissel (67). Studies including mutants such as the ys3 maize line, which is defective in PS release but not in biosynthesis of PS,

Table 5 Effect of Anion-Channel Antagonists (Anthracene-9-carboxylic acid, ethacrynic acid; each 100 FM) and of Brefeldin A (Exocytosis Inhibitor; 45 PM) on Release of Phytosiderophores from Roots of Fe-Deficient Barley and Maize

Anthracene-9- carboxylic Ethacrynic

acid acid Brefeldin-A Barley

EpiHMA Inmol h" g I

F W I

87.42a

SE 12.8

M, _dlLe '.

DMA [nmol h" g" FW] 30.73a

SE 8.2

6.03b 70.9:l 8.48b

4.9 6.8 6.6

0.3b 9.69c 52.56a

0.3 4.4 11.9

Inhibitors applied during the 2-h period ofexudate collection into distilled water. starting at the begln- ning of the light period. I n each row, significant differcnces are indicated by diffcrcnt characters.

could be a powerful tool to elucidate the mechanisms of PS exudation (Basso and Romheld, unpublished).

D. Other Micronutrients and Heavy Metals

Mobilization of micronutrients such as Zn, Mn, Cu, and CO and of heavy metals (Cd, Ni) in soil extraction experiments with root exudates isolated from various axenically grown plants is well documented (61,204-206) and has been relatcd to the presence of complexing agents.

1. Role of Phytosiderophores

Formation of stable chelates with phytosiderophores occurs with Fe but also with Zn, Cu, CO, and Mn (Fig. 8) (39,207,208) and can mediate the extraction of considerable amounts of Zn, Mn, Cu, and even Cd i n calcareous soils (204,209).

There is increasing evidence that PS release in graminaceous plants is also stimu- lated i n response to Zn deficiency (210-212), but possibly also under Mn and Cu deficiency (2 13). Similar to Fe deficiency, the tolerance of different gramina- ceous plant species to Zn deficiency was found to be related to the amount of released PS (2 1 1,2 12), but correlation within cultivars of the same species seems to be low (214). It is, however, still a matter of debate as to what extent PS release is a specific response to deficiencies of the various micronutrients. Gries et al. (2 13) reported that exudation of PS in Fe-deficient barley was about 15- 30 times greater than PS release in response to Zn, Mn, and Cu deficiency. In contrast, PS exudation in Zn-deficient bread wheat was i n a similar range as PS

release under Fe deficiency in barley (205,21 l ) . Walter et al. (215) demonstrated that Zn deficiency-induced PS release in bread wheat is probably an indirect response, caused by impaired iron metabolism; this is also supported by the data of Rengel and Graham (216). In contrast. Gries et al. (2 17) suggested that there was a specific response to Cu deficiency in Hordeivmus europaeus L. Root uptake rates of PS complexes with Cu, Zn, and CO were found to be much lower than uptake of FelII-PS chelates ( I 5 1 ) but may still be sufficient due to a lower demand for micronutrients (217). In contrast, based on studies with the maize y s l mutant, which is defective in Fe-PS uptake, v. Wiren (218) proposed two pathways of Zn uptake in grasses, including uptake of the free Zn cation and uptake of the Zn-PS complex via the Fe-PS transport system (Fig. 8F).

2. Role of Carboxylates, Rhizosphere pH, and Redox State

Mobilization of micronutrients (Mn, Zn, Cu), heavy metals (Cd), and even ura- nium i n the rhizosphere has been also related to rhizosphere acidification and to complexation with organic acids (e.g., citrate) in root exudates (219-224). This view is further supported by intense mobilization of Mn, Zn, Cu, and Cd observed i n soil extraction experiments with leachates from rhizosphere soil or with or- ganic acid mixtures according to the root exudate composition of plant species such as Lrrpirlus trlbus, Htlketr uruiuiatu, and Spinuceu oleruceu under P-deficient conditions, where exudation of carboxylates and protons is particularly expressed (27,33,34,102). However, only limited information exists about the plant avail- ability and uptake of the metal-carboxylate complexes. Solution culture experi- ments in the presence of complexing agents revealed that plant uptake is corre- lated mainly with the activity of free uncomplexed metal ions in solution (225- 229). This implies that utilization of chelated metals requires liberation of the metal ligands from the carboxylate complex, which may be mediated by rhizo- sphere acidification and reduction of metal species such as Mn and Cu (99,227).

Similar to Fe acquisition in strategy I plants, Mn mobilization in the rhizo- sphere of soil-grown plants is a result of the combined effects of rhizosphere acidification, complexation with organic ligands and reduction of Mn oxides ( l).

Phenolics and organic acids in root exudates (especially malate) are involved i n both complexation and reduction of Mn (230,23 l ) . In cluster-rooted plant species such as Llrpirurs a 1 b u . s and members of the Proteaceae, particularly intense exuda- tion of organic acids and phenolics in response to P deficiency is frequently also associated with enhanced Mn mobilization in the rhizosphere and accumulation of high or even toxic Mn levels in the shoot tissue (27,3 1,232). Similarly, Mn toxicity was indirectly induced by the iron-deficiency response in flax grown i n a calcareous soil high in extractable Mn but low in Fe (233). Besides mobilizing effects of plant root exudates, Mn availability in the rhizosphere is also strongly affected by the activity of microorganisms involved in Mn oxidation and Mn

reduction, which, in ^turn, depend on root exudates as a carbon source (1). Utiliza- tion of Cu complexes with humic acids and citrate has been reported for red clover, especially under P-deficient conditions (234). The authors suggested that this was the result of liberation of complexed Cu in the rhizosphere, mediated by an increased reductive capacity of the roots, which was also identified as an adaptive mechanism of Cu deficiency in Pisum sativum (227).

Despite increased citrate accumulation in roots of Zn-deficient rice plants, root exudation of citrate was not enhanced. However, in distinct adapted rice cultivars, enhanced release of citrate could be observed in the presence of high bicarbonate concentrations in the rooting medium, a stress factor, which is fre- quently associated with Fe and Zn deficiency in calcareous soils (235) (Hajibo- land, unpublished). This bicarbonate-induced citrate exudation has been related to improved Zn acquisition in bicarbonate-tolerant and Zn-efficient rice geno- types (Fig. 9) (235). Increased exudation of sugars, amino acids, and phenolic compounds in response to Zn deficiency has been reported for various dicotyle- donous and monocotyledonous plant species and seems to be related to increased

elevated bicarbonate concentration in soil solution

Increase in synthesis of citrate in roots

root exudation

/ \

accumulation

of citrate citrate in roots

enhanced Zn mobilization in the rhizosphere

disorder of root metabolism

improved inhibition of

root growth root growth

leakiness of membranes (19). Zinc has essential functions in the stabilization of membranes (236,237) and in preventing oxidative membrane damage as a metal component of superoxide dismutase, which is part of the free-radical scavenging system of higher plants (238). It is, however, as yet unknown whether this kind of Zn deficiency-induced root exudation has any impact on mobilization of Zn or other micronutrients in the rhizosphere.

Comparatively high mobility of Cd in soils associated with high rates of uptake and accumulation in some plant species is an important aspect from the ecotoxicological point of view. Cd mobilization in soils can be mediated by rhizo- sphere acidification (223) but to some extent also by complexation with carboxyl- ates (99,221) or phytosiderophores (209). A comparison of high and low Cd- accumulating genotypes of durum wheat revealed higher levels of carboxylates in the rhizosphere soil of the Cd accumulator (222). Based on these findings, it was concluded that plant availability of Cd may be increased by complexation with root-derived carboxylates. In contrast, Gerke (99) suggested that carboxylate complexation of Cd might decrease plant availability, since only free Cd?+ seems to be taken up by plant roots (229,239). Wallace (225) demonstrated that, in soil- plant systems, solubility and transport to the root uptake sites are likely to be the limiting steps in uptake of cationic microelements by plants.