Production of Reactive Oxygen Intermediates (O₂^˙−, H₂O₂, and ^˙OH) by Maize Roots and Their Role in Wall Loosening and Elongation Growth

Abstract

Cell extension in the growing zone of plant roots typically takes place with a maximum local growth rate of 50% length increase per hour. The biochemical mechanism of this dramatic growth process is still poorly understood. Here we test the hypothesis that the wall-loosening reaction controlling root elongation is effected by the production of reactive oxygen intermediates, initiated by a NAD(P)H oxidase-catalyzed formation of superoxide radicals (O₂^˙−) at the plasma membrane and culminating in the generation of polysaccharide-cleaving hydroxyl radicals (^˙OH) by cell wall peroxidase. The following results were obtained using primary roots of maize (Zea mays) seedlings as experimental material. (1) Production of O₂^˙−, H₂O₂, and ^˙OH can be demonstrated in the growing zone using specific histochemical assays and electron paramagnetic resonance spectroscopy. (2) Auxin-induced inhibition of growth is accompanied by a reduction of O₂^˙− production. (3) Experimental generation of ^˙OH in the cell walls with the Fenton reaction causes wall loosening (cell wall creep), specifically in the growing zone. Alternatively, wall loosening can be induced by ^˙OH produced by endogenous cell wall peroxidase in the presence of NADH and H₂O₂. (4) Inhibition of endogenous ^˙OH formation by O₂^˙− or ^˙OH scavengers, or inhibitors of NAD(P)H oxidase or peroxidase activity, suppress elongation growth. These results show that juvenile root cells transiently express the ability to generate ^˙OH, and to respond to ^˙OH by wall loosening, in passing through the growing zone. Moreover, inhibitor studies indicate that ^˙OH formation is essential for normal root growth.

The primary roots of seedlings typically grow at a rate of 1 to 2 mm h⁻¹. This growth is brought about by a narrow zone of less than 10-mm length behind the apical meristem. Isodiametric cells delivered from the meristem move through this growing zone within a few hours, whereby they undergo elongation by more than 10-fold until they reach their final length. In primary roots of maize (Zea mays) seedlings, the favorite experimental system in most studies on root growth, a maximum local growth rate of 50% length increase per hour has been determined in the middle of the growing zone (4–5 mm behind the root tip; Spollen and Sharp, 1991; Pritchard et al., 1993; Peters and Felle, 1999). For comparison, the maximum rate of diffuse growth of the coleoptile amounts to about 5%/hour under similar conditions.

Despite many attempts in the past, the mechanism of the dramatic growth reaction of roots and its hormonal control is still poorly understood (Pritchard, 1994; Dolan and Davies, 2004). It is now generally accepted that steady-state elongation growth of plant organs is effected by an increase in the plastic extensibility of longitudinal cell walls at constant (or decreasing) turgor pressure (Cosgrove, 1993; Pritchard et al., 1993). The hypotheses proposed so far for explaining, in biochemical terms, the wall-loosening process in the growing zone of roots are largely based on circumstantial and/or controversial experimental evidence. Although several apoplastic enzymes catalyzing the degradation of cell wall polymers have been described and characterized in vitro, there is no evidence that these enzymes are actually involved in cell wall loosening in vivo (Hoson, 1993; Cosgrove, 1999). Xyloglucan endotransglycosylase has been demonstrated in the growing zone of roots (Pritchard et al., 1993; Vissenberg et al., 2003), but there is likewise no indication that this enzyme possesses wall-loosening activity (McQueen-Mason et al., 1993). Moreover, the substrate xyloglucan is only a minor component in the cell walls of grasses, such as maize (Carpita, 1996), and may therefore play no major role as a target for wall-loosening enzymes in these plants. A similar argument can be raised in the case of α-expansins, i.e. proteins that are capable of loosening cell walls at acid pH by weakening noncovalent bonds within the xyloglucan-cellulose network of cell walls (Cosgrove, 2000a; Whitney et al., 2000). However, grasses differ from dicots by containing β-expansins, in addition to α-expansins, which have not yet been clearly defined with respect to substrate specificity and pH optimum. Extractable expansin activity has been demonstrated in the walls of the growing zone in maize roots and has been suggested to play a role in the regulation of root cell expansion in response to water stress (Wu et al., 1996).

The wall-loosening activity of expansins has been linked to acid growth, i.e. cell extension by acid cell wall pH (Cosgrove, 2000b), although it is presently not clear whether all expansins function in a pH-dependent manner. Originally established for the auxin-promoted growth of shoot organs such as coleoptiles, this hypothesis has often been claimed for explaining root growth phenomena, including gravitropic bending, long before the discovery of expansins (Pritchard, 1994). It is founded on the observation that root growth can be enhanced by acid buffers (pH 2–4; Edwards and Scott, 1974; Evans, 1976) or fusicoccin-mediated cell wall acidification (Lado et al., 1976), and is accompanied by proton secretion in the growing zone (Mulkey and Evans, 1981; Mulkey et al., 1982; Peters and Felle, 1999). In apparent agreement with the acid growth hypothesis, application of auxin (≥1 μm) stops root growth and causes alkalinization (Evans et al., 1980) and stiffening (Büntemeyer et al., 1998) of the cell walls in the growing zone. However, the inhibitory effects of auxin on growth and cell wall extensibility cannot be reversed by buffer- or fusicoccin-induced cell wall acidification (Evans, 1976; McBride and Evans, 1977; Lüthen and Böttger, 1993). Moreover, in obvious conflict with the older findings, it has recently been reported that elongation growth and cell wall extensibility of maize roots are practically independent of medium acidity in the range of pH 4 to 9. More acidic or alkaline pH conditions inhibited growth (Lüthen and Böttger, 1993; Büntemeyer et al., 1998). From a comparison of oligogalacturonide- and auxin-induced cell wall alkalinization and growth responses in cucumber (Cucumis sativus) roots, Spiro et al. (2002) concluded that auxin-induced alkalinization is not sufficient to account for the mechanism by which auxin inhibits root growth.

In this article, we explore an alternative possibility, based on the idea that the wall-loosening reaction underlying root elongation growth is effected by the production of reactive oxygen intermediates (ROIs), intermediate reduction products of O₂ en route to water, especially hydroxyl radicals (^˙OH), in the cell wall. ^˙OH represents a short-lived, highly reactive molecule that cleaves cell wall polysaccharides in a nonenzymatic reaction (Fry, 1998) and can, in this way, cause wall loosening in isolated cell walls as well as in living tissues (Schopfer, 2001). In experiments with maize coleoptiles and leaves, evidence has been gathered for the concept that cell elongation growth results from the polysaccharide-splitting action of ^˙OH produced in the cell wall in a peroxidase-catalyzed reaction from superoxide anion (O₂^˙−) and H₂O₂, which in turn are products of the monovalent reduction of O₂ by a NAD(P)H oxidase in the plasma membrane (Schopfer, 2001; Rodríguez et al., 2002; Schopfer et al., 2002; Liszkay et al., 2003). This concept has received support from a recent report demonstrating that an Arabidopsis mutant in which root and root hair elongation is impaired (root hair defective 2) is also impaired in the generation of ROIs in the root due to the disruption of a NAD(P)H oxidase gene of the gp91^phox type (Foreman et al., 2003). These results were interpreted in terms of a role of ROIs in the regulation of cell expansion through the activation of Ca²⁺ channels. Here we test the hypothesis that ^˙OH, produced in the cell wall from its precursors O₂^˙− and H₂O₂, is directly responsible for cell expansion in the growing zone of maize roots.

RESULTS AND DISCUSSION

O₂^˙−, H₂O₂, and ^˙OH Are Produced in the Growing Zone

The absence of a cuticle at the surface of roots allows the penetration of reagents without necessitating mechanical perturbations. We used nitroblue tetrazolium chloride (NBT), a reagent forming an insoluble blue formazan product upon reduction (Bielski et al., 1980), as a histochemical probe for O₂^˙−. Figure 1, A to D, shows the time course of staining by NBT in an intact maize root. The reaction becomes visible after about 5 min, first in the central growing zone (4–5 mm behind the root tip) and, during the next 15 min, includes the distal growing zone. Very little staining occurs in the meristematic region and the root cap. At higher magnification, staining of the cell walls in the epidermis can be detected (Fig. 1G). Staining of freshly cut cross-sections through the growing zone demonstrates that O₂^˙− formation is preferentially localized in the epidermis and the vascular tissues (Fig. 1I). Basically similar results were obtained using 3,5,3′,5′-tetramethylbenzidine-HCl (TMB) as a probe for H₂O₂ in the presence of endogenous peroxidase (Ros Barceló, 1998a), although this chromogen produced a more diffuse staining pattern due to the solubility of the blue oxidation product formed (Fig. 1, E and J). Finally, a mixture of 3,3′-diaminobenzidine-HCl (DAB) and H₂O₂ was used for localizing peroxidase activity (Thordal-Christensen et al., 1997) in the epidermal cell wall and, to a lesser extent, in the cortex walls of the growing zone (Fig. 1, F, H, and K). These results show that intact, unstressed maize roots are capable of producing O₂^˙− and its dismutation product, H₂O₂, in the apoplastic space of the growing zone, preferentially in the epidermis and vascular tissues. With the assay reactions used, no staining was observed in the root region proximal to the growing zone, except in immature root hairs that were strongly stained at the growing tip. The demonstration of peroxidase in the cell walls of the growing zone satisfies a further condition for the generation of ^˙OH in this part of the root.

Figure 1.

Histochemical demonstration of O₂^˙− production, H₂O₂ production, and peroxidase activity in the growing zone of maize roots. A to D, G, and I, O₂^˙− production was visualized by incubating a root of an intact seedling in NBT for 5 (A), 10 (B), 15 (C), and 20 (D and G) min. Precipitation of blue formazan in the walls of epidermal cells (G) indicates apoplastic formations of O₂^˙−. A cross-section through the growing zone shows that O₂^˙− formation is strongest in the epidermis and vascular tissues (I). E and J, H₂O₂ production was visualized by incubating a root, or a root section, in TMB for 180 min. F, H, and K, Peroxidase activity was demonstrated similarly with DAB + H₂O₂ (15 min). Bars, 5 mm (A–F), 0.2 mm (G and H), and 0.5 mm (J and K).

Corresponding histochemical tests with a number of other species (e.g. cucumber, Helianthus annuus, Lycopersicon esculentum, Glycine max, and Arabidopsis) confirmed that apoplastic ROI production in the growing zone of roots represents a common feature of seed plants (data not shown). NBT staining of root tips of Medicago truncatula was previously reported in a study on the role of ROI in root nodule formation (Ramu et al., 2002).

Na₂,3′-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT) represents an O₂^˙− probe with properties similar to NBT, except that it produces a soluble reduction product that can be photometrically measured (Sutherland and Learmonth, 1997). We used XTT for measuring the secretion of O₂^˙− in the apical 10-mm region of maize roots. A typical time course of the test reaction is depicted in Figure 2. Table I shows that the reaction could be inhibited by superoxide dismutase (SOD), the SOD substitute Mn-desferrioxamine (Mn-DFA), and the NAD(P)H oxidase inhibitor ZnCl₂, but not by KCN, which inhibits peroxidases but not NAD(P)H oxidase (Frahry and Schopfer, 2001). The XTT assay made it possible to determine the effect of auxin on the O₂^˙− production of roots. Figure 3 shows that the inhibition of elongation growth and the medium alkalinization (Evans et al., 1980) induced by 1 μm indole-3-acetic acid (IAA) in the roots of intact maize seedlings are accompanied by an equally rapid reduction in the rate of O₂^˙− production. These results support the idea of a functional role of apoplastic O₂^˙− generation by a plasma membrane NAD(P)H oxidase in cell elongation in the growing zone of the root.

Figure 2.

Time course of apoplastic O₂^˙− production in maize roots. Root tips (10 mm) of intact seedlings were incubated in citrate buffer (10 mm, pH 6.0) containing 0.5 mm XTT. The increase in absorbance represents the reduction of XTT to formazan by O₂^˙−.

Table I.

Effect of O₂^˙− scavengers (SOD, Mn-DFA) and inhibitors of NAD(P)H oxidase (ZnCl₂) or peroxidase (KCN) on the release of O₂^˙− from the apical 10-mm region of maize roots

Treatment	Relative O2˙− Production Rate
	%
Control	100 ± 15
+ SOD (50 μg mL−1)	54 ± 11
+ Mn-DFA (1 mm)	31 ± 3
+ ZnCl2 (10 mm)	49 ± 12
+ KCN (1 mm)	102 ± 8

Roots of intact seedlings were preincubated for 1 h in salt medium, followed by 20 min in citrate buffer containing test substances, before measuring O₂^˙− production by root tips (10 mm) in the presence of test substances.

Figure 3.

Effect of auxin on elongation growth (A), medium pH (B), and O₂^˙− production (C) in maize roots. Elongation growth was measured in roots of intact seedlings incubated in salt medium with or without 1 μm IAA. pH changes were measured in salt medium surrounding the root tips (10 mm) of intact seedlings in the presence or absence of 1 μm IAA. O₂^˙−production was measured in the root tips (10 mm) of intact seedlings, as shown in Figure 2, after incubating them for various times with or without 1 μm IAA.

The chemical determination of ^˙OH in biological systems is aggravated by its extremely short lifetime (approximately 1 ns) and its promiscuous reactivity toward organic molecules. For this reason, the demonstration of ^˙OH generation, in contrast to the generation of O₂^˙− and H₂O₂, by plant tissues has only rarely been attempted (Kuchitsu et al., 1995; von Tiedemann, 1997; Schopfer et al., 2001, 2002; Fry et al., 2002). We tested ^˙OH production in maize roots using electron paramagnetic resonance (EPR) spectroscopy in combination with ethanol/α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) as a spin-trapping reagent (Ramos et al., 1992).

In this system, the reaction of ^˙OH with ethanol produces a carbon-centered hydroxyethyl radical, the long-lived POBN adduct of which demonstrates a characteristic EPR spectrum allowing the estimation of ^˙OH captured by the trap from the peak height (signal size) under standardized measuring conditions. In preliminary experiments, we found that the removal of the mucilage covering the root tip, together with the root cap, considerably improved the signal size of such measurements, but had no effect on the elongation rate of the roots. Figure 4 shows typical EPR spectra obtained by incubating the apical 10-mm region of decapped maize roots in a medium containing the spin-trapping reagents. The signal size increased with the incubation time, reaching a constant level after about 90 min (Fig. 5). The EPR signal could be eliminated, or strongly reduced, by killing the roots before the incubation or including ^˙OH scavengers, such as thiourea, adenine, or Na-salicylate, in the medium. Moreover, Table II shows that ^˙OH production could also be inhibited with O₂^˙− scavengers (CuCl₂, SOD), a H₂O₂ scavenger (catalase; CAT), and a peroxidase inhibitor (NH₂OH). These results are consistent with the concept that ^˙OH is formed in a peroxidase-catalyzed reaction from apoplastic O₂^˙− and H₂O₂ produced by a NAD(P)H oxidase located in the plasma membrane of root cells. Moreover, ^˙OH production by the roots was drastically increased by exogenous NADH, and this effect could be inhibited by KCN, supporting the involvement of apoplastic peroxidase in ^˙OH production (Liszkay et al., 2003). Interestingly, ^˙OH production by the roots could also be enhanced by fusicoccin, a fungal toxin that activates the plasma membrane H⁺-ATPase, causing a hyperpolarization of the plasma membrane (Marrè et al., 1974). This result makes sense if one takes into account that the apoplastic reduction of O₂, at the expense of cytosplasmic NAD(P)H by plasma membrane NAD(P)H oxidase, represents an electrogenic process depending on the membrane potential (Babior, 1999). This aspect will be taken up in a forthcoming publication.

Figure 4.

EPR spectra of hydroxyethyl/POBN adduct diagnostic for ^˙OH. The incubation media contained, in addition to spin-trapping reagents, 10 μm FeSO₄ + 1 mm H₂O₂ (Fenton reagent, reference spectrum; A); root tips (10 mm) of intact maize seedlings without (B) or with (C) 1 mm thiourea (^˙OH scavenger), or root tips killed with 20% trichloracetic acid before the incubation (D). The incubation time was 5 min (A) or 1 h (B–D).

Figure 5.

Kinetics of ^˙OH production in maize roots. Root tips (10 mm) of intact seedlings were incubated with spin-trapping reagents. Signal size of EPR spectra were measured in aliquots of the incubation medium, as shown in Figure 4.

Table II.

Effect of scavengers, inhibitors, and promoters of ^˙OH formation by maize roots

Treatment	Relative Amount of ˙OH
	%
Control	100 ± 10
+ Thiourea (1 mm)	32 ± 2
+ Adenine (3 mm)	44 ± 11
+ Na-salicylate (1 mm)	45 ± 7
+ CuCl2 (50 μm)	0 ± 0
+ SOD (100 μg mL−1)	36 ± 6
+ CAT (100 μg mL−1)	27 ± 21
+ NH2OH (1 mm)	54 ± 8
+ NADH (50 μm)	770 ± 7
+ NADH (50 μm) + KCN (1 mm)	190 ± 10
+ Fusicoccin (0.5 μm)	144 ± 22

Root tips (10 mm) of intact seedlings were preincubated for 30 min with ^˙OH scavengers (thiourea, adenine, Na-salicylate), O₂^˙− scavengers (CuCl₂, SOD), a H₂O₂ scavenger (CAT), a peroxidase inhibitor (NH₂OH), a peroxidase promoter (NADH ± peroxidase inhibitor KCN), or a H⁺-ATPase promoter (fusicoccin) followed by 1 h in the spin-trapping reaction medium also containing the respective test substances.

Generation of ^˙OH Causes Wall Loosening in the Growing Zone of the Root

The measurement of creep kinetics of cell walls kept under tension in an extensiometer provides direct information on their extensibility properties as affected by wall-loosening agents such as ^˙OH (Schopfer, 2001). ^˙OH can be experimentally produced from H₂O₂ exposed to a reductant, such as ascorbate, in the presence of catalytic amounts of a suitable one-electron-transferring mediator, e.g. Fe²⁺ ions (Fenton reaction; Halliwell and Gutteridge, 1999). To direct the action of the short-lived ^˙OH produced in this reaction effectively to load-bearing polymers in the cell walls, it is convenient to first bind the Fe ions to the cell walls before starting ^˙OH production by adding H₂O₂ + ascorbate (Schopfer, 2001). Figure 6 shows that cell walls taken from the growing zone of the maize root can be induced to extend under tension by treating them with ^˙OH generated with the Fenton reaction. Scavengers of ^˙OH, such as thiourea or salicylate, inhibit the induction of cell wall creep, confirming that ^˙OH is the effective wall-loosening agent (data not shown). It is interesting to note that a sizeable creep response can be induced even in the absence of added Fe ions, indicating that the formation of ^˙OH can also apparently be catalyzed by endogenous metal ions, perhaps by Fe contained in the porphyrin group of cell wall peroxidase.

Figure 6.

Induction of cell wall extension (creep) by generating ^˙OH in the walls of maize root segments with the Fenton reaction. Ten-millimeter-long killed segments dissected 1 mm below the root tip were stretched in the extensiometer and preincubated in buffer with 1 mm FeSO₄. After 30 min, ^˙OH production was initiated in the washed segments by adding buffer with 50 mm H₂O₂ + 50 mm ascorbate (upper curve). Controls were run by omitting either FeSO₄, ascorbate, or H₂O₂ from the incubation buffer.

The ability of cell wall peroxidase to promote the increase in cell wall extensibility was investigated using NADH as a reducing substrate that is effectively oxidized by this enzyme, forming ^˙OH in the presence of H₂O₂ (Chen and Schopfer, 1999). As in related experiments with other plant tissues (Liszkay et al., 2003), it was necessary to provide the reaction with supplementary H₂O₂ for raising ^˙OH production to a level sufficient to cause significant wall loosening (Fig. 7). These experiments demonstrate that the peroxidase located in the cell walls of the elongation zone of the root is potentially capable of catalyzing the formation of ^˙OH mediating wall loosening.

Figure 7.

Induction of cell wall extension (creep) by generating ^˙OH in the walls of maize root segments by endogenous cell wall peroxidase. Root segments were treated as in Figure 6, except that the preincubation was in buffer and ^˙OH production was initiated by adding 2 mm NADH + 10 mm H₂O₂ (upper curve). Controls were run by omitting either NADH or H₂O₂ from the incubation buffer.

If the formation of ROIs, including ^˙OH, is important for wall loosening in the growing zone, one would expect that this region of the root is specifically sensitive to the wall-loosening action of experimentally produced ^˙OH. This prediction was tested by dissecting root segments of 10-mm length, 1, 2, 3, etc. mm behind the tip, thus excluding an increasing fraction of the growing zone from the test segments. Figure 8 shows that the extent of cell wall creep of these segments induced by the Fenton reaction continuously decreases when the position of the segment is shifted stepwise toward the root base, reaching a minimum value when the segment is taken 6 to 8 mm behind the root tip. Thus, the region most sensitive to the wall-loosening action of ^˙OH closely matches the growing zone of the root.

Figure 8.

Spatial distribution of the wall-loosening response induced by the Fenton reaction in the maize root. Ten-millimeter-long segments were dissected at various distances from the root tip, beginning with 1 mm. Induction of cell wall extension was measured as in Figure 6. The increase in extension reached after 30 min is plotted on the ordinate.

Inhibition of Endogenous ^˙OH Formation Causes Suppression of Elongation Growth

The experiments described so far prompt the question of whether ^˙OH, endogenously produced in the growing zone of the root, is causally involved in the growth process under natural conditions. To investigate this question, we studied the effect of various scavengers and inhibitors known to eliminate O₂^˙−, H₂O₂, or ^˙OH, or prevent their formation by blocking the activity of the plasma membrane NAD(P)H oxidase or cell wall peroxidase. As representative examples, Figure 9 shows the effects of benzoate (^˙OH scavenger; Halliwell and Gutteridge, 1999), Mn-DFA (O₂^˙− scavenger; Beyer and Fridovich, 1989), diphenylene iodonium-HCl (DPI; NAD(P)H oxidase inhibitor; Cross and Jones, 1986), and salicylhydroxamate (SHAM; peroxidase inhibitor; Martinez et al., 1998; Kawano and Muto, 2000) on the growth kinetics of roots of intact maize seedlings. Table III summarizes the results of corresponding experiments with several reagents of similar actions (Schopfer et al., 2002; Liszkay et al., 2003, and refs. cited therein); the inhibition of peroxidases by 4-amino benzoic acid hydrazide (ABAH) and methimazol has been described by Burner et al. (1999) and Wagner et al. (2000), respectively. These data show that root growth can be inhibited by numerous agents interfering with the production of ^˙OH at different points of the reaction chain O₂ → O₂^˙− → H₂O₂ → ^˙OH. Evidently, the specificity of action of these reagents cannot be taken for granted in each single case (Ros Barceló, 1998b). However, taken together, these inhibitor studies support the notion that the elongation growth of maize roots depends on the endogenous generation of ^˙OH by cell wall peroxidase and that this reaction, in turn, depends on the apoplastic supply of O₂^˙− and its dismutation product, H₂O₂, by an NAD(P)H oxidase-type enzyme in the plasma membrane.

Figure 9.

Inhibition of root growth of maize seedlings by an ^˙OH scavenger (K-benzoate), an O₂^˙− scavenger (Mn-DFA), an NAD(P)H oxidase inhibitor (DPI), or a peroxidase inhibitor (SHAM). Roots of intact seedlings were incubated with the indicated concentrations of test substances for up to 6 h.

Table III.

Effect of scavengers of O₂^˙−, H₂O₂, ^˙OH, or inhibitors of NAD(P)H oxidase and peroxidase on root elongation growth

Treatment	Elongation Rate
	%
Control	100 ± 1
+ ˙OH scavengers
Adenine (3 mm)	18 ± 8
Na-benzoate (3 mm)	33 ± 10
Na-salicylate (1 mm)	22 ± 10
His (30 mm)	41 ± 15
+ O2˙−scavengers
Mn-DFA (10 mm)	18 ± 5
CuCl2 (30 μm)	37 ± 8
Tiron (10 mm)	37 ± 4
SOD (100 μg mL−1)	67 ± 10
Mn-TMPa (100 μm)	18 ± 4
+ H2O2 scavengers
Na-pyruvate (30 mm)	32 ± 6
KI (30 mm)	47 ± 8
+ NAD(P)H oxidase inhibitors
DPIb (50 μm)	20 ± 4
ZnCl2 (10 mm)	35 ± 8
+ Peroxidase inhibitors
KCNc (1 mm)	27 ± 10
NaN3 (0.3 mm)	12 ± 4
NH2OH (1 mm)	29 ± 6
SHAM (3 mm)	20 ± 4
1,10-Phenanthroline (100 μm)	28 ± 4
2,2′-Bipyridyl (1 mm)	29 ± 6
ABAH (3 mm)	38 ± 15
Methimazol (10 mm)	37 ± 6

Root tips (10 mm) of intact maize seedlings were incubated in various inhibitor solutions at concentrations producing inhibition in the range of 20% to 40%. In most cases, close to 100% inhibition was obtained at 3-fold higher concentrations (except SOD because of restricted penetration through the cell walls). Elongation rate was measured as in Figure 9.

^aAlso can scavenge H₂O₂ (Day et al., 1997).

^bAlso inhibits peroxidase (Frahry and Schopfer, 1998).

^cAlso inhibits SOD (Asada et al., 1974).

CONCLUSIONS

The biological role of ROIs in plants is traditionally associated with damaging or signal-transfer reactions in situations of biotic or abiotic stress and cell death (Van Breusegem et al., 2001; Mittler, 2002; Overmyer et al., 2003). Here we show that juvenile, unstressed cells transiently express the ability to generate ROIs, including ^˙OH, in passing through the growing zone of the root. The close spatial correlation between apoplastic ROI production and cell elongation along the root strongly suggests a causal relationship between these events. In addition, pharmacological experiments provide evidence that wall loosening, a basic condition for cell extension, can be induced by ^˙OH generated in the cell walls in a peroxidase-mediated reaction from O₂^˙− and H₂O₂. Finally, it can be shown that normal cell extension in the growing zone depends on ^˙OH formation. These results support the hypothesis that the biochemical mechanism of cell growth involves wall loosening by ^˙OH. Further work is necessary to test this hypothesis at the quantitative level.

MATERIALS AND METHODS

Plant Material

Maize (Zea mays L. cv Perceval; Asgrow, Bruchsal, Germany) seedlings were grown on damp paper towels in a vertical position (root apex downward) in rectangular petri dishes at 25°C for 2.5 d in red light (0.7 W m⁻²; Mohr et al., 1964). Under these conditions, seedlings with straight roots of 30- to 40-mm length, growing at a rate of about 1.5 mm h⁻¹, were obtained. Light had no significant effect on root elongation. Experiments were performed with roots of intact seedlings or root sections at 25.0 ± 0.3°C under normal laboratory light.

Special Chemicals

Mn-DFA (green complex) was prepared as described by Beyer and Fridovich (1989). Other chemicals include DFA, DPI, and methimazole (Sigma, Deisenhofen, Germany); Mn-5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine (Mn-TMP), Tiron, and POBN (Aldrich, Deisenhofen, Germany); ABAH (myeloperoxidase inhibitor-I; Calbiochem, Schwalbach, Germany); CAT and SOD (Roche Biochemicals, Mannheim, Germany); NBT (Serva, Heidelberg); and XTT (Polysciences, Eppelheim, Germany). Fusicoccin was a gift from Dr. Lercari (Pisa, Italy).

Determination of Elongation Growth

Roots of intact seedlings were incubated in vertically oriented glass tubes (3.5-mm i.d.) with a funnel-shaped top in which the seed could be fixed. Length changes were measured with an accuracy of ±0.5 mm with the help of a millimeter scale attached to the tube. CO₂-free air was bubbled (40 mL min⁻¹) through the incubation medium (3 mL; 1 mm KCl + 0.1 mm CaCl₂ + test substances, pH adjusted to 6.0 with KOH or HCl).

Determination of Cell Wall Extensibility

Creep measurements were performed with a custom-built constant-force extensiometer essentially as described by Hohl and Schopfer (1992). Ten-millimeter-long root segments were killed by freezing and thawing and extended with a load of 10 g in 50 mm Na-succinate/NaOH buffer (pH 6.0) during a preincubation period of 30 min before inducing a creep response by changing the medium to buffer containing test substances.

Determination of H⁺ Secretion

The roots of 10 intact seedlings were placed in a vial containing 5 mL aerated (60 mL min⁻¹) salt medium (1 mm KCl + 0.1 mm CaCl₂, pH 6) covering about 10 mm of the root tip. The H⁺ concentration in the medium was continuously measured with a semi-micro pH electrode.

Determination of O₂^˙− Production

The roots of intact seedlings were preincubated in 5 mL salt medium (1 mm KCl + 0.1 mm CaCl₂, pH 6). After 1 h, the seedlings were transferred to buffered salt medium (10 mm K-citrate, 1 mm KCl, 0.1 mm CaCl₂, pH 6) with or without IAA or other test substances. O₂^˙− production was measured with the XTT assay, as described by Frahry and Schopfer (2001). The roots of five intact seedlings were placed in a short test tube containing 1.5 mL aerated reaction mixture (10 mm K-citrate buffer, pH 6.0; 1 mm KCl; 0.1 mm CaCl₂; 0.5 mm XTT; test substances; 1 μm IAA in the case of IAA-pretreated roots), covering about 10 mm of the root tip. The reaction mixture (total volume 5 mL) was pumped through the flow cell of a spectrophotometer and the absorbance increase due to the formation of colored formazan was recorded at 470 nm for 10 min. Absorbance was transformed into molar concentration using an extinction coefficient of 2.16 × 10⁴ L mol⁻¹ cm⁻¹ (Sutherland and Learmonth, 1997).

Determination of ^˙OH Production

A modified version of the spin-trapping system described by Ramos et al. (1992) was used (Liszkay et al., 2003). After a 30-min preincubation in salt medium (1 mm KCl + 0.1 mm CaCl₂) with test substances (pH 6), the roots of 20 seedlings were placed in a vial containing 1 mL reaction medium (850 mm ethanol, 50 mm POBN, 20 mm K-phosphate buffer, test substances, pH 6.0) covering about 10 mm of the root tip. Before transferring the roots into the reaction medium, the root cap with the adhering mucilage was removed with a scalpel using a stereomicroscope. After incubating for 0.5 to 2 h on a shaker, EPR spectra of the hydroxyethyl-POBN adduct were immediately measured at room temperature in an aliquot of the reaction mixture, as described by Liszkay et al. (2003). The size of EPR signals was calculated from the maximum signal-to-noise ratio of recorder traces and corrected, if necessary, by subtracting reagent blanks determined in parallel.

Histochemical Assays

The production of O₂^˙− in vivo was visualized by incubating intact roots or hand-cut root sections in 10 mm K-citrate buffer (pH 6.0) containing 0.5 mm NBT (Frahry and Schopfer, 2001). H₂O₂ production in vivo was assayed after Ros Barceló (1998a), using 10 mm K-citrate buffer (pH 4.0) containing 1 mm TMB (from a 25-mm stock solution in ethanol). Peroxidase activity in vivo was assayed using 10 mm K-citrate buffer (pH 6.0) containing 2.5 mm DAB and 1 mm H₂O₂ (Frahry and Schopfer, 2001). In principle, TMB and DAB are exchangeable chromogenic electron donors converted to a soluble blue (TMB) or an insoluble brown (DAB) reaction product in the presence of H₂O₂ and peroxidase. However, because of its much higher sensitivity, the TMB assay is preferable for demonstrating H₂O₂ despite of its lower spatial resolution at the cellular level.

Statistics

Data represent typical examples from measurements repeated 4 to 8 times or means of 4 to 10 independent experiments ±se.

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