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Keywords:

  • coumarins;
  • diphenylene iodonium;
  • hydrogen peroxide (H2O2);
  • peroxidase;
  • potassium iodide;
  • root hairs;
  • salicylhydroxamic acid (SHAM);
  • superoxide (O2•–)

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The respective distribution of superoxide (O2•–) and hydrogen peroxide (H2O2), two reactive oxygen species (ROS) involved in root growth and differentiation, was determined within the Arabidopsis root tip. We investigated the effect of changing the levels of these ROS on root development and the possible interactions with peroxidases.
  • • 
    H2O2 was detected by confocal laser-scanning microscopy using hydroxyphenyl fluorescein (HPF). Both O2•– accumulation and peroxidase distribution were assessed by light microscopy, using nitroblue tetrazolium (NBT) and o-dianisidine, respectively. Root length and root hair length and density were also quantified following ROS scavenging.
  • • 
    O2•– was predominantly located in the apoplast of cell elongation zone, whereas H2O2 accumulated in the differentiation zone and the cell wall of root hairs in formation. Treatments that decrease O2•– concentration reduced root elongation and root hair formation, while scavenging H2O2 promoted root elongation and suppressed root hair formation.
  • • 
    The results allow to precise the respective role of O2•– and H2O2 in root growth and development. The consequences of their distinct accumulation sites within the root tip are discussed, especially in relation to peroxidases.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Oxygen supply is crucial for root, not only for cell respiration, but also for the formation of reactive oxygen species (ROS). These, although mostly considered as key actors in oxidative burst, can also play important physiological roles in plants (Mittler et al., 2004), mainly in the apoplast, where they can be produced by several enzymes. The plasma membrane NADPH oxidase is responsible for the one-electron reduction of oxygen at the surface of cells, yielding superoxide anion (O2•–), an important factor for root growth and root hair development (Foreman et al., 2003). The superoxide ion may be further converted into H2O2 spontaneously or by superoxide dismutase. Hydrogen peroxide can also be produced by other enzymes such as apoplastic oxalate oxidase (Calişkan & Cuming, 1998), diamine oxidase (Federico & Angelini, 1986), or class III peroxidases (Elstner & Heupel, 1976). Hydrogen peroxide is involved in many developmental and physiological processes (Gapper & Dolan, 2006; Kwak et al., 2006). It is necessary for the growth of root hairs (Foreman et al., 2003), and is also essential in the peroxidase-mediated formation of lignin (Ros Barceló, 1997). It is at the origin of hydroxyl radical (OH) formation by peroxidases (Chen & Schopfer, 1999). This radical has a loosening effect on cell walls and is therefore very important for cell elongation (Liszkay et al., 2004).

The tip of roots is a zone of active ROS production (Liszkay et al., 2004). It comprises cells in very different states within a very short distance, including meristematic and elongating cells, and cells undergoing various kinds of differentiation (Scheres et al., 2002). A precise determination of the localization of the different ROS would be useful to better understand their physiological roles and the interaction they may have with various apoplastic proteins, including peroxidases.

The heme-containing class III plant peroxidases (E.C. 1.11.1.7) have complex relations with ROS. They are known above all as H2O2-reducing enzymes, able to oxidize or polymerize various hydrogen donors while converting H2O2 into water, but they can also promote the formation of H2O2, OH or O2•–, provided that an appropriate strong reductant is present. NAD(P)H (Mäder & Amberg-Fisher, 1982), indoleacetic acid (IAA) (Smith et al., 1982), saturated fatty acids (Bolwell et al., 2002) or cysteine (Pichorner et al., 1992) are among the molecules shown to be active in peroxidase-mediated ROS generation. The catalytic pathway followed by a peroxidase (use of H2O2 or formation of ROS) is therefore dependent on its chemical environment (Passardi et al., 2004). Peroxidases are present in all organs and almost all tissues, but they are particularly abundant in roots. The reason for this preferential accumulation has not been elucidated. They have been shown to be implicated in a great deal of physiological processes, including growth, cell wall differentiation or responses to various biotic and abiotic stresses (Penel et al., 1992). They have long been considered as being exclusively involved in growth limiting reactions, because they are able to oxidize the growth-promoting hormone auxin and to stiffen cell walls by cross-linking their constituents. However, recent work has shown that decreasing the level of expression of two very homologous peroxidase-encoding genes of Arabidopsis reduces root elongation, while overexpression promotes root length (Passardi et al., 2006). A correlation between peroxidase overexpression and growth has also been found in aspen (Kawaoka et al., 2003).

In the present work, several substances known to react with ROS were applied to Arabidopsis seedlings and the consequences on root development and peroxidase activity were assessed. The precise distribution of O2•– and H2O2 within the root tip was studied and the consequences for apoplastic peroxidase functions are discussed.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant material

Seeds of Arabidopsis thaliana L. (Heynh.) (ecotype Columbia) were sown in square boxes containing half-concentrated MS medium in 0.8% agar. The boxes were kept vertically in a growth chamber at 24°C under a 16-h photoperiod and 180 µmol m−2 s−1 of light intensity.

Effect of chemical treatments on root development and peroxidase activity

Potassium iodide (KI), potassium benzoate, Mn desferal (Mn-DFA, green complex) prepared as described by Beyer & Fridovich (1989), H2O2 (Merck, Lucerne, Switzerland), diphenylene iodonium (DPI; Acros Organics, Geel, Belgium), esculetin (6,7-dihydroxycoumarin), umbelliferone (7-hydroxycoumarin) and salicylhydroxamate (SHAM; Sigma, Buchs, Switzerland) were added to the culture medium from appropriate stock solutions at concentrations specified in each case. Seedlings were grown in the presence of the various substances for 6–7 d.

The length of the roots was obtained by measuring 40–50 roots from several independent batches. Root hair lengths were obtained by measuring 200 hairs from several seedlings on images obtained with a MZ 16 Leica stereomicroscope (Leica Microsystems, Heerbrugge, Switzerland) equipped with a DC300F Leica camera. The IM1000 Leica software was used for the measures. Root hair densities were also determined on these images.

Soluble proteins were extracted by grinding the roots used for length measurements in 20 mm N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.0 containing 1 mm ethylene glycol-bis(beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). Each extract was assayed for total protein content (Coomassie Blue Reagent; Bio-Rad, Marnes-la-Coquette, France) and for total peroxidase activity, using guaiacol/H2O2 (Penel & Greppin, 1994).

Nitroblue tetrazolium staining

Whole seedlings were used 6–8 d after germination. Roots were stained for 15 min in a solution of 2 mm nitroblue tetrazolium (NBT) in 20 mm phosphate buffer pH 6.1. The reaction was stopped by transferring the seedlings in distilled water. Chemical pretreatments were applied for 60 min before NBT staining. The roots were observed with a MZ 16 Leica stereomicroscope. Pictures were taken as detailed earlier. Settings were identical for all the pictures in an experiment. Each experiment was repeated at least four times with similar results. The Photoshop (Adobe Systems Inc., San Jose, CA, USA) histogram function was used to assess the mean staining intensity of the elongation zone (0, white; 255, black).

Hydroxyphenyl fluorescein staining

Seedlings were preincubated in phosphate buffer (pH 6.1) with or without substance interfering with ROS production and incubated for 2 min in the same buffer containing 5 µm 3′-(p-hydroxyphenyl) fluorescein (HPF; Alexis Biochemical, Lausen, Switzerland). Root tips were then mounted on a microscope slide in a drop of buffer and covered with a coverslip supported by pieces of coverslip to prevent squashing the root. They were immediately observed with a × 20 water objective (numerical aperture: 0.71). Fluorescent images were collected with a Leica SP2 confocal laser-scanning microscope coupled to an inverted Leica microscope DMIRE 2. The 488-nm line of an argon ion laser was selected for HPF excitation and the fluorescence detection was in the range of 506–586 nm. The images were acquired with a scan speed of 400 Hz and line average of 8. Figure 5 shows typical examples from experiments repeated at least four times with roots from different batches of seedlings.

image

Figure 5. Distribution of hydrogen peroxide (H2O2) in Arabidopsis root visualized by 3′-(p-hydroxyphenyl) fluorescein (HPF) fluorescence. (a) Whole root tip; (b) lateral root with a part of the main root; (c) close-up on the differentiation zone. White arrows show the forming root hairs; mz, meristematic zone; ez, elongation zone; dz, differentiation zone, fdz, fully differentiated zone. Bars, (a,b) 150 µm, (c) 75 µm.

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Images of Fig. 6 were obtained with an Axioplan 2 Zeiss microscope equipped with a narrow band excitation filter (485 ± 2 nm) and a FITC-suited emission filter (> 515 nm, Filter set 09; Carl Zeiss, Inc., Newark, DE, USA).

image

Figure 6. Distribution of hydrogen peroxide (H2O2) visualized by 3′-(p-hydroxyphenyl) fluorescein (HPF) fluorescence in Arabidopsis roots of seedlings grown for 7 d on half-strength MS medium (a), supplemented with 100 µm esculetin (b) or 100 µm umbelliferone (c). Bar, 100 µm.

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Staining of peroxidase activity

Seedlings were used 6–8 d after germination. They were stained in a solution containing 1 mm o-dianisidine and 10 mm H2O2 for 2 min and then transferred into water. Pretreatments were applied by dipping seedlings for 60 min in various solutions. Pictures were taken as described for NBT staining.

In vitro H2O2 formation

The formation of H2O2 was followed in 1 ml of 20 mm phosphate buffer pH 6.1, containing 0.2 µm anionic peroxidase from zucchini (APRX), purified as previously described (Penel & Greppin, 1994) and 1.25 µm of the fluorescent probe HPF. The reaction was started by the addition of 100 µm NADPH and proceeded at room temperature. Fluorescence was followed with a SFM 25 Kontron spectrofluorimeter (Zürich, Switzerland) with an excitation at 490 nm and an emission at 515 nm. Fluorescence was adjusted to 100 with a solution of 0.5 µm fluorescein. The effect of various chemicals was tested by adding them before NADPH. Each experiment was repeated at least three times with same results.

Statistical analysis

When necessary, the effect of treatments was compared with untreated controls by the one-way anova test using analyse-it (Analyse-It Software Ltd, Leeds, UK).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Effect of chemical treatments on root development and peroxidase activity

Arabidopsis seeds were sown on media containing various substances known to react with ROS and thereby modify their content in plants. Esculetin and umbelliferone were also tested because of the faculty of coumarins to modulate peroxidase activity (Sirois & Miller, 1972; Krylov & Dunford, 1996). After 7 d of culture on vertical plates, the effect of the treatments was assessed on root morphology and peroxidase activity. Root growth and the formation and elongation of root hairs were profoundly affected by some substances (Table 1, Fig. 1). Manganese-desferal green complex (Mn-DFA), a scavenger of O2•– (Beyer & Fridovich, 1989) and H2O2, as well as the two coumarins esculetin and umbelliferone reduced considerably root growth when supplied at a sufficient concentration. By contrast, KI, a scavenger of H2O2, had a stimulatory effect. Pyruvate, another known scavenger of H2O2 only active at moderate concentrations (Desagher et al., 1997), exhibited also a slight but significant growth stimulation at 500 µm. Salicylhydroxamic acid, at a concentration at which it interacts with peroxidases but not with the mitochondrial alternative oxidase (Spreen Brouwer et al., 1986), also reduced root elongation. A reduction was observed with benzoate, an efficient scavenger of hydroxyl radical (Chen & Schopfer, 1999). In addition to their effects on the length of roots, some of the above-mentioned chemicals exhibited a spectacular influence on root hair development (Table 1, Fig. 2). When supplied in excess, KI, Mn-DFA, SHAM and H2O2 reduced or suppressed the elongation of hairs. The coumarins had more complex effects. Esculetin did not modify the density of root hairs, but stimulated their elongation at low concentrations and inhibited it at higher concentrations. Umbelliferone increased both the elongation of root hairs and their number per surface unit, but only at rather high concentrations. This latter effect was probably due to the fact that root cells were shorter. The roots were also much thicker (Fig. 2). Pyruvate had no effect (data not shown) and diphenylene iodonium (DPI), an inhibitor of the plasma membrane NADPH oxidase, reduced root hair length, as already reported (Foreman et al., 2003), but increased root hair density at 10 µm.

Table 1.  Root hair length and density of Arabidopsis seedlings grown for 7 d in the presence of various substances
TreatmentµmRoot hair length (µm)Root hair density (number mm2)
  1. nd, Not determined; Mn-DFA, manganese-desferal; DPI, diphenylene iodonium; H2O2, hydrogen peroxide; KI, potassium iodide; SHAM, salicylhydroxamic acid.

  2. The length is the mean (± SD) of 200 fully developed root hairs. (Mean ± SD) root hair density was determined on 20 roots.

None 287 ± 20104 ± 16
Umbelliferone   1272 ± 71109 ± 29
 100409 ± 77301 ± 83
Esculetin   1501 ± 117110 ± 9
 100 14 ± 2123 ± 23
Mn-DFA 250 80 ± 26 67 ± 17
DPI  10136 ± 2156 ± 3
 100105 ± 0 94 ± 19
H2O2 500249 ± 35108 ± 45
1000188 ± 59177 ± 27
KI  10203 ± 15113 ± 21
 100Bulgesnd
SHAM  50123 ± 35128 ± 36
image

Figure 1. Root length of Arabidopsis seedlings grown for 7 d in vertical plates containing various concentrations of different substances interacting with reactive oxygen species (ROS). Each point represents the mean (± SD) of 40–65 roots analysed from three independent assays. *, Significant increase with respect to control (P < 0.0001); **, significant increase with respect to control (P < 0.0015). (a) Closed circles, benzoate; open circles, diphenylene iodonium (DPI); triangles, esculetin. (b) Closed circles, hydrogen peroxide (H2O2); open circles, potassium iodide (KI); triangles, manganese desferal (Mn-DFA). (c) Closed circles, salicylhydroxamic acid (SHAM); open circles, umbelliferone; triangles, pyruvate.

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image

Figure 2. Effect of reactive oxygen species (ROS) scavengers and other substances on the development of root hairs. The pictures show a representative view of the root hair zone from Arabidopsis seedlings grown for 7 d on a medium containing the following substances: (a) control; (b) 100 µm hydrogen peroxide (H2O2); (c) 1 mm potassium iodide (KI); (d) 50 µm salicylhydroxamic acid (SHAM); (e) 100 µm manganese-desferal (Mn-DFA); (f) 50 µm benzoate; (g) 200 µm esculetin; (h) 2 µm esculetin; (i) 200 µm umbelliferone. Bar, 100 µm.

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The activity of soluble peroxidases was measured in roots used for growth measurements. The results showed that some of the substances inhibiting root elongation (benzoate, esculetin, H2O2 and SHAM) reduced root peroxidase activity (Fig. 3), while KI increased it at concentrations promoting root elongation. Esculetin also enhanced peroxidase activity at low, growth-promoting, concentrations and decreased it at higher concentrations, which inhibited root elongation. Diphenylene iodonium enhanced peroxidase activity, but reduced root growth, while Mn-DFA and pyruvate had no clear effect.

image

Figure 3. Soluble peroxidase activity of Arabidopsis seedlings grown for 7 d in vertical plates containing various concentrations of different substances interacting with reactive oxygen species (ROS). Each point represents the mean (± SD) of 20 roots analysed from three independent assays. (a) Closed circles, benzoate; open circles, diphenylene iodonium (DPI); triangles, esculetin. (b) Closed circles, hydrogen peroxide (H2O2); open circles, potassium iodide (KI); triangles, manganese desferal (Mn-DFA). (c) Closed circles, salicylhydroxamic acid (SHAM); open circles, umbelliferone; triangles, pyruvate.

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Localization of superoxide

The results presented above showed that substances modifying O2•–, H2O2, or OH• content can interfere with the development of roots. Since O2•– is often the first reduced form of oxygen to be generated in plant tissues, leading to the subsequent formation of H2O2 and OH•, we studied its distribution in Arabidopsis roots with NBT, a widely used indicator, which forms a dark blue formazan precipitate in contact with superoxide (Bielski et al., 1980). The blue/violet color denoting the presence of O2•– appeared at the tip of the main root and was particularly strong in the elongation zone, especially in the inner tissues and extended more or less to the meristematic zone (Fig. 4). It was also often observed in the stele all along the root. The walls of other cells appeared also slightly stained. This pattern was observed in well-aerated roots, but it decreased in roots deprived of oxygen (data not shown). The effect of the substances tested on root development was also estimated on NBT staining after an incubation period that allowed the chemicals to penetrate into the roots. It appeared that Mn-DFA, SHAM, and DPI reduced O2•– formation, while esculetin and umbelliferone had no significant effect (Table 2).

image

Figure 4. Distribution of O2•– visualized by nitroblue tetrazolium (NBT) staining in Arabidopsis root tip. Bar, 100 µm.

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Table 2.  Intensity of nitroblue tetrazolium (NBT) staining in the tip of Arabidopsis roots treated with various substances
TreatmentsmmStaining intensity (± SE)
  1. Mn-DFA, manganese-desferal; SHAM, salicylhydroxamic acid; DPI, diphenylene iodonium.

  2. Numbers with the same superscript letters indicate significant difference (P < 0.0001).

Control 186 ± 5a,b,c
SHAM5111 ± 17a
Mn-DFA1129 ± 18b
Umbelliferone0.1195 ± 13
Esculetin0.1215 ± 3
DPI0.1123 ± 12c

Localization of hydrogen peroxide

Hydroxyphenyl fluorescein, a probe that becomes fluorescent in the presence of H2O2 and peroxidases, or OH• alone (Setsukinai et al., 2003), was used to detect H2O2 in Arabidopsis roots. This probe is more specific and less prone to auto-oxidation than the widely used 2′,7′-dichlorofluorescein (Setsukinai et al., 2003). It was found that after a 2-min incubation, the cell walls of a region encompassing the end of the elongation zone and the beginning of the differentiation zone appeared fluorescent (Fig. 5). No autofluorescence could be detected in the absence of HPF (data not shown). The cell wall fluorescence was particularly strong in expanding root hairs (Fig. 5b) and in the elongation part of lateral roots (Fig. 5c). Conversely, there was no fluorescence in the meristematic zone, as well as in the older regions of the root (Fig. 5c). Increasing the incubation time to 10 min and HPF concentration to 10 µm did not significantly increase the fluorescence.

The effect of a peroxidase inhibitor (SHAM), a scavenger of OH (benzoate), a scavenger of H2O2 (KI), and an inhibitor of the plasma membrane NADPH oxidase (DPI) was tested on the fluorescence of roots incubated with HPF. Cell wall fluorescence was strongly reduced by KI in both the elongating region and the root hairs (data not shown), in accordance with its scavenging effect on H2O2. Diphenylene iodonium also inhibited HPF fluorescence, as did SHAM. In the case of SHAM, the fluorescence was almost undetectable in the root hair zone, but it appeared in cell walls of the meristematic region, which did not exhibit fluorescence in the absence of this substance. Benzoate did not modify the level of fluorescence, neither its distribution (data not shown). This suggests that the fluorescence is not related to the HPF probe oxidation by OH. Roots of seedlings grown on umbelliferone for 7 d exhibited a very strong HPF fluorescence, mostly at the level of the hair formation zone (Fig. 6), indicating the presence of much H2O2. By contrast, roots grown in the presence of esculetin had a very reduced level of fluorescence.

Distribution of peroxidase activity

Peroxidases react either with H2O2 to fulfill their classical peroxidase cycle or with O2– to form oxyperoxidase (Passardi et al., 2004). This latter compound gives quite another orientation to peroxidase function and may yield H2O2 or OH (Halliwell, 1977). Since ROS are formed in the apoplast, where a great part of peroxidases are located, an interaction between the oxygen derivatives and peroxidases seems to be highly probable. This prompted us to look for the distribution of peroxidase activity along the Arabidopsis root. It was visualized by incubating living roots in a medium containing o-dianisidine as hydrogen donor and H2O2 (Fig. 7). It was found that roots were readily and entirely stained. The omission of H2O2 in the reaction medium suppressed the staining (data not shown), indicating that it was not caused by other enzymes such as laccases. A pretreatment with 1 mm SHAM strongly decreased peroxidase staining in the root tip, but not in the other parts. At a concentration of 5 mm, SHAM inhibited peroxidase activity throughout the root (data not shown). The other treatments had a slightly strengthening effect, except DPI.

image

Figure 7. Roots of Arabidopsis seedlings stained for peroxidase activity with o-dianisidine/hydrogen peroxide (H2O2). Seedlings were pretreated with various substances interacting with reactive oxygen species (ROS) for 60 min before peroxidase staining. (a) Control; (b) 100 µm diphenylene iodonium (DPI); (c) 100 µm esculetin; (d) 5 mm potassium iodide (KI); (e) 300 µm manganese desferal (Mn-DFA); (f) 1 mm salicylhydroxamic acid (SHAM); (g) 100 µm umbelliferone. Bar, 100 µm.

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Peroxidase-mediated H2O2 formation in vitro

Hydroxyphenyl fluorescein fluorescence observed in living roots revealed the simultaneous presence of H2O2 and peroxidases, with a maximum in the differentiation zone. The in vivo situation of apoplastic peroxidases can be mimicked in vitro by adding NADPH to a purified peroxidase. In that case, NADPH provides the electrons normally given by the plasma membrane NADPH oxidase. Since no purified Arabidopsis peroxidase was available in sufficient amount for this experiment, we used APRX, a purified peroxidase from zucchini, previously shown to be strongly expressed in elongating tissues (Dunand et al., 2003). It appeared that APRX and NADPH induced the conversion of HPF into fluorescein. This reaction was dependent on the formation of H2O2, since catalase completely abolished the fluorescence (Fig. 8a). In this reaction, two connected processes took place (Fig. 8b): the first was the generation of H2O2 by APRX and NADPH, and the second was the conversion of HPF into a fluorescent product, fluorescein, by APRX and the H2O2 produced in the first step. Umbelliferone and Mn-DFA activated the production of H2O2 or the conversion of HPF into fluorescein, while esculetin, superoxide dismutase, KI, SHAM or DPI had more or less inhibitory effects.

image

Figure 8. Fluorescence of 3′-(p-hydroxyphenyl) fluorescein (HPF) measured in 1 ml of phosphate buffer pH 6.1 containing 1.25 µm HPF and 0.2 µm anionic peroxidase from zucchini (APRX). The reaction was started by the addition of 100 µm NADPH. The following additions were tested: 50 µm diphenylene iodonium (DPI); 100 µm umbelliferone; 100 µm esculetin; 1000 units ml−1 beef liver catalase; 300 µm manganese desferal (Mn-DFA); 1 mm potassium iodide (KI); 1 mm salicylhydroxamic acid (SHAM); and 20 µg ml−1 bovine erythrocyte superoxide dismutase (SOD).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The connection between the formation of ROS in the apoplast and many developmental and physiological processes is well established (Joo et al., 2001; Foreman et al., 2003; Liszkay et al., 2004; Shin & Chachtman, 2004; Gapper & Dolan, 2006; Kwak et al., 2006). In most cases, the transplasma membrane NADPH oxidase seems to be a key player in the formation of oxygen derivatives (Mittler et al., 2004). However, many aspects of ROS formation and mechanism of action remain to be elucidated. A more precise determination of the localization of each ROS could help to better discern their respective role. In the present work, we tried to get an accurate picture of the localization of O2•– and H2O2 in the root tip of Arabidopsis seedling, a region which includes cell division, elongation and differentiation within a very short distance (Scheres et al., 2002).

Different substances able to modify O2•– and H2O2 content have been used. The effect of these treatments on O2•– and H2O2 accumulation and the consequences on root development have been assessed. The main results are summarized in Table 3. Staining with NBT indicated that O2•– is mainly present in the cell walls of young elongating or dividing cells and in the steele. Chemical treatments reducing its concentration (DPI, Mn-DFA) also decreased root and root hair elongation. As for H2O2, its localization was accurately determined with HPF, a fluorochrome that requires the simultaneous presence of peroxidase and H2O2 to become fluorescent (Setsukinai et al., 2003). Since peroxidases are present throughout the root (Fig. 8), the fluorescence observed after HPF staining most likely reflected the distribution of H2O2, which appeared to be rather abundant in the differentiation zone and in growing root hairs, but almost absent in the meristematic and the elongation zones. This is in line with the report showing that H2O2, as a precursor of OH, is involved in root hair formation (Foreman et al., 2003). The fact that roots of seedlings grown in the presence of KI, an efficient scavenger of H2O2, had no hairs is also consistent with this idea, as well as the low HPF fluorescence observed in such roots. These roots were also significantly longer, suggesting that H2O2 reduces root growth. This was confirmed by the inhibition of root elongation by exogenously supplied H2O2 and the inhibitory effect of umbelliferone, a coumarin that promotes H2O2 formation in vivo and in vitro. It was found that H2O2 also increased root hair density. Another explanation of the effect of KI could be that roots compensate the loss of absorbing surface resulting from the absence of hairs by increasing their length. Pyruvate, another H2O2 scavenger (Desagher et al., 1997) also increased root elongation at appropriate concentrations.

Table 3.  Recapitulation of the main effects observed on root development and reactive oxygen species (ROS)
TreatmentsRoot lengthRoot hair lengthRoot hair densityO2•– in elongation zoneH2O2 in differentiation zoneFormation of H2O2 in vitro
  1. Mn-DFA, manganese-desferal; DPI, diphenylene iodonium; H2O2, hydrogen peroxide; KI, Potassium iodide; SHAM, salicylhydroxamic acid.

  2. +, Increase; –, decrease; 0, no effect; +/–, increase at low concentration/decrease at high concentration; +/0, increase at low concentration/no effect at high concentration; nd, not determined.

Umbelliferone++0++
Esculetin+/–00
Mn-DFAnd+
DPI+/0ndnd
H2O2+ndndnd
KI+nd
SHAM0

Unlike controls, roots grown in the presence of SHAM exhibited some H2O2 in the cell walls of their meristematic and elongation zones. Salicylhydroxamic acid is known to inhibit the activity of plant peroxidases (Fig. 7; Aitken et al., 2001); however, it also mediates decomposition of the oxygenated form of these same peroxidases (oxyperoxidase), thus promoting the formation of H2O2 (Askerlund et al., 1987). Since oxyperoxidase is readily formed in the presence of O2•– (Nakajima et al., 1991), it should be present in the elongation zone. Thus, HPF fluorescence observed in this zone in the presence of SHAM (Fig. 5) could result from the release of H2O2 (Nakajima et al., 1991) and OH• (Chen & Schopfer, 1999) from oxyperoxidase, thereby promoting cell elongation by loosening cell walls (Liszkay et al., 2004). In this context, it is also interesting to note that SHAM increases oxygen uptake by roots (Spreen Brouwer et al., 1986). Finally, the very strong HPF fluorescence of roots grown with umbelliferone contrasted with the weak signal observed in esculetin-treated roots (Fig. 6). This result parallels the effect of the two coumarins on the in vitro formation of H2O2 by APRX and NADPH (Fig. 8) and is consistent with their effect on root hair formation (Fig. 2, Table 1). It shows that very similar molecules differing only in the absence or presence of a hydroxyl group may have antagonistic effects on the development of root and on redox reactions. Since umbelliferone has a stimulating influence on peroxidases (Krylov & Dunford, 1996), it is difficult to determine whether it acted on peroxidase-mediated H2O2 generation, on conversion of HPF by peroxidases/H2O2 or on both. However, its promotive effect on root hair elongation suggests that it increased H2O2 production in vivo.

Peroxidases are present in the apoplast of the three zones of Arabidopsis root tip, but these zones do not contain the same type of ROS. The elongation zone and, to a lesser extent, the meristematic zone are rich in O2•–, while H2O2 predominates in the differentiation zone, where cell elongation ceases. This difference has certainly a great influence on the role played by peroxidases. In the first two zones, the combination of O2•– and peroxidases should produce OH necessary for cell wall loosening, as shown by Schopfer and collaborators (Chen & Schopfer, 1999; Liszkay et al., 2004). Conversely, the simultaneous presence of H2O2 and peroxidases in the differentiation zone should orientate peroxidases towards reactions leading to growth arrest by cross-linking cell wall constituents (Fry, 1986) and to diversification of cell wall composition, for example by lignin deposition (Ros Barceló, 1997). It remains to be determined which of the 73 Arabidopsis peroxidase genes (Tognolli et al., 2002) are expressed in these zones and participate in these reactions. It has already been shown that the overexpression of a peroxidase encoding gene in Arabidopsis enhanced root growth (Passardi et al., 2006). As already mentioned, peroxidases can be involved in the generation or in the interconversion of ROS. The in vitro assays of Fig. 8 brought confirmation that a peroxidase produces H2O2 in the presence of NADPH (Halliwell, 1977; Mäder & Amberg-Fisher, 1982) and that HPF is a suitable fluorescent probe to follow this formation. In contrast to previous works, in which peroxidases generated H2O2 only in the presence of Mn2+ and phenolics as cofactors, APRX is active alone. It can be assumed that this in vitro reaction between APRX and NADPH mimics the situation prevailing in the apoplast between the plasma membrane NADPH oxidase, oxygen and some peroxidases. The in vitro inhibitory effect of SOD indicated that O2•– was a necessary intermediate in the peroxidase-mediated generation of H2O2 (Fig. 8). Surprisingly, Mn-DFA stimulated the formation of H2O2 in vitro. This result could be explained by the fact that this compound releases Mn2+ ions (Gray & Carmichael, 1992), known to have a promoting effect on this reaction (Halliwell, 1977). Diphenylene iodonium, usually regarded as a rather specific inhibitor of the NADPH oxidase (Foreman et al., 2003), also reduced HPF fluorescence in vitro in the presence of a purified peroxidase and NADPH. This means that this inhibitor could somehow interfere in vivo with a process catalysed by apoplastic peroxidases in addition to its effect on NADPH oxidase.

In conclusion, the present work provides evidence that O2•– and H2O2 have both distinct accumulation zones and different roles in the extremity of the growing Arabidopsis root. Hydrogen peroxide appears to be involved in growth restriction and root hair formation, as illustrated by the opposite effects of KI and umbelliferone. By contrast, O2•– seems to be necessary for root elongation. It also interferes with root hair development. Both O2•– and H2O2 are substrates of peroxidases. They can also be produced by these enzymes. It appears therefore that peroxidases, owing to their great catalytic versatility, play most likely a prominent role in apoplastic ROS metabolism.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the Swiss National Science Foundation (grant 3100A0-109325). We thank Christoph Bauer from the Bioimaging Plaform of the NCCR (Geneva University) for his help with confocal microscopy imaging. The skillful technical assistance of Mireille de Meyer and Dale Brighouse is gratefully acknowledged.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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