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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. 126.96.36.199) 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.
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- Materials and Methods
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)
|Treatments||Root length||Root hair length||Root hair density||O2•– in elongation zone||H2O2 in differentiation zone||Formation of H2O2 in vitro|
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.