Both the structure and the chemical composition of the hypodermal cell layers of roots were examined in G. maxima and P. australis, monocotyledonous wetland species of Poaceae. Both species possess an exodermal hypodermis with suberized cell walls as defined by Haberlandt (1918) and von Guttenberg (1968). The P. australis hypodermis is composed of a sclerenchymatous ring and multilayered exodermis, while in G. maxima a two-layered exodermis is present (Fig. 1). The exodermal layers of P. australis develop suberin lamellae very early, and Casparian bands are obscured close to the root tip (Soukup et al., 2002). Instant or almost instant deposition of suberin lamellae over Casparian bands seems to be a general feature of the exodermis, having also been found in other species tested to date (Peterson & Perumalla, 1984; Seago et al., 1999; Ma & Peterson, 2000). In roots of G. maxima, however, in the inner hypodermal layer, the Casparian bands and band plasmolysis remained discernible for a considerable distance behind the root tip (e.g. Fig. 2b). Later formation of complete suberin lamellae and additional cell wall material deposition released the attached plasmalemma from the cell wall of the inner exodermal layer. The outer exodermal layer seems to be impregnated mostly in its inner tangential and partially radial cell walls. The heaviest deposition of suberin was in the adjacent tangential cell walls of the hypodermal layers, and several parallel lamellae seem to be formed in this part of the cell wall. The pattern thus differed from the regular multilayered lamellae of P. australis.
In most work published to date, the localization of suberin in tissue was confirmed by staining with sudan red or fluorol yellow lipophilic dyes. However, both stains failed to detect G. maxima exodermal lipidic cell wall material, the presence of which was confirmed biochemically. The staining of the endodermis can be seen as a positive control, and the striking difference in staining demonstrates the different properties of suberized cell walls not only between species but also between the endodermal and exodermal layers of G. maxima roots (Fig. 1e,f). The limitation of histochemical detection has been noted also by De Simone et al. (2003), and was interpreted as being attributable to low amounts of suberization. However, in the current case the quality and not the quantity of suberin is the main difference between the species, as even in the very apical tissues of P. australis suberin lamellae are stainable, but they are not stainable in the basal parts of G. maxima. We can only hypothesize that differences in the composition and/or molecular and spatial arrangements of suberin within the cell wall or differences in its molecular context in the cell wall may be factors affecting lipid staining. Obviously, negative results for sudan staining should not always be regarded as decisive.
Suberin of hypodermal layers
The presence of a lipophilic (aliphatic) domain of suberin is generally connected with barrier properties (Vogt et al., 1983; Schreiber et al., 1999) described for the periderm (Groh et al., 2002), and the endodermis and exodermis (Zimmermann & Steudle, 1998; Barrowclough et al., 2000; De Simone et al., 2003). The monomeric composition of suberin significantly differed between the two species (Fig. 3, Supplementary Table S1). The ω-OH alkanoic and 2-OH alkanoic acids were the most abundant aliphatic monomers in our species. Interestingly, 2-OH alcanoic acids have been reported to be a significant monomer of suberin in maize (Zeier et al., 1999b) and, in the endodermal Casparian bands, in Clivia minimata but not in other species tested to date (Schreiber et al., 1999; Zeier et al., 1999a; Bernards, 2002). No dioic acids were detected in the hypodermal cell walls of G. maxima but were present in P. australis. α,ω-dioic acid is considered to be an aliphatic compound typical of, and unique to, suberin and was even suggested as a marker to differentiate between suberized and cutinized tissue (Matzke & Riederer, 1991). The only other known exception, in which dioic acids are absent, is the suberin of endodermal Casparian strips in C. minimata (Zeier & Schreiber, 1997), which remain in the first developmental stage, i.e. without complete suberin lamellae (Clarkson & Robards, 1975). Changes in suberin monomer composition were also found to accompany differentiation of the exodermis along the growing root. Lengthening of the aliphatic chains of major monomers was recorded for both genera. Suberin deposits with higher proportions of longer chain aliphatic monomers in older parts of the root (Fig. 4), reported also by Zeier et al. (1999b) for different developmental stages of the endodermis, thus seem to be a more general feature of suberized tissues. Another developmentally related trend, recorded only for P. australis, was an increase in the relative proportions of unsaturated fatty acids towards the base. The above-mentioned developmental changes in the composition as well as in the content of suberin were not restricted to particular developmental stages of the exodermis or to the short subapical region (‘differentiation zone’) of the root, but took place along considerable lengths of the root behind the root tip in both species.
The content of aliphatic suberin in hypodermal layers of both P. australis and G. maxima reached values comparable in order of magnitude to values measured for other plant species (for comparison, see De Simone et al., 2003; Schreiber et al., 2005a). However, considerable variation exists among tested species. In the case of Amazonian trees (De Simone et al., 2003), suberin contents measured in the first 30 mm were comparable to, or even higher than, those measured in the basal parts of the roots of our species. The more than three times higher content of aliphatics in the P. australis exodermis compared with G. maxima seems to be connected not only to a higher concentration within the cell wall material but also to a different pattern of suberin deposition and a different number of suberized exodermal cylinders. We did not find any significant difference in overall suberin content and composition between roots grown in stagnant and circulating nutrient solutions. This conclusion might be related to constitutive development of the exodermis in both species.
Barrier properties of the exodermis
The barrier properties of the exodermis have been described in the literature dealing with water transport (Ranathunge et al., 2003), ion uptake and penetration (Gierth et al., 1998), apoplastic tracers (Peterson et al., 1982; Soukup et al., 2002) and movement of plant growth regulators (Seago et al., 1999; Hose et al., 2000). The properties and development of hypodermal layers are strongly affected by environmental conditions in some species (Enstone & Peterson, 1998; Degenhardt & Gimmler, 2000; Hose et al., 2001; Soukup et al., 2004) and affect the communication of root tissues with the near rhizosphere. Barriers formed subapically in hypodermal layers of wetland plants or plants from flooded soil might, by restricting ROL, be beneficial for longitudinal oxygen transport in roots (Armstrong, 1979). They can also regulate the passive transport into and out of the root of other compounds from solution in close contact with the roots. In most wetland plants tested to date, the proportion of tissues that are not shielded from the often harsh rhizosphere conditions within flooded soil seems to be minimized by the very early development of the exodermis (Seago et al., 1999, 2000a,b). Modifications of exodermal layers even precede modifications of the endodermis and appear closer to the root tip in P. australis (Soukup et al., 2002) and G. maxima. The inverse developmental sequence has been recorded for nonwetland plants, where the exodermis develops some considerable distance behind the root tip (Ferguson & Clarkson, 1976; Perumalla & Peterson, 1986). However, even in nonwetland plants the very early differentiation of the exodermis can be induced in some plants as a consequence of cultivation in rooting medium low in oxygen (Soukup et al., 2004; Enstone & Peterson, 2005) and reversed when plants are transferred into well-oxygenated soil (Soukup et al., 2004). This seems not to be the case for G. maxima and P. australis roots, where the exodermis developed close to the tip as a constitutive feature regardless of oxygen availability and also in well-aerated soil (Soukup et al., 2002).
In the literature, the restriction of ROL and barrier formation have been linked to various features of tissues external to the aerenchyma. The dense arrangement of the hypodermal cylinder (Connell et al., 1999), thickening of hypodermal cell walls (Colmer et al., 1998b; Armstrong et al., 2000) and suberization and/or lignification (Armstrong et al., 1994a) were suggested to have these functions. In both species tested in our work, the barriers were shown, using microelectrodes (for P. australis, see Armstrong et al., 2000), to be in the suberized exodermis. As no other observed anatomical features other than suberin impregnation of the hypodermal cell wall were recorded in the short region (at c. 30 mm) of sharp ROL decrease along the root, the key role of this marked increase in setting up barrier properties seems obvious. The tracing of the location of the barrier to the suberized exodermis and not the thickened and lignified cell walls of sclerenchymatous hypodermis was further supported by periodic acid penetration tests. The thickened and lignified hypodermal cell walls of the sclerenchymatous ring of P. australis did not provide a significant barrier to diffusion of periodic acid and is also unlikely to be a barrier to diffusion of smaller molecules such as oxygen. Recently, De Simone et al. (2003) reported a correlation between suberin content and improved survival of tropical trees in flooded soil, thus confirming experimentally for the first time the connection between suberization and the barrier to ROL in roots. Our data provide further, more direct evidence supporting this conclusion.
Major differences between the ROL profile patterns along roots grown in stagnant and nonstagnant solutions were found in G. maxima, but not in P. australis (Afreen-Zobayed, 1996). The absence of statistically significant differences in suberin content and the measured initial reduction in ROL of the subapical region of roots from both treatments show that even in nonstagnant cultivation some barrier is formed in the apical region. Reasons for the basipetal increase in ROL might include some of the following. (a) The emerging laterals puncture the exodermis and open up a radial pathway across the epidermis and hypodermal layers. We can only speculate as to whether, for example, the difference in the effectiveness of resealing after the emergence of laterals might result in a difference in the spatial pattern of ROL among treatments. (b) The ROL from laterals developed in nonstagnant medium might be higher than that from laterals developed in stagnant medium. (c) The creation of the ROL barrier in the near subapical region, which is the most metabolically active part of the root, was so quick in G. maxima that it took place during measurement (within several hours) in stagnant medium, but the barrier did not form in the more differentiated distal parts of the root. The evaluation of the functional properties of the root exodermis might be affected by cultivation treatments and/or by disregarding the presence of gaps in otherwise impermeable parts of the root, which are associated with the pre-emergent stage of lateral root development, as seen in this and other studies (Armstrong et al., 2000; Soukup et al., 2002). Such interruptions are below the spatial resolution of most measurement techniques of root radial hydraulic conductivity, solute penetration and ROL, which are almost exclusively performed on those parts of the roots still bearing no laterals. These factors therefore should be taken into account.
Although suberization sufficient to create a ROL barrier appeared in the immediately subapical regions of the roots of both species, the efficiency of the exodermis in oxygen retention might be attributable in part to synergism between respiratory demand and radial diffusive resistance (Armstrong et al., 2000), an effect magnified in apical parts by high respiration rates. However, suberin content further significantly increased in the basipetal direction. A correlation between the total amount of suberin in hypodermal layers and the ability of the plant to withstand flooding (De Simone et al., 2003) thus might not always necessarily reflect only the oxygen barrier properties of the root, as the suberin content necessary to form ROL barrier is several times exceeded at some distance from the root tip, at least in the species tested. The correlation might be also related to other environmental factors of flooded soil. One of these factors might be the presence of phytotoxic compounds, which are characteristic of flooded soils (Ponnamperuma, 1984). The periodic acid penetration into root tissues across the exodermis illustrates this function of the exodermis and interspecific differences. In comparison with the tight barrier in P. australis, the penetration of the exodermis of G. maxima by periodic acid may indicate a lower selectivity of passive uptake via roots from the rhizosphere in G. maxima. Such a difference could explain the approximately three times higher accumulation of aluminium from the rooting medium in root tissues and the greater degree of middle cortex injury in G. maxima compared with P. australis (Vlkováet al., 2003). However, the causal connection requires further evidence.
In addition to interspecific differences in exodermis structure, composition and function, these comparisons demonstrate the variation in barrier permeability to different substances. Studies on other species have indicated that water intake is not prevented by exodermal suberization, for example in rice (Ranathunge et al., 2005; Schreiber et al., 2005b), and appears to contrast with tight ROL barrier properties (Colmer et al., 1998, 2003a). There may be several reasons for this. One possible reason is that the aerated hydroponics used in most studies (e.g. Ranathunge et al., 2005; Schreiber et al., 2005b) did not, in the species used, induce ROL barriers as strong as those developed under wetland conditions or in deeply hypoxic stagnant media. Another may relate to the positions along the root that were measured; the work on hydraulic conductivity has been neglectful of more basal regions. Recently, Garthwaite et al. (2006) observed that the induction of a ROL barrier with a stagnant nutrient solution did not significantly affect the hydraulic conductivity of Hordeum marinum roots, but the barrier to ROL was not as complete as those found here or previously in rice in stagnant anaerobic media. In contrast, Groh et al. (2002) demonstrated that the diffusional permeance of oxygen in suberized phellem of several woody species was at least an order of magnitude lower than that for water. The mechanism underlying the difference in permeability is not clear, but molecular size may play a role in transport. The water molecule, with a mean van der Waal diameter of c. 280 pm (Finney, 2001), might permeate the apoplast more easily than the oxygen molecule, with a van der Waal diameter estimated at 428 pm.
Schreiber et al. (2005b) suggested that gross values of suberin content in hypodermal layers do not necessarily reflect the barrier properties of impregnated cell walls, and this suggestion is supported by the current data. The spatial arrangement of suberin deposition within the cell wall and cylinder of the exodermis should be further studied at sufficient resolution to provide the data required to elucidate the transport and pathways of various solutes across such apoplastic barriers and to explain the reported interspecific differences.