- Top of page
- Materials and Methods
Low oxygen (O2) concentrations can impede root growth in waterlogged soils. Adaptations to overcome low soil O2 include biochemical, physiological, morphological and anatomical traits, with root aeration being critical for plant survival (Armstrong, 1979; Colmer & Voesenek, 2009). Traits important for internal root aeration (and hence tolerance to waterlogging) are development of aerenchyma and formation of a barrier to radial O2 loss (ROL) (Armstrong, 1979; Colmer, 2003). Aerenchyma formation in primary cortical tissues has been extensively studied in monocotyledonous species (Drew et al., 2000; Seago et al., 2005; Fagerstedt, 2010). In dicotyledonous species, however, in addition to primary aerenchyma, secondary aerenchyma is sometimes produced from a phellogen: internally as phelloderm (secondary cortex) or externally as phellem (Jackson & Armstrong, 1999; Stevens et al., 2002).
Secondary aerenchyma in the form of aerenchymatous phellem has been reported in several dicotyledonous wetland species when under flooded conditions (Justin & Armstrong, 1987; Lempe et al., 2001). This type of aerenchyma consists of a white spongy tissue, with large volumes of intercellular gas spaces that form externally from a phellogen and is homologous to cork (Shimamura et al., 2010). Under O2-deficient conditions, aerenchymatous phellem has been observed in outer tissues of lower parts of stems, upper parts of roots and even in nodules; examples are found in Viminaria juncea (Walker et al., 1983), Neptunia plena (James et al., 1992), Lotus uliginosus (James & Sprent, 1999), soybean (Glycine max; Shimamura et al., 2002, 2003, 2010; Thomas et al., 2005), Sesbania species (Shiba & Daimon, 2003) and Lythrum salicaria (Stevens et al., 1997). In Glycine max, aerenchymatous phellem in the stem base and tap root contributes to internal O2 transfer to roots and nodules (Shimamura et al., 2010). In Lythrum salicaria, the roots can form aerenchymatous phellem (Stevens et al., 1997), and aerenchymatous phellem in stems of flooded L. salicaria has been shown to provide a pathway for O2 transport between the shoot base and roots (Stevens et al., 2002). However, although removal of a ring of phellem at the stem base disrupted the continuity of the phellem and the concentration of O2 in gas samples from roots declined by 30%, this had no impact on the total dry mass of flooded plants (Stevens et al., 2002). It would be useful to have further information on the function of root aerenchymatous phellem and its role in O2 transport for waterlogging tolerant species, especially for those that are of interest to agriculture.
Melilotus siculus , common name messina, is an annual legume native to saline marshy areas of the Mediterranean Basin (Marañòn et al., 1989). This species is of great interest to agriculture for development as an annual pasture legume on waterlogged and saline land (Nichols et al., 2008). An earlier study evaluated 19 Melilotus species and found M. siculus was very tolerant to low O2 in the root-zone, with no reduction in root or shoot mass after 4 wk treatment with stagnant deoxygenated agar solution (Rogers et al., 2008). Melilotus siculus developed relatively high (around 14%) lateral root porosity (Rogers et al., 2008). Our preliminary observations were that M. siculus develops an aerenchymatous phellem layer when grown in stagnant nutrient solution, and particularly interesting is the extensive phellem development along the upper portions of the main root axis (tap root) and the base of some lateral roots that emerge from the main root. The present study evaluated the function of root phellem for O2 transport and the role of this aerenchymatous tissue in the waterlogging tolerance of M. siculus. In addition, we have determined the origin of the phellem and possible routes whereby atmospheric O2 could diffuse via the plant to the rhizosphere.
- Top of page
- Materials and Methods
In contrast to the primary cortical aerenchyma of roots, aerenchymatous phellem has rarely been studied, despite its prevalence in roots of several wetland species (Jackson & Armstrong, 1999). The present study of aerenchymatous phellem in roots of M. siculus, a species that occurs in wetlands (Marañòn et al., 1989) and tolerates low O2 in the root-zone (Rogers et al., 2008), demonstrated that: (i) root dry mass is not reduced by low O2 concentrations, although rooting depth is constrained; (ii) the roots have a high porosity owing to the presence of aerenchymatous phellem and honeycomb schizogenous (expansigenous) aerenchyma in the primary cortex [correction added after online publication 8th February 2011: in the preceding text, parentheses have been removed for clarity.]; (iii) the root aerenchymatous phellem increases in volume in response to low O2 treatment; (iv) the aerenchymatous phellem enables transport of O2 from the shoot base to roots, and even a small proportion of hypocotyl phellem exposed to air can be a major entry point for O2 to diffuse into the root system. The cross-sectional area of phellem is particularly high for the main root and hypocotyl of stagnant-treated plants, and disruption of the continuity of the hypocotyl phellem impairs O2 transport into roots. These morphological and anatomical adaptations almost certainly contribute to the high waterlogging tolerance of M. siculus.
Our data for M. siculus indicate a clear association between the amount of aerenchymatous phellem in roots and increased root porosity. Growth of M. siculus under stagnant conditions caused an increase in the amount of phellem (e.g. threefold increase in the cross-sectional area of the root phellem at the widest point) and a 1.2 to 2.5-fold increase in root-porosity (for roots covered with phellem). Roots of M. siculus with phellem had porosities up to 25%, a value comparable to many other wetland species, although not in the highest range (Justin & Armstrong, 1987; Colmer, 2003). As few gas spaces were observed in the stele, we would expect porosity of the phellem alone to be much greater than 25%. The main root axis of plants grown in aerated solution had lower porosity than stagnant-treated roots, particularly towards the root tip, as the phellem layer only extended c. ¼ down for aerated roots, whereas it covered c. ¾ of the length of stagnant roots. Exposure of soybean to 10 d flooding (water level 2–3 cm above the soil surface) also stimulated development of aerenchymatous phellem in the upper 8 cm portion of the tap root, which had a porosity of 22% under flooded conditions but only 0.4% under drained conditions (Thomas et al., 2005). Submergence of the stem base can also induce formation of aerenchymatous phellem in stems. Flooding of Lythrum salicaria to a depth of 4–8 cm above the soil surface for up to 73 d caused enlargement of the basal area of stems associated with an increase in aerenchymatous phellem (Lempe et al., 2001), so that stem bases of L. salicaria with phellem can have porosity values > 30% (Stevens et al., 1997).
Transverse sections of M. siculus roots showed that aerenchymatous phellem contains significant amounts of gas-filled spaces (Fig. 4) that function as aerenchyma to enable internal O2 diffusion (Figs 5, 8). Anatomical studies of aerenchymatous phellem in stems and roots of other species has also revealed extensive gas spaces in this type of phellem (Walker et al., 1983; Stevens et al., 1997; James & Sprent, 1999; Lempe et al., 2001; Shiba & Daimon, 2003; Thomas et al., 2005). Furthermore, the present anatomical observations for M. siculus showed that aerenchyma was present in the phellem of roots from both stagnant and aerated treatments, so the increased overall main root porosity between stagnant vs aerated treatments is therefore likely caused mainly by differences in the amounts of root phellem per total root volume (i.e. increase in thickness of the layers of aerenchymatous phellem).
Figure 8. Pathways whereby atmospheric O2 may reach rhizospheres via internal gas-space of Melilotus siculus. *– and 1°cortex when still present in younger plants; aer, aerenchymatous. Assuming hypocotyl and base of main root are not submerged.
Download figure to PowerPoint
The importance of the aerenchymatous phellem for internal transport of O2 from the shoot to the root was demonstrated for M. siculus, both qualitatively (methylene blue dye) and quantitatively (root-sleeving O2 electrodes) and correlated with anatomical findings. Interestingly, the function of phellem in hypocotyl-to-root O2 transport was the same for both aerated and stagnant-treated plants. For plants from both treatments transferred into an O2-free rooting medium, disrupting the hypocotyl phellem diminished O2 supply to lateral roots, with measured values at lateral root tips approaching zero within 30–40 min after phellem removal. Based on the fast declines in ROL from M. siculus roots in anoxic media after phellem removal, it is clear that phellem is important for internal root aeration. Furthermore, submerging this phellem, or covering it in nontoxic cream impaired O2 transport into roots. However, exposure of only 10% of its length to the air was hardly distinguishable from full exposure in terms of effects on root ROL. Surface gas-films were also prominent on submerged stems and on hypocotyl and root phellem, so it seems likely that with the hypocotyl and shoot bases submerged, O2 could then diffuse via gas-films (Colmer & Pedersen, 2008) as well as internally from the emergent shoots. It was also interesting to find chloroplasts, particularly in the hypocotyl and upper tap root (Fig. 4b); photosynthesis in these regions might also help to maintain root O2 concentrations during shallow flooding. Nevertheless, our key results demonstrate that even a partially emergent hypocotyl may well be the main region for atmospheric O2 to enter the submerged root system (Fig. 6) and that this could be particularly advantageous during events of shallow flooding with a fluctuating water table.
The present work on M. siculus extends an earlier experiment in which phellem on the stem of Lythrum salicaria was removed with a consequent 30% decline in root O2 concentrations (Stevens et al., 2002). However, the decline in O2 is clearly more severe in the present study of M. siculus with removal of hypocotyl phellem. Other previous studies on functioning of hypocotyl and/or root phellem have focused on soybean, a species that is sensitive to flooding. Shimamura et al. (2003) covered the stem phellem of waterlogged soybean plants with petroleum jelly and this treatment decreased the root dry mass of waterlogged plants by 70% (compared with controls without petroleum jelly), presumably owing to a restriction of O2 transport via aerenchyma in the hypocotyl phellem. A subsequent study by Shimamura et al. (2010) used 18O2 tracer gas to show that the stem phellem was an important entry point for O2 transport to roots of soybean in waterlogged pots. However, as soybean takes several days to form aerenchymatous phellem in response to flooding, Shimamura et al. (2010) suggested that formation of aerenchymatous phellem under aerobic conditions could improve tolerance of soybean to flooding events, as plants suffered from flooding stress until sufficient aerenchymatous phellem developed. M. siculus forms aerenchymatous phellem more readily than soybean as phellem formed on the hypocotyl and roots of plants in aerated solution culture in the present experiments, albeit to a smaller extent than for stagnant-treated plants (Table 1). More information is required on the ease with which aerenchymatous phellem forms in response to wet conditions in M. siculus; a key question is whether it forms in plants grown in drained field conditions, or whether in such conditions only nonaerenchymatous corky phellem forms.
The role of phellem for providing O2 to nodules during waterlogging and/or submergence was not investigated in the present study, but is likely to be another important function of the phellem found on root and stem nodules of legumes (Walker et al., 1983; James & Sprent, 1999; Shimamura et al., 2002, 2003). Phellem has been observed on nodules at the root–hypocotyl junction of field-grown M. siculus (P. Nichols, pers. comm.), and further work is needed to determine if phellem assists N2 fixation in waterlogged soils by maintaining O2 (and N2) supply to nodules.
Our present findings clearly demonstrate the importance of an aerenchymatous phellem layer for providing gas-space continuity for internal O2 movement from shoot and hypocotyl to roots of a waterlogging tolerant legume. The aerenchymatous phellem formed in the hypocotyl and roots of M. siculus increases in response to stagnant solutions (and waterlogged soils), indicating the adaptive value as this tissue would enhance internal O2 movement to distal portions of the root system when in waterlogged soil.