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- Materials and Methods
Methane (CH4) production and emission from wetlands are controlled by factors such as water table, temperature, soil redox potential and vegetation (Schimel, 1995; Grünfeld & Brix, 1999; Dinsmore et al., 2009). Plants affect CH4 emissions from wetlands in several ways. They provide litter and root exudates as a carbon source for methanogenic Archaea, and transport of gases via root and shoot aerenchyma frequently dominates wetland O2 and CH4 fluxes (Joabsson et al., 1999; Brix et al., 2001; Ström et al., 2003). The primary function of aerenchyma in wetland plants is to transport oxygen from the atmosphere to the roots (Armstrong, 1979). This process results in radial oxygen loss (ROL) from the roots to the rhizosphere (Colmer, 2003), which can reduce CH4 production and increase CH4 oxidation. Thus, in some cases, the presence of aerenchymatous vegetation reduces CH4 emission from wetlands (Grünfeld & Brix, 1999; Roura-Carol & Freeman, 1999; Dinsmore et al., 2009; Fritz et al., 2011).
Aerenchyma, on the other hand, can also provide a conduit for CH4 from the rhizosphere to the atmosphere, bypassing the oxidizing soil layers (Chanton & Dacey, 1991; Beckett et al., 2001; Colmer, 2003). This pathway can result in greater CH4 emissions from areas inhabited by aerenchymatous plants (Sorrell & Boon, 1994; Schimel, 1995; Shannon et al., 1996; Greenup et al., 2000; Ström et al., 2006). Schimel (1995), in particular, found that the composition of the plant community, based on cover of aerenchymatous plants, is a better predictor of CH4 flux from arctic wet meadow tundra than both water table and CH4 production rates. Greenup et al. (2000) likewise found substantial differences in CH4 emissions within the same peatland, with six times higher emissions from plots with the aerenchymatous plant Eriophorum vaginatum compared with plots without this plant, correlating well with below-ground biomass. Furthermore, they placed glass tubes in the soil to mimic the conduit effect and exclude the organic material supply of aerenchymatous plants, and elevated CH4 emissions were also observed here. Plant-mediated gas transport contributes up to 95% of the total CH4 flux from wetlands (Schütz et al., 1989; Chanton & Dacey, 1991; Grünfeld & Brix, 1999), depending on specific physicochemical conditions and intrinsic differences in species' ability to transport CH4. For example, the presence or absence of pressurized convective gas flow (Brix et al., 1992) can both increase and decrease CH4 emissions, as it can accelerate internal CH4 flux (Sorrell & Boon, 1994), but also increase rhizosphere oxidation and activity of methanotrophic bacteria (Grünfeld & Brix, 1999).
Whilst it seems likely that the large variation between species in plant-mediated CH4 emissions is a function of differences in airspace structure and tissue permeability to gases, understanding of this is hampered by contradictory evidence in the literature regarding the location and significance of the main transport resistances. The high porosities often found in roots of wetland graminoids of 30–50% (Justin & Armstrong, 1987) do not in themselves seem to be rate-limiting for plant gas transport, but it is difficult to generalize about which aspects of root and shoot morphology and physiology predominantly limit fluxes, based on the existing literature. Pioneering work by Sebacher et al. (1985) was able to categorize the CH4 transport capacity of a large number of species based on their growth form and CH4 transport capacity, but limiting factors for CH4 transport were not directly tested. In Carex-dominated wetlands, Morrissey et al. (1993) observed reduced CH4 emissions after induced stomatal closure, and concluded that stomatal conductance is an important control of CH4 flux. Their findings are supported by Schimel (1995), who found elevated CH4 emission from Carex aquatilis after experimentally removing the leaf blade barrier by clipping the leaves. On the other hand, the transfer of gases between the rhizosphere and the root aerenchyma has also often been proposed as a rate-limiting step for CH4 transport in graminoids (Schimel, 1995; Beckett et al., 2001). Many wetland plants have extensive barriers to gas diffusion in the exodermal root tissue (Colmer, 2003; Garthwaite et al., 2008). The barrier is a strategy for conserving oxygen within roots in anoxic soils, but also restricts CH4 flux in the opposite direction (Beckett et al., 2001). Mathematical models on the root-rhizosphere scale have also highlighted root wall permeability as an important factor limiting plant CH4 fluxes (Beckett et al., 2001; Segers & Leffelaar, 2001). Still other studies have identified high resistances to diffusion at the root–shoot junction (Groot et al., 2005).
To be able to quantify the effect of aerenchymatous plants on CH4 emissions, it is important to understand which specific anatomical and morphological factors govern the transport of CH4. Given the divergent literature on factors limiting CH4 fluxes in wetland plants, it is surprising that very few studies have attempted to use manipulative experiments to test predictions regarding rate-limiting resistances to transport. Thus, the objective of this study was to make a detailed empirical investigation of the factors controlling CH4 transport in Juncus effusus. We chose this as a representative, model species as it is now globally widespread in wetlands in all temperate biomes, and is typical of the graminoid taxa that dominate most minerotrophic wetlands. Specifically, we aimed to test the predictions implicit in mathematical modelling (Beckett et al., 2001), that root characteristics such as branching patterns and barriers to diffusion (Colmer, 2003) are the predominant factors controlling diffusive CH4 transport in wetland plants.
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We used plant characteristics and shoot and root manipulations to clarify details about CH4 transport in J. effusus. Unlike some wetland plants, such as Typha sp. and Phragmites australis (Brix et al., 1992), J. effusus has no pressurized gas flow in its aerenchyma (Yavitt & Knapp, 1998; H. Brix & B. K. Sorrell, unpublished). Hence the transport of CH4 in J. effusus can be described with diffusion models similar to those developed for oxygen transport in plants (Armstrong & Beckett, 1987; Sorrell, 1994; Beckett et al., 2001). Some root manipulations significantly affected CH4 transport in this study, whereas shoot manipulations had little effect. Only removing root material with permeable surfaces from the plants reduced the CH4 transport, indicating that the amount of root surface permeable to gases is the most important limiting factor for CH4 transport through J. effusus. We used length of the fine roots rather than their surface area in our model, because of the difficulty in root scans of accurately distinguishing and estimating the permeable surface area of the complex, fine laterals, and length apparently functioned well as a proxy for permeable area, given the high explanatory power of the model.
The majority of the root surface permeable to CH4 consisted of the laterals of the fine roots, with a small contribution of the tips of the coarse roots, leading to strong correlations between fine root development and CH4 transport in our study. The low permeability to gas exchange over the root surfaces occurs because many wetland plants, primarily monocotyledons, develop a barrier to ROL along their roots to avoid oxygen deprivation and also to exclude phytotoxins (Armstrong, 1979). This barrier, consisting of lignin and suberin (Armstrong & Beckett, 1987; Visser et al., 2000; Garthwaite et al., 2008), reduces the loss of oxygen from the internal air spaces to the reducing rhizosphere and enhances the diffusion of oxygen within the root towards the apex (Armstrong & Beckett, 1987; Sorrell, 1994; Colmer, 2003). The early experiments and diffusion models summarized by Armstrong (1979) suggested that the greatest resistance to oxygen transport in plants is in the root/rhizosphere transition zone. More recently, Visser et al. (2000) found that, specifically in J. effusus, roots have a relatively strong constitutive barrier to ROL in their basal parts, but the distal parts just a few cm behind the apex are more permeable. ROL in J. effusus increases with increasing oxygen demand in the root environment, but quickly becomes saturated at higher demands, suggesting that the area permeable to oxygen quickly becomes the limiting factor (Sorrell, 1999). These results agree with the methylene-blue experiment and the flux results presented here, and suggest that the sites of CH4 uptake are the same as for oxygen leakage in reducing environments. Several authors have previously presented the rhizosphere–root interface as the limiting resistance for CH4 transport in some graminoid species (Chanton & Dacey, 1991; Schimel, 1995), as well as model simulations (Beckett et al., 2001), but to our knowledge this is the first time that this concept has been supported by experimental manipulation.
Shoot manipulations, by contrast, had little effect on CH4 transport in our study. If the ability of CH4 to escape from the plant was limited by a high resistance in the shoot walls, cutting the shoots and leaving the cut ends open would result in an increase in CH4 transport as observed for Carex species (Schimel, 1995; Kelker & Chanton, 1997). Likewise, the cutting of the shoots to increasingly lower heights, 150, 100 and 50 mm, with subsequent sealing, did not alter the CH4 transport significantly. From these results we conclude that the shoot tissues are very permeable to CH4, and that all the CH4 escapes from the plant at 50 mm from the base or below. CH4 release is also independent of stomatal conductance in Oryza sativa, where CH4 escapes through micropores in the basal parts (Nouchi et al., 1990). Basal CH4 release also occurs in Pontederia cordata and Sagittaria lancifolia, but through petioles (Harden & Chanton, 1994). In contrast to our study, Sebacher et al. (1985) found very low CH4 release rates in the field from J. effusus, as well as other aerenchymatous plants, and suggested that the relatively small transport rates were restricted by the impermeable outer layers of the shoots, but they also recognized that differences in root structure may be involved. Our study strongly suggests that it is root parameters, including access to sediment methane, not shoot permeability, that control CH4 transport in J. effusus. By inducing stomatal closure or by cutting off the leaf blades with or without subsequent sealing of the cut ends, some studies have found significant resistance in the above-ground parts of Carex sp. (Morrissey et al., 1993; Schimel, 1995; Kelker & Chanton, 1997). In our study, the effect of stomatal closure was not addressed specifically, but since virtually all the CH4 escapes through the base of the shoot, it is unlikely that above-ground parts or stomatal physiology of J. effusus offer any significant resistance to CH4 transport. The high correlation between shoot surface area and CH4 transport was a general effect of plant size on CH4 transport rather than any direct control of CH4 release by shoot morphology, and underlines the importance of combining modelling work with experimental manipulations. The differences in controlling factors for CH4 transport among wetland graminoids might originate from differences in plant architecture as suggested by Schimel (1995). The morphology of J. effusus differs from taxa such as Carex sp., having vestigial leaves and erect leafless shoots that constitute the main photosynthetic organs.
The only shoot manipulations that significantly decreased CH4 transport were when all shoots but one were cut and sealed at the base, allowing CH4 to escape only via a single shoot, after which CH4 transport decreased on average to 56% of that of the intact plant. It seems plausible that the decrease in transport rate from this shoot manipulation was caused by limited connectivity between roots and stems through the rhizome of J. effusus, and that not every root is connected to every shoot. The formation of a new shoot in J. effusus is coupled to the formation of new roots at the elongated rhizome. These newly formed roots and shoots might be interconnected, but connection to the older roots and shoots might be limited. Examples of studies where the rhizome is shown to limit CH4 transport in graminoids are sparse, but Groot et al. (2005) found high resistance in the root–shoot transition of rice tillers when the roots and shoots had been cut. This implies that the connectivity in the rhizome might be limiting for the transport of CH4 if some of the shoots in a tussock are damaged or waterlogged such that gas phase continuity with the rhizome is compromised.
The results presented here will be useful in the evaluation of CH4 emissions in the field. Modelling of CH4 emissions from different land use types requires information of the abiotic and biotic factors controlling the emissions. Walter & Heimann (2000) developed a process-based model to quantify CH4 emissions from natural wetlands. The model estimates the CH4 concentration in a one-dimensional soil profile, and estimates the flux of CH4 from molecular diffusion, ebullition and transport through aerenchymatous plants. For the latter the rooting depth of plants is used as a site-specific model parameter. Locating the specific sites for CH4 uptake in the root system, as was done here for J. effusus, will possibly help to improve these kinds of models. The amount of permeable root surface coinciding with high concentrations of soil water CH4 would then be the sites of particular importance.
We conclude that CH4 in J. effusus is taken up by the laterals of the fine roots and the apical part of the coarse roots, and the same barriers reported for radial oxygen loss going from the atmosphere to the rhizosphere also apply to CH4 diffusion in the opposite direction. Our results empirically confirmed what is predicted by the models of Beckett et al. (2001), that is, that the area of permeable root surface, derived from the proxy of root length of fine roots and number of coarse roots, is the most important controlling factor for CH4 transport in plants. Furthermore, we found evidence of limited connectivity in the rhizome of J. effusus, but further studies are needed to clarify its significance in this species and other similar graminoids. In such plants, we can now confidently confirm that roots provide the primary resistance to diffusion and limit plant CH4 transport, while the shoot resistance is insignificant, with all CH4 escaping from the basal parts of the shoot. Interspecific differences in sites for CH4 uptake by plants, as well as plant-specific controlling anatomical factors for CH4 transport, are important in future studies of aerenchymatous plants in their natural habitat explaining the microsite variability in CH4 emissions.