Experimental laboratory studies

The earliest laboratory study reporting an emission of CH4 from leaves was conducted in the late 1950s at the Academy of Sciences of Georgia (Tbilisi) on emissions of volatile organic compounds (VOCs) from leaves of willow and poplar tress (Sanadze and Dolidze, 1960). In that study, mass spectrometric analysis was used to scan the volatile emissions of leaves incubated in 1.5 litre glass containers. Based on their mass spectra they concluded that plants released CH4 as well as ethane, propane, isoprene and several other VOCs, but their subsequent work focused on isoprene and provided no explanation for their detection of CH4 or any follow-up studies on CH4 release.

It was not until the study of Keppler et al (2006), described above, that the scientific community began to examine the question of vegetation as a direct source of CH4 in some detail. The first subsequent experimental study by Dueck et al (2007) grew six plant species hydroponically from seed for nine weeks inside a hermetically sealed plant growth chamber provided with 13C-labelled CO2 in order to create isotopically labelled plant material. Shoots of four species were then sealed into a continuous-flow gas exchange cuvette with a visible light (300 or 600pmol m-2 s-1) and a corresponding air temperature of

25 or 35°C, respectively. They observed CH4 emissions -10ng g-1 h-1 to 42ng g-1 h-1, some 18 times lower than the average rates reported by Keppler et al (2006), and which were not statistically different from zero. In a further experiment, they found no release of 13C-labelled CH4 into the sealed growth chamber from the labelled plant material over six days. Both experiments led to their conclusion that there was no evidence for substantial aerobic CH4 emission by terrestrial plants.

Beerling et al (2008) measured CH4 and CO2 exchange rates of corn (Zea mays) and tobacco (Nicotiana tabacum) using a high-accuracy gas analysis system inside a controlled environment room. They found no evidence for aerobic CH4 emissions during alternating three-hour periods of 700pmol m-2 s-1 visible light and darkness over a 12-hour experiment at 25°C. They concluded that there was no evidence for a link between aerobic CH4 emissions from leaves and photosynthetic or respiratory metabolism but noted that an emission might be linked to a non-enzymatic process driven by non-photosynthetic radiation such as ultraviolet (UV) wavelengths which would not have been detected by their experimental system. It was not until researchers used irradiation containing a UV wavelength component, that several observations of CH4 release by vegetation under aerobic conditions were demonstrated (McLeod et al, 2008; Vigano et al, 2008).

Vigano et al (2008) measured the effect of UV radiation and elevated temperature on the emission of CH4 from dry and detached fresh leaves from over 20 species and plant structural components including pectin, lignin and cellulose. They used a range of lamps to provide exposures to variable amounts of UV-A (320-400 nanometres (nm)), UV-B (290-320nm) and UV-C (<290nm) radiation, mostly at least five times higher than ambient spectrally unweighted UV irradiances. They demonstrated that CH4 emissions were linearly related to UV irradiance and were almost instantaneous after irradiation, indicating a direct photochemical process. By conducting one experiment with dry grass for 35 days and other experiments using both ambient and CH4-free air, they were able to provide some evidence that the CH4 originated within the plant material and emissions were not explained by desorption from plant surfaces. They also used a dry leaf of 13C-labelled wheat (Triticum aestivum) from the study of Dueck et al (2007) and were able to demonstrate that 13C-labelled CH4 was emitted at 32ng g-1 leaf dry weight h-1 under an unweighted UV irradiance of three times the typical tropical conditions. Their CH4 emissions rates from UV-A and UV-B lamp irradiation of plant material ranged from zero to 393ng g-1 dry weight h-1 (with all but one value for cotton flowers being below 200ng g-1 dry weight h-1). The highest emissions were detected at UV levels that exceeded typical ambient levels but the study clearly indicated a mechanism for aerobic CH4 release from plant material under some experimental conditions.

McLeod et al (2008) also performed experiments to expose detached fresh leaves to UV radiation from filtered lamps and pectin-impregnated glass fibre sheets to both lamps and natural sunlight. They found a linear response of CH4

emission from pectin, up to 750ng g-1 h-1, with each lamp type and tested a range of common and idealized spectral weighting functions. They identified a function that decayed one decade in 80nm wavelength, which gave a significant linear regression between weighted UV and CH4 for all lamp sources and sunlight. These irradiances, which included sunlight in Edinburgh during September (Figure 6.2), were within the range of ambient UV

Figure 6.2 (a) Methane production from citrus pectin at 30°C showing linear regression with ultraviolet irradiance weighted with the inset spectral weighting function, using lamps filtered with cellulose diacetate (CA), a UV-opaque filter or sunlight in Edinburgh (55°55'N, 3°10'W) between 6 and 21 September 2006; (b) Equipment used for the temperature-controlled laboratory irradiation of pectin-impregnated glass fibre sheets; (c) Equipment used for the temperature-controlled exposure of pectin-impregnated sheets to natural sunlight

Source: (a) adapted from McLeod et al (2008)

Figure 6.2 (a) Methane production from citrus pectin at 30°C showing linear regression with ultraviolet irradiance weighted with the inset spectral weighting function, using lamps filtered with cellulose diacetate (CA), a UV-opaque filter or sunlight in Edinburgh (55°55'N, 3°10'W) between 6 and 21 September 2006; (b) Equipment used for the temperature-controlled laboratory irradiation of pectin-impregnated glass fibre sheets; (c) Equipment used for the temperature-controlled exposure of pectin-impregnated sheets to natural sunlight

Source: (a) adapted from McLeod et al (2008)

exposures, especially in tropical regions. Methane emissions from pectin were virtually eliminated by chemical removal of methyl ester groups from the pectin molecule or by adding a scavenger of singlet oxygen to the pectin. The latter observation was the first indication that reactive oxygen species (ROS) might be involved in a mechanism for aerobic CH4 production in vegetation. Their experiments also demonstrated that UV irradiation of dry pectin not only produced CH4 but also ethene, ethane and CO2 and the study included UV irradiation of freshly detached leaves of tobacco (Nicotiana tabacum) that released CH4 (and also ethene and ethane) at a rate of 12ng g-1 leaf dry weight per hour over 45 hours. Although these studies were performed using pure pectin or detached fresh leaves they clearly demonstrated a mechanism for aerobic CH4 production driven by levels of UV radiation, including sunlight, within the ambient range.

Methane emissions under aerobic conditions were investigated in detached leaves and stems of 44 indigenous species of the Inner Mongolian Steppe using gas-tight serum bottles by Wang et al (2008). The study included ten herbaceous hydrophytes (wetland-adapted plants) from low-lying areas and 34 xerophytes (arid-adapted plants) including shrubs and herbs from upland areas. Plants were sampled in the early morning, separated into stems and leaves and samples sealed in triplicate serum bottles for incubation in CH4-free air and in darkness for 10-20 hours at 20-22°C. Gas samples were withdrawn (and replaced with CH4-free air) by syringe and the CH4 flux calculated from the initial rate of change of CH4 concentration. The study found that nine species emitted CH4 in the range 0.5-13.5ng g-1 d.wt. h-1 but 80 per cent of the species tested emitted no detectable CH4. The 20 per cent proportion of species emitting CH4 were about the same for hydrophytes, xerophytes, C3 plants and C4 plants but whereas 78 per cent of shrubs emitted CH4, only 6 per cent of herbaceous plants did so. They observed the highest emission rates of 6.8-13.5ng CH 4 g 1 d.wt. h 1 in two hydrophytes - Glyceria spiculosa and Scirpus yagara - and these emitted from stems and not from detached leaves. The xerophytes examined included seven out of nine shrub species that emitted CH4 from detached leaves but not stems, while none of the herbaceous xerophytes emitted CH4. The authors also examined the carbon isotope ratio (S13C value) of the emitted CH 4 in order to distinguish between a plant and a soil-derived source.

Kirschbaum and Walcroft (2008) performed laboratory studies to investigate whether reported CH4 emissions might have been caused by CH4 desorption from sample surfaces after prior exposure to higher concentrations and to examine CH4 emissions from living detached leaves and intact plant materials. They tested detached leaves of wormwood (Artemesia absinthum), yarrow (Achillea millefolium), dandelion flowers (Taraxacum officinale), broadleaf (Griselina littoralis), five finger (Pseudopanax arboreus) and a mixture of local grasses for six days sealed inside 5.7 litre Plexiglas chambers exposed indoors to low visible (and no UV) light (5pmol quanta m-2 s-1 from fluorescent lamps) and flushed at the start with CH4-free air. They also used intact seedlings of corn (Zea mays) grown in pots containing vermiculite as an inert rooting medium. Measured rates of CH4 emission were zero or very small ranging from -0.25 ± 1.1ng CH4 kg-1 d.wt. s-1 for Z. mays to 0.1 ± 0.08ng CH4 kg-1 d.wt. s-1 for mixed detached grasses. These values are much lower than those reported by Keppler et al (2006). As organic materials have a high adsorption capacity for CH4 and its desorption was a possible experimental artefact that might explain observed vegetation emissions. Kirschbaum and Walcroft (2008) also conducted experiments with stacks of cellulose filter papers (as organic adsorbers). These were exposed first to ambient CH4 concentration and then sealed inside chambers for six days and the time-course of CH4 release determined. They found no significant desorption of CH4 and concluded that desorption is not a quantitatively important artefact contributing to observed aerobic CH4 fluxes.

Experiments on CH4 emissions from plants were also conducted by Nisbet et al (2009) who examined genome sequences of plant material and found no evidence that enzymes necessary for classical methanogenesis pathways found in microorganisms were present in plants. They conducted experiments that demonstrated the influence of CH4 dissolved in soil water (described below) and also measured the release of CH4 from plant material enclosed in flasks. They grew thale cress (Arabidopsis thaliana) and the green alga (Chlamydomonas reinhardtii) on an agar medium under aseptic conditions in 2 litre flasks and found no increase in CH4 concentration above ambient with a throughflow of 5mL min-1. They also examined detached leaves of Z. mays and whole plants of rice (Oryza sativa) grown on vermiculite in closed 15 litre glass flasks under low levels of visible light (cool-white fluorescent lamps, 180pmol m-2 s-1) and found no detectable CH4 emissions. Their conclusion from these experiments was that plants grown under controlled conditions in the laboratory under artificial light are not capable of producing CH4.

The effects of temperature and UV radiation on non-plant material and pectin were also demonstrated by Bruhn et al (2009). They exposed detached leaves of six species, fruit tissues and purified pectin inside glass vials to a range of temperatures in darkness, to visible light (400-700nm photosynthetically active radiation (PAR) at 400pmol photons m-2 s-1 and to UV-A and UV-B radiation from two types of lamp. They found that detached green leaves of Betula populifolia released CH4 under PAR irradiation at 31.7ng g-1 d.wt. h-1 at 4°C over 24 hours and 4.5ng g-1 d.wt. h-1 at 30°C throughout one week. Dry pectin also released CH4 at 80°C in darkness at rates comparable to those observed by Keppler et al (2006) but also at similar rates at a much lower temperature of 37°C when dissolved in water. At a high temperature of 80°C, all tested plant material released CH4 but with two orders of magnitude variation between species.

Bruhn et al (2009) also found that CH4 emission from twigs of Picea abies and from pectin was stimulated by UV radiation and the effect of UV-B wavelengths was greater that UV-A (Figure 6.2). The UV-B irradiance had a linear relationship with CH4 emission, corresponding to the observations of

McLeod et al (2008). By digesting pectin with the enzyme pectin methyl esterase prior to temperature or irradiation treatments, they demonstrated a reduction in CH4 emissions thus supporting the observations of Keppler et al (2006) and McLeod et al (2008) that methyl groups of pectin are a source of CH4 from vegetation under aerobic conditions.

Qaderi and Reid (2009) recently reported effects of temperature, UV-B radiation and water stress on CH4 emissions (and plant growth and CO2 exchange) from the leaves of six crops: faba bean (Vicia faba), sunflower (Helianthus annuus), pea (Pisum sativum), canola (Brassica napus), barley (Hordeum vulgare) and wheat (T. aestivum). They grew one-week-old seedlings for one week in controlled environment growth chambers under two temperature regimes (24°C day/20°C night and 30°C day/26°C night), three levels of UV-B radiation using filtered lamps (zero, ambient, enhanced) and watered either to excess (well watered) or to wilting point (water stressed). Emissions from freshly detached leaves into CH4-free air inside plastic syringes were measured over the range 57-210ng CH4 g-1 d.wt. h-1. Significantly higher CH4 emissions were observed from plants subject to higher temperature (1.14 times higher) or water stress (1.21 times higher) compared to plants grown under lower temperature or watered to excess. The zero and enhanced levels of UV-B increased CH4 emissions compared to the ambient UV-B treatment. However, unlike previous studies (McLeod et al, 2008; Vigano et al, 2008; Bruhn et al, 2009) in which emissions were concurrent with UV exposure, the measurements of Qaderi and Reid (2009) were after cessation of the stress treatments, including UV, and may therefore represent different aspects of the plant response. They also found that CH4 emissions from attached leaves of P. sativum were 1.89 times higher than for detached leaves and that emissions increased with incubation time over four hours, suggesting that CH4 was being produced by the plants and not simply diffusing from CH4 stored in the leaves.

Although some of the studies outlined above clearly demonstrated that UV irradiation and elevated temperature can lead to CH4 formation in plant foliage and from pectin, further understanding of the process was only revealed by studies using stable isotopes and biochemical analyses described later in this chapter.

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