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a School of Natural Resources & Environment, Univ. of Michigan, Ann Arbor, MI 48109
b Dep. of Earth, Ecological and Environmental Sciences, Univ. of Toledo, Toledo, OH 43606-3390
* Corresponding author (drzak{at}umich.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glucosidase, N-acetylglucosaminidase, cellobiohydrolase, phenol oxidase, and peroxidase enzymes, which are involved in plant and fungal litter decomposition. Microbial biomass and respiration were not significantly altered by elevated CO2 and O3. Cellobiohydrolase activity significantly increased under elevated CO2; however, this response was eliminated by elevated O3. N-acetylglucosaminidase activity also increased under elevated CO2, but elevated O3 did not significantly alter this response. We found no difference in the metabolism of amino acids, organic acids, and simple carbohydrates, suggesting our experimental treatments did not alter the use of these substrates by soil microorganisms. Our analysis indicates that changes in plant growth in response to elevated CO2 and O3 alters microbial metabolism in soil.
Abbreviations: FACE, free-air CO2 and O3 enrichment • MBc, microbial biomass C • MUB, methylumbelliferone • PCA, principal components analysis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ozone is a greenhouse gas that is accumulating in the lower atmosphere, and elevated O3 has the potential to alter soil microbial communities through its influence on plant litter production. Elevated O3 can decrease photosynthesis (Pye, 1988; Coleman et al., 1995a) and net C gain (Skärby et al., 1987; Reich et al., 1990) in many woody plants, which should modify litter production. For example, high levels of O3 often decrease root growth to a greater extent than foliage or stem growth (Manning et al., 1971; Blum and Tingey, 1977; Hogsett et al., 1985). Elevated O3 also can modify the biochemical composition of fine roots, wherein starch and soluble sugar concentrations can decline following exposure to elevated O3 (Andersen et al., 1991). It is possible that decreased growth and biochemical changes under elevated O3 could potentially mitigate increases in plant growth and changes in tissue biochemistry resulting from elevated atmospheric CO2. However, we do not understand how these atmospheric gases will interact to alter the input of organic substrates to soil, which could potentially modify microbial activity in soil.
In a recent experiment, we observed that elevated CO2 significantly increased the biomass of living and dead fine roots, but the magnitude (83–113%) of this response differed between tree species and was eliminated by elevated O3 (King et al., 2001). Because microbial growth is constrained by the type and amount of organic substrates entering soil (Babiuk and Paul, 1970; Smith and Paul, 1990), the aforementioned changes in belowground plant growth could potentially alter both substrate availability and microbial activity. The presence of substrates can induce the synthesis of specific extracellular enzymes (Paul and Clark, 1996), and we reasoned that the activity of key enzymes involved with plant litter decomposition should respond to changes in fine-root litter under elevated CO2 and O3. We also reasoned that changes in microbial activity under elevated CO2 and O3 would differ among plant taxa. To test these ideas, we measured extracellular enzyme activities and the metabolism of labile, root-derived substrates beneath contrasting temperate tree species grown under experimental CO2 and O3 treatments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microbial Biomass Carbon and Respiration
Two 30-g subsamples of nonrhizosphere soil from each ring section were used to determine MBc and respiration using the CHCl3 fumigation-incubation technique. One subsample was fumigated for 24 h with ethanol-free chloroform in a vacuum desiccator, while the second subsample was incubated without chloroform (control). Following the 24-h incubation, the desiccator containing the fumigated samples was evacuated eight times to remove remaining chloroform. Each fumigated sample was inoculated with 0.5 g of the corresponding control sample. Each fumigated and control sample was sealed in a 1-L mason jar and incubated at 25°C for 14 d. Following the 14-d incubation, the headspace gas of the jars was analyzed for CO2 using a Tracor 540 gas chromatograph (Tremetrics Corp., Austin, TX). Microbial biomass was determined by subtracting the amount of CO2 in the control sample from that in the fumigated sample. The difference was divided by a correction factor (Kc = 0.41) to estimate MBc (Voroney and Paul, 1984). Microbial respiration was measured as the increase in CO2 in unfumigated samples over the 14-d incubation.
Enzyme Assays
Soil microorganisms synthesize extracellular enzymes based on the concentration of substrates present in the soil (Paul and Clark, 1996). Therefore, changes in plant-derived substrates entering soil under elevated CO2 and O3 should alter the activity of enzymes used in their degradation. We used methylumbelliferone (MUB) linked substrates to determine the rate at which the microbial community in nonrhizosphere soil metabolized six classes of plant-derived compounds (sensu Sinsabaugh et al., 1999). We assayed the activities of 1,4--glucosidase, cellobiohydrolase, phosphatase, 1,4-ß-N-acetylglucosaminidase, and leucine-aminopeptidase.
We suspended 1.0 g of nonrhizosphere soil from each composite sample in 60 mL of 50 mM acetate buffer (pH 5.0). The slurry was mixed with a tissue homogenizer (Polytron Devices Inc., Paterson, NJ) and diluted with additional buffer to 125 mL. The suspensions were stored in 125-mL Nalgene screw-cap bottles for up to 30 min prior to analysis. Sixteen replicates of three separate enzyme assays were conducted on individual 96-well microplates. Each plate also contained eight replicates of a blank, a 4-MUB standard, a negative control, and a quench standard. Plates were incubated at 25°C and 25 µL of 200 mM NaOH was added to each well to terminate the enzymatic reactions. Fluorescence resulting from the cleavage of 4-MUB from utilized substrate was determined with an f-Max fluorimeter (Molecular Devices Corp., Sunnyvale, CA). Excitation energy was 355 nm and emission was measured at 460 nm. Enzyme activities are reported as nmol 4-MUB g-1 h-1.
We used colorimetric assays to determine the activity of phenol oxidase and peroxidase, both of which oxidize phenols and contribute to lignin degradation. A 25 mM L-3,4-dihydroxyphenylalanine (L-DOPA) solution was prepared in acetate buffer to assay the activity of these enzymes. The procedure for measuring the activity of these enzymes was similar to that described above. Clear microplates (LabSystems; Helsinki, Finland) were used for the colorimetric assay, and each contained sixteen replicates of each soil. There were eight blank replicates for each soil (soil with no substrate), as well as eight replicates of a reference standard. For the peroxidase assay, 25 µL H2O2 was added to each well. Following an 18 h incubation, absorbance was read on an EL-800 plate reader (Biotek Instruments Inc., Winooski, VT) at 450 nm. Activity was reported as µmol L-DOPA converted per gram per hour. The results of all enzymatic assays are expressed on a dry weight basis.
Metabolism of Root-Derived Substrates
We used a subset of BIOLOG GN substrates (BIOLOG, Hayword, CA) that are found as constituents of root exudate to assess their metabolism in rhizosphere soil (sensu Campbell et al., 1997; Table 2). Two 10-g samples of rhizosphere soil were diluted in 100 mL of a 0.85% (w/v) NaCl solution and placed on an electric shaker for 30 min at 250 rpm. Each sample underwent a second dilution; 10 mL from each sample were placed into 100 mL of NaCl solution. We did not adjust our dilutions for differences in inoculum density, because an initial survey indicated that microbial biomass was equivalent across treatments in both rhizosphere and nonrhizosphere soil (D.R. Zak, unpublished data, 1998). From the second dilution, 150 µL was used to inoculate individual wells of BIOLOG GN plates, which were subsequently incubated at 25°C for 72 h; absorbance at 595 nm was measured following inoculation and after 72 h (EL-800; Biotek Instruments, Winooski, VT). At each measurement time, the absorbance value of the control well was subtracted from the absorbance value of all other wells. Using these corrected values, we subtracted the initial absorbance values from those after the 72-h incubation to calculate overall color development, a measure of substrate metabolism.
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= 0.05. We used an identical ANOVA model to test for significant differences in the use of root-derived substrates on BIOLOG GN plates. In addition, we conducted a principal components analysis (PCA) to ordinate CO2, O3, and species treatment combinations by microbial growth on amino acids, organic acids, and simple carbohydrates. Separate PCAs were performed on the corrected absorbance values obtained in May, July, and October.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Enzyme Activities
The effect of elevated CO2 on phosphatase activity in nonrhizosphere soil varied significantly among species; however, date, CO2, and O3 were not significant as main effects. Phosphatase activity was not influenced by CO2 in nonrhizosphere soil beneath aspen and aspen–birch, but activity was significantly lower beneath aspen–maple (Fig. 1
; Table 3).
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-glucosidase activity did not differ in the ambient and elevated CO2 treatment. However, activity in July was significantly higher in the elevated CO2 treatment (Fig. 3A)
. There were no significant effects of time or O3 on -glucosidase activity, nor was there a significant interaction between these factors and species (Table 3). Species also was not significant as a main effect.
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On each sampling date, cellobiohydrolase activity was significantly greater under elevated CO2 compared with the rate at ambient CO2 (Table 3; Fig. 3C). Ozone and CO2 also interacted to significantly influence cellobiohydrolase activity. Activity under elevated CO2 and ambient O3 was 43.4% greater than activity under elevated CO2 and elevated O3 (Fig. 4) . Carbon dioxide, O3, and species were not significant as main effects (Table 3).
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Metabolism of Root-Derived Substrate
Metabolism of labile, root-derived substrates in rhizosphere soil (Table 2) was little affected by CO2, O3, species, and their interactions (data not shown). However, time was a significant main effect in our ANOVA, with greater metabolism of most substrates occurring in July. Nonetheless, we did find that acetic acid, citric acid, and malonic acid were used to a significantly greater extent in May than the other sampling dates. Principal components analysis of the metabolism (color development) of root-derived substrates on BIOLOG GN plates provided no separation among CO2 and O3 treatments for the aspen and aspen–maple ring sections on all sampling dates. However, in October, PCA of growth on root exudates showed marginal separation along PC 1 for rhizosphere soil collected beneath the aspen–birch species combination grown under elevated CO2 (Fig. 5A)
. In this analysis, PC 1 accounted for 36% of the total variance and PC 2 accounted for 20%. The loadings computed for each substrate showed that L-serine, L-aspartic acid, L-asparagine, and 
-amino-butyric acid weighed most heavily on the positive segment of PC 1. Citric acid also received a high score on this axis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many studies have observed increases in belowground plant productivity under elevated CO2 (Poorter, 1993; Rogers et al., 1994; Curtis and Wang, 1998), a finding which has been hypothesized to foster a larger or more active microbial population in soil (Zak et al., 2000b). However, our results, along with numerous others (Jones et al., 1998; Kampichler et al., 1998; Niklaus, 1998; Hungate et al., 2000), have found no significant response of microbial biomass to elevated CO2. A review of 47 published reports on responses of soil C and N cycling to elevated CO2 concluded that microbial biomass can exhibit both large increases and declines beneath woody taxa (i.e., 52% decline to a 121% increase), even though microbial respiration generally increases in response to elevated CO2 (Zak et al., 2000b). After 2 yr of CO2 and O3 enrichment of the atmosphere, we were not able to detect a significant response of microbial biomass and respiration to elevated CO2, nor did we find significant species-specific responses to either CO2 or O3. This could be due, in part, to the relatively young age of the plants (i.e., 2 yr old) and the fact that they likely had not fully colonized both above- and belowground growing space. Several observations made in the subsequent growing season support this idea: elevated CO2 significantly increased fungal activity and the metabolism of 13C-cellobiose and 13C-N-acetylglucosamine; moreover, the response of microbial metabolism to CO2 and O3 was greatest beneath aspen and aspen–birch (Phillips et al., 2002). Given the aforementioned results, soil microbial activity appears to be responding in concert with plant growth in our experiment, even though we initially could not detect a significant increase in microbial biomass.
Cellulose comprises up to 40% of plant tissue (Wilke et al., 1983), and greater root-litter production in response to our experimental treatments should increase cellulose input to soil. In our study, cellobiohydrolase activity increased under elevated CO2, but this increase was only marginally significant 2 yr after initiation of our experimental treatments (Fig. 2D), a response that was consistent in subsequent growing seasons (H. Chung and D.R. Zak, unpublished data, 2001). Concurrent research at our FACE experiment has found a 113% increase in fine-root biomass for aspen, and an 84% increase in fine-root biomass in the aspen–birch species combination (King et al., 2001). These results suggest a greater amount of cellulose entered soil under elevated CO2, and this is consistent with the finding that elevated CO2 significantly increased the metabolism of 13C-cellobiose by soil fungi in our experiment (Phillips et al., 2002). In combination, increases in root-litter input, greater rates of cellobiohydrolase activity, and more rapid metabolism of labeled cellobiose all indicate that elevated CO2 has enhanced cellulose degradation in soil. In a similar study, Mayr et al. (1999) found a significant increase in cellobiohydrolase activity beneath plants growing under elevated CO2, whereas Dhillion et al. (1996) reported a nonsignificant increase in the activity of this enzyme. These results all suggest an overall increase in cellulose metabolism by microbial communities in nonrhizosphere soil beneath plants exposed to elevated CO2.
In our experiment, greater cellobiohydrolase activity under CO2 was mitigated by elevated O3 (Fig. 4), a response consistent with that of fine-root litter production (King et al., 2001). These observations suggest that changes in root-litter production altered substrate availability and hence the activity of this soil enzyme. The fact that elevated O3 also eliminated enhanced rates of 13C-cellobiose metabolism under elevated CO2 further supports this contention (Phillips et al., 2002). We are not aware of other studies that have directly evaluated changes in root-litter production and cellobiohydrolase activity under elevated O3. Nonetheless, ample evidence indicates that elevated O3 can decrease photosynthesis and impair stomatal function (Tjoelker et al., 1995), which can lower the allocation of C to root growth (Scagel and Andersen, 1997). Moreover, Kress and Skelly (1982) reported a 41% decrease in biomass of Acer saccharum seedlings, and Wang et al. (1986) reported a 17% decrease in biomass of Populus tremuloides seedlings subjected to elevated O3. A decrease in plant growth and allocation to roots under elevated O3 should, in turn, lead to a decline in cellulose input to soil. Such a response would be consistent with our observations of fine-root litter production, cellobiohydrolase activity, and the metabolism of 13C-cellobiose (King et al., 2001; Phillips et al., 2002).
N-acetylglucosaminidase is a soil enzyme involved in chitin degradation (Alexander, 1977), and we observed a significant increase in the activity of this enzyme under elevated CO2 (Fig. 2C). Similar results were found in alpine grassland in which N-acetylglucosaminidase activity under elevated CO2 increased by nearly 30% (Mayr et al., 1999). Although we presently do not have data on specific fungal communities in our experiment, an increase in N-acetylglucosaminidase activity under elevated CO2 may result from a greater production of fungal cell wall litter (Miller et al., 1998). This would be consistent with other studies that have observed greater mycorrhizal infection (%), and higher levels of mycorrhizal and saprophytic fungal biomass under elevated CO2 (Rillig et al., 1998; Klironomos et al., 1996). N-acetylglucosaminidase in our study displayed a nearly two-fold increase with elevated CO2 (Fig. 2C), and the magnitude of this response was similar in the presence and absence of elevated O3 (i.e., no CO2 x O3 interaction). The relative response of N-acetylglucosaminidase to our experimental treatments was similar to that of 13C-N-acetylglucosamine metabolism, which significantly increased under elevated CO2 (Phillips et al., 2002). In combination, these results indicate that chitin degradation was enhanced by elevated CO2, but further work will be required to determine if increases in fungal-litter production are responsible for this observation.
Leucine aminopeptidase is an enzyme involved in the degradation of proteins, and we found no significant difference in the activity of this enzyme under ambient and elevated CO2 (Table 3, Fig. 2A). This result is consistent with Mayr et al. (1999), who found leucine aminopeptidase was unaffected by elevated CO2. We also found no significant influence of CO2, O3, or species combination on the activity of phenol oxidase, an enzyme involved with lignin degradation (Table 3). In the context of our experiment, this implies that protein and lignin inputs from root mortality were unchanged across all treatments. Clearly, chemical analyses of root tissue are needed to support to this contention.
Assaying the metabolism of simple substrates is one approach of assessing physiological capabilities of microbial communities under experimental conditions (Zak et al., 1994). Carbon dioxide, O3, sampling date, and species had no influence on the metabolism of amino acids, organic acids, and simple carbohydrates by microbial communities inhabiting rhizosphere soil. These results are consistent with elevated CO2 studies in Mediterranean (Dhillion et al., 1996) and model tropical ecosystems (Insam et al., 1999), which observed no change in labile substrate metabolism under elevated CO2. In contrast, Mayr et al. (1999) found differences in the metabolic capabilities in an undisturbed, late-successional alpine grassland exposed to elevated CO2. In our experiment, rhizosphere microbial communities equivalently metabolized a range of amino acids, carbohydrates, and organic acids found as constituents of root exudates across all treatments. However, the activity of enzymes involved with plant and fungal cell wall degradation responded significantly to our experimental treatments in nonrhizosphere soil and were consistent with the responses of fine-root litter production.
In summary, we found that greater belowground plant growth under elevated CO2 significantly increased the activities of cellobiohydrolase and N-acetylglucosaminidase. In contrast, elevated O3 counteracted the CO2 effect on cellobiohydrolase activity, but elevated O3 had no significant influence on N-acetylglucosaminidase activity. Because these enzymes play a key function in the degradation of plant and fungal litter in soil, their response to our experimental treatments may presage a change in decomposition and the flow of substrates through soil food webs. This is consistent with greater rates of 13C-cellobiose and N-acetylglucosamine beneath plants growing under elevated CO2 (Phillips et al., 2002). Although the tree taxa we studied broadly differ in life-history traits, we have no initial evidence to suggest that changes in belowground growth in response to elevated CO2 and O3 will elicit fundamentally different responses by soil microbial communities (Phillips et al., 2002). Clearly, further work would be required to understand the long-term implication of our observations on the microbial processing of organic matter and its storage in soil. Notwithstanding this caveat, our observations suggest that changes in plant growth induced by high concentrations of CO2 and O3 in the Earth's atmosphere can mediate physiological changes in soil microbial communities, which, in turn, have the potential to alter soil C and N cycling in forests. Understanding how changes in plant growth in response to elevated CO2 and O3 impact the long-term dynamics of C and N in soil remains an important challenge.
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ACKNOWLEDGMENTS |
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Received for publication March 9, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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