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- Materials and Methods
Plant species can have distinct effects on ecosystems, often linked conceptually to the quality of plant litter, with important implications for ecosystem function (Paul & Clark, 1996; Binkley & Giardina, 1998). Of particular interest are plant effects on soil carbon (C) process rates, which are mediated by the quality and quantity of detrital inputs and detrital losses resulting from microbial decomposition. While the biotic and abiotic controls on net primary production (NPP) and hence detritus production are well understood, and detailed models exist to describe the disappearance of surface detritus during decomposition, there is a poor understanding of how litter quality and quantity interact to regulate soil C formation (Scott, 1998; Giardina et al., 2001; Joffre et al., 2001).
These knowledge gaps may be attributed to a reliance upon species differences in tissue quality (e.g. pine vs maple litter) to understand decomposition processes (Melillo et al., 1982; Berg & Lundmark, 1985; McClaugherty & Berg, 1987; Aber et al., 1990; Magill & Aber, 1998), and a greater focus on mass loss than on understanding the fate of detrital residues. Further, ecosystem models have focused on the quantity and dynamics of above-ground litter, but mineral soil C dynamics may be correlated most strongly with below-ground inputs (Zak & Pregitzer, 1998; Zak et al., 2000; Giardina et al., 2004), of which the quality is measured less frequently. In order to improve understanding of how plant inputs and tissue quality influence soil C formation, model systems are needed that: simultaneously vary lignin quantity or quality without changing other plant properties that can influence decomposition rates; and allow for tracing the fate of C from plants into soils. The genetic alteration of lignin biosynthesis in aspen (Li et al., 2003) coupled with 13C isotope techniques (Amundson et al., 1998; Collins et al., 1999; Liu et al., 2003; Loya et al., 2003; Pataki et al., 2003; Giardina et al., 2004; Hobbie et al., 2004; Steinmann et al., 2004; Heath et al., 2005) offers a powerful approach for understanding how plants influence ecosystem processes.
Significant progress has been made in understanding lignin biosynthesis in both herbaceous and woody species through the characterization of the genes and enzymes regulating this complex process (Higuchi, 1997; Whetten et al., 1998). Genetic transformation of lignin biosynthesis in trees could therefore be used to better understand how altering specific ‘tissue quality’ genes impacts ecosystem processes. In aspen trees, stem lignin is derived primarily from syringyl (S)–guaiacyl (G) monolignols, which are synthesized from coniferyl and sinapyl alcohols. By modifying genes that encode for enzymes along the lignin biosynthetic pathway, genetic alteration of both lignin quantity and S : G ratios can now be achieved in aspen. Specifically, transgenic aspen produced by antisense suppression of the gene encoding 4-coumarate:CoA ligase (4CL) results in up to a 45% reduction of stem lignin concentrations with an associated 15% increase in stem cellulose concentrations (Hu et al., 1999), while overexpression of the gene encoding coniferaldehyde 5-hydroxylase (CAld5H) increases stem lignin S : G ratio. Simultaneous suppression of 4CL and overexpression of CAld5H can lead to a 38–52% reduction in stem lignin concentrations and a 22–64% increase in the stem lignin S : G ratio (Li et al., 2003).
For our research, we used three transgenic lines of aspen (Populus tremuloides; Table 1) derived from a well-characterized, naturally occurring aspen clone from the northern Great Lakes region (line 271 – the control line in this study). Two of the modified lines express reduced stem lignin concentrations with or without increased cellulose concentrations or elevated S : G lignin ratios: (1) low lignin concentration (35%), normal cellulose concentration, normal S : G lignin ratio (line 23; ‘low lignin/normal cellulose’); and (2) low lignin concentration (50%), high cellulose concentration (30%), slightly elevated (20%) S : G lignin ratio (line 141; ‘low lignin/high cellulose’). Because syringyl-rich lignin is substantially easier to separate from cellulose during pulping for paper production than guaiacyl-rich lignin (Li et al., 2000), with important economic and ecological benefits, we examined a third modified line that expresses normal lignin and cellulose concentrations but a 150% increase in the S : G ratio of lignin (line 93; ‘high S : G lignin’; Table 1).
Table 1. Aspen (Populus tremuloides) stem tissue chemistry (Hu et al., 1998) of transgenic and control plants used in glasshouse mesocosm experiment
| ||Plant line number|
|4CL|| ||√||√|| |
|CAld5H||√|| ||√|| |
|Stem lignin (%)||21.1||14.4||10.7||22.2|
|S : G of stem lignin|| 5.5|| 2.2|| 2.7|| 2.2|
|Stem cellulose (%)||44.7||44.8||53.3||41.4|
In order to understand the impacts of altered lignin biosynthesis on resource capture and growth we measured total plant leaf area, specific leaf area, leaf-level physiology and biomass accumulation for each aspen line over 8 months of growth in a glasshouse. After 8 months, we harvested plants and measured above- and below-ground biomass in order to understand the impacts of altered lignin biosynthesis on biomass partitioning. To understand plant effects on soils, we measured soil microbial biomass at harvest, and, because aspen were grown in a C4 prairie soil, we used 13C/12C isotope methods to measure how altered lignin biosynthesis in aspen affected soil C formation.
In designing this study, we developed several hypotheses about the effects of altered lignin biosynthesis on: the lignin content of leaves and fine roots (Hypothesis 1); plant growth and above- and below-ground allocation (Hypothesis 2); and given Hypotheses 1 and 2, below-ground C cycling and soil C formation (Hypothesis 3). For Hypothesis 1, we predicted that alteration of lignin quality or quantity in the stems of aspen plants would result in parallel changes in leaf and fine root lignin – the tissues that dominate detritus return to ecosystems in early stages of stand development in forests. For Hypothesis 2, we predicted that in low lignin lines, the reduced C cost of lower lignin production would result in more C being available for plant growth (Herms & Mattson, 1992) such that low lignin aspen (lines 23 and 141) would accumulate more biomass than control (271) and high S : G lignin (93) lines (Hypothesis 2a). We further predicted that high S : G lignin aspen (93) would grow more slowly than other lines (Hypothesis 2b) because of the higher C costs of syringyl production (Amthor, 2003). For Hypothesis 3, we predicted that the combination of lower lignin concentration in roots (Hypothesis 1), which would lead to faster root decomposition rates but greater root-derived detritus to soil (Hypothesis 2a), would lead to little difference in rates of soil C formation between low lignin (23 and 141) and control (271) lines (Hypothesis 3a). Further, because the syringyl component of plant lignin is more reactive than the guaiacyl component, high syringyl lignin should decompose faster than lignin with a normal structure (Hedges et al., 1985; Chefetz et al., 2000). As a result, we predicted that aspen with high S : G lignin (Hypothesis 1) combined with reduced below-ground inputs (Hypothesis 2b) would result in the lowest formation rates of aspen-derived soil C (Hypothesis 3b).
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- Materials and Methods
The role of individual genes in the regulation of plant–environment interactions is an emerging field within ecology (Jackson et al., 2002). Important advances have included identification of genes regulating disease resistance (Bent et al., 1994; Bent, 1996), ecophysiology (Martienssen, 1998; Byrne et al., 2000; Jackson et al., 2002; Bergmann et al., 2004) and plant nutrition (Gallais & Hirel, 2004; Franco-Zorilla et al., 2004). While the focus of these studies has been on the genetic basis for plant adaptations to the environment, the effects of individual genes on ecosystem processes have received much less attention (Pilate et al., 2002). By combining 13C isotope techniques with aspen trees expressing modifications to lignin biosynthesis, we were able to examine how alteration of individual genes affected plant growth and physiology, and in turn below-ground ecosystem processes.
Lignin biosynthesis and tissue chemistry
We hypothesized that alterations of stem lignin concentrations would result in changes in the lignin concentration of leaves and fine roots (Hypothesis 1). However, no differences among transgenic aspen in stem lignin concentrations were observed in fine roots and leaves (Table 2), indicating that transgenic alterations to plant stem tissue may not result in parallel changes to leaves and fine roots. It is well established that lignin formation and resulting structure varies widely across cell types within a plant (Whetten et al., 1998) and that controls on lignin biosynthesis may be tissue specific (Lagrimini et al., 1997). Further, lignin biosynthesis may occur along several pathways (Whetton & Sederoff, 1995; Whetten et al., 1998), with molecular and enzymatic regulation of lignin biosynthesis in one tissue type (e.g. stem wood) potentially differing from that in another tissue (e.g. leaves and fine roots).
Stem lignin concentrations for the four lines examined here were higher than previously reported values for transgenic aspen with altered lignin biosynthesis (Hu et al., 1998, Table 1). However, we did not remove bark tissue from our stem samples before lignin analysis, while Hu et al. (1998) removed bark before stem tissue analyses. Because bark is rich in polyphenolic compounds, our lignin numbers may be higher than if only wood had been examined.
Across lines, leaf lignin concentrations averaged 21 ± 0.5%. This value is similar to lignin concentrations previously observed in field grown trees and now widely incorporated into ecosystem models simulating northern forests (Running & Gower, 1991, lignin concentration fixed at 22%). These values are slightly lower than lignin concentrations reported in leaf litter studies (Melillo et al., 1982; Aber et al., 1990; Berg & Ekbohm, 1991), though higher concentrations (25% to 30%) in leaf litter studies may relate to leaf mass loss resulting from retranslocation or leaching of labile compounds out of leaves during and after senescence (Pregitzer et al., 2006).
Our root lignin concentrations averaged 42% ± 0.9, which is on the high end of published estimates for broadleaved species. Camiréet al. (1991) reported fine root lignin concentrations of 25.4% for a hybrid poplar (Populus nigra L. × Populus trichocarpa Torr & Gray), which matches mean root lignin concentrations identified in reviews of root tissue quality (Silver & Miya, 2001; broadleaf mean lignin concentration = 28%; Chen et al., 2002, broadleaf mean = 22%; Gordon & Jackson, 2000, temperate broadleaf mean = 26%). Other studies, however, have reported root lignin concentrations of 35% (Puttsepp, 2004) and 49% (Hendricks et al., 2000). Differences across studies could result from differences in root age or size, growth environment, methodology, or a combination of these factors.
Lower C concentrations in the stem tissue of low lignin lines (23 and 141) compared with control aspen may relate to the fact that lignin has a high C content, such that a reduction in lignin concentrations in stem tissue could lead to a reduction in C concentrations. In line with the lack of difference in lignin concentrations of leaves and fine roots, we observed no differences in the C concentrations of leaves and fine roots among the four lines of aspen. Low lignin aspen (23 and 141) did have higher concentrations of N in leaf tissues compared with control aspen, and for low lignin/high cellulose (141) aspen, higher N concentration of leaves were associated with higher Amax values (Table 3), perhaps indicating that higher N concentrations in low lignin lines were not caused by luxury consumption but rather by increases in chlorophyll. By contrast, the significantly higher N concentrations in leaves, roots, and stems of high S : G lignin plants compared with control plants was likely a result of luxury consumption of N in high S/G plants (93), as shown by overall low rates of light saturated photosynthesis compared with the other lines.
Plant physiology, growth and carbon allocation
Across plants and lines, Amax values of transgenic and control aspen from this study are within the range of Amax rates reported for aspen in the Great Lake region (Whitehead & Gower, 2001), and they overlap with ambient CO2 treatment Amax values reported by Takeuchi et al. (2001) for field-grown, mid-canopy leaves of control (271) trees (10–20 µmol CO2 m−2 s−1) at the Aspen FACE facility in Rhinelander, WI, USA. The range of Amax for upper canopy leaves for the 3-yr-old field-grown aspen were c. 3 µmol CO2 m−2 s−1 higher than the mid-canopy leaves, but photosynthetic photon flux density (PPFD) reaching upper canopy leaves of field grown trees (maximum light of > 1500 µmol PPFD m−2 s−1) is higher than maximum PPFD encountered in our glasshouse (maximum light of c. 600 µmoles PPFD m−2 s−1).
Our hypothesis that low lignin/normal cellulose (23) and especially the low lignin/high cellulose plants (141) would allocate more C to growth (Hypothesis 2a) was not supported by our results. There are a limited number of studies that have attempted to quantify the effects of altered lignin biosynthesis on the growth of aspen, but results have been conflicting. In an earlier glasshouse study, Hu et al. (1999) observed larger leaves and longer internodes for transgenic aspen expressing a 45% reduction in stem lignin concentration and a 15% increase in stem cellulose concentration compared with the unaltered control aspen. Notably, these authors did not examine below-ground biomass. By contrast, our results show that significant reductions in stem lignin concentrations in Populus may not substantially or even positively alter leaf area, height growth, above-ground or total biomass accumulation, particularly if the S : G ratio of lignin is also increased. Pilate et al. (2002) found no differences in height growth between 4-yr-old field grown control aspen and aspen with slightly reduced stem lignin concentrations, though this study did not examine increased S : G ratio plants. Given the greatly reduced growth performance of high S : G lignin plants (93), the modest increase in S : G ratio in low lignin/high cellulose plants (141) may explain the reduced growth performance of this line.
In Hypothesis 2b, we predicted that high S : G lignin (93) aspen would show reduced growth compared with the other lines because of higher C costs for syringyl production (Amthor, 2003). The results of this study support our hypothesis as aspen with high S : G lignin had significantly reduced height, leaf area, and above and below-ground biomass. Syringyl monolignols have a 17% higher molecular mass than guaiacyl and contain 132 g C mol−1 while guaiacyl monolignols contain 120 g C mol−1. Lignin biosynthesis from syringyl monolignols through the most efficient biosynthetic pathway has a higher glucose requirement than lignin biosynthesis from guaiacyl monolignols (Amthor, 2003). Overall, however, these differences are too small to account for the observed reduction in growth for high S : G plants. While genetic alteration of lignin quality in high S : G lignin plants (93) resulted in a large reduction in leaf area, SLA and photosynthetic rates, with impacts on growth, the molecular basis for these changes remain unclear.
Carbon allocation patterns to above and below-ground tissues differed across lines, though we are uncertain of the mechanism controlling these differences and whether changes represent the direct or indirect effects of altered lignin biosynthesis. Nonetheless, genetically related changes in above- and below-ground plant growth have important implications for industrial plantings of reduced lignin plants. In earlier studies, differences in root architecture in conifers (Coutts et al., 1999) and reduced biomass partitioning to roots in Populus (Harrington & DeBell, 1996) increased susceptibility to wind throw damage. In our study, if observed reductions in biomass partitioning to roots in low lignin lines persists in field settings, the commercial benefits of reduced lignin in stem wood and higher stem wood production could be offset by the risk of increased loss to wind throw. We caution that C allocation to roots and root to shoot ratios can change with stand development, and C allocation to roots in pots may differ from allocation under field conditions. Clearly, longer-term studies are needed to confirm whether observed differences among control and low lignin lines persist, increase or disappear with time.
Soil microbial biomass and carbon formation
Across plants, the values of microbial biomass in transgenic and control aspen from this study are on the high end of the range of published microbial biomass values for soils from the Konza Prairie where our soils were collected. Vinton & Burke (1997) reported microbial biomass values for field moist Konza soils of 0.24 mg biomass C g−1 soil, while Zak et al. (1994) reported 1.47 mg biomass C g−1 soil, for field moist soils. Following establishment of prairie vegetation, Baer et al. (2003) reported microbial biomass C-values that ranged from 1.78 to 2.84 mg biomass C g−1 soil for field moist Konza prairie soils. Though differences or similarities are difficult to interpret, the mean values we observed for Konza soils in our glasshouse experiment may indicate that below-ground C supply was higher in our mesocosm pots than in field environments where moisture and nutrient availability are likely to be lower. Given the optimal growth conditions for plants and microbes, differences are not surprising. Overall, patterns of microbial biomass across lines could in part be explained by a significant relationship between total root C and microbial biomass C (MB = Root C × 0.22 + 1.5; R2 = 0.25, P = 0.04), indicating that the mechanisms driving reduced microbial biomass may include reduced below-ground inputs that relate to reduced root biomass.
We originally hypothesized that low lignin aspen would have higher below-ground production than control aspen (Hypothesis 2a) but combined with high microbial respiration of labile low lignin roots (Hypothesis 1), these lines would accumulate similar amounts of new soil C (Hypothesis 3a). However, altering lignin biosynthesis in stem tissue did not affect the lignin concentrations of leaves and fine roots, and the low lignin lines did not allocate more biomass below-ground than control lines. Instead we observed modestly lower root biomass C in low lignin aspen (23 and 141), which may have contributed to lower accumulation rates of aspen-derived soil C relative to control aspen. The reason for the observed shift in biomass partitioning in low lignin lines is unclear. However, the changes we observed in biomass partitioning, below-ground allocation, and processing of below-ground inputs indicate that ecosystem responses to genetic alterations are more complex than could be predicted from information on the function of single genes. For example, in line with hypothesis 3b, increasing the S/G of lignin (line 93) reduced growth, leaf area and physiology, below-ground biomass allocation, and ultimately rates of soil C formation in soils (Figs 1–3).
Overall, the detrital decomposition pathways leading to soil C formation are very complex (Stevenson, 1994), with nonlignin-based pathways potentially exerting an important influence on formation rates. Variation in rates of soil C formation was most strongly related to total root C (soil C formation = 0.44 × (total root C) − 0.48; R2 = 0.36; P < 0.01), indicating that below-ground inputs (rhizodeposition, fine root and mycorrhizal turnover) can exert an important influence on soil C formation, independent of tissue quality effects. While most of the variation in this relationship is not explained, these results are in line with previous field studies documenting positive correlations between soil C and root biomass (Jobbagy & Jackson, 2000), fine root turnover (Pregitzer et al., 1995) or rhizosphere C flux (Giardina et al., 2004).
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We combined 13C isotope-based techniques with modified aspen to examine, within a common genome, the effects of altered stem lignin concentration and/or altered lignin quality on plant performance, including leaf photosynthesis, above- and below-ground biomass growth, and leaf area growth. We also examined how changes in the plant tissue chemistry and performance led to changes in soil properties including microbial biomass, soil surface CO2 efflux and organic C formation in soil.
Changes in stem lignin concentration in the two low lignin lines (23 and 141) did not reduce lignin in fine roots or leaves, did not affect above-ground growth, but compared with the control line (271), did reduce biomass C in roots and formation rates of new soil C. By contrast, increasing stem lignin S : G ratio had a strong negative effect on leaf area, height growth and biomass accumulation, and negatively affected soil C formation. Taken together, our results indicate that trees expressing reduced lignin content could simultaneously provide comparable growth rates while reducing the economic and environmental costs of lignin removal in the pulping process. By contrast, increasing the S : G ratio of lignin, while also of economic and environmental interest because of increased ease of lignin removal, appears to negatively affect growth of this species. Changes in susceptibility to pest and pathogens or climate events were not examined here. Clearly, any benefits of reduced lignin in wood will need to be weighed against potential reductions in rates of soil C formation and, because of changes in biomass partitioning to roots, potentially increased susceptibility to wind throw.
Overall, we show that these C4 grassland soil mesocosms provide a powerful tool for assessing and screening above and below-ground plant performance and for testing basic questions in plant biology. We caution that our short-term results need to be validated in longer glasshouse studies and in field trials across a range of soil types.