1. Introduction: Globally, atmospheric concentrations of CO 2 are rising, and are expected to increase forest productivity and carbon storage. However, forest response to atmospheric CO 2 enrichment has long been thought to be controlled by nitrogen (N) availability. In particular, CO 2 -mediated increases in forest productivity, and corresponding increases in N demand have been predicted to exacerbate any existing N limitation within an ecosystem. When nutrients are limiting, the immobilization of N in plant biomass or soil carbon may provide a negative feedback to plant growth by further decreasing soil N availability, and eventually limit positive productivity responses to elevated CO 2. Luo et al. (2004) have termed the process by which elevated [CO 2 ] causes the available N in an ecosystem to be allocated to long-lived biomass pools (pathway 1) or immobilized to increased soil carbon stocks (pathway 2), progressive N limitation (PNL). Thus, in order to predict the role of rising [CO 2 ] on future carbon storage in forested ecosystems, it is necessary to first determine whether a forested ecosystem is limited by the availability of N. This requires the addition of excess N, where predicted responses include: 1. Increased leaf [N] 2. Increased stem productivity 3. Increased LAI 4. Decreased root proliferation Methods: The on-going ORNL (Oak Ridge National Lab) FACE (Free-Air CO 2 Enrichment) experiment was initiated in 1998 in a 15-m tall, 10-year old, closed-canopy sweetgum stand. The FACE experimental design consists of five 25-m diameter plots. FACE apparatus were installed in four of the plots (Figure 2a), and two FACE rings blow elevated concentrations of atmospheric CO 2 (approximately 550 ppm). A fertilization experiment was initiated in a sweetgum stand adjacent to ORNL FACE (Figure 2a,b) in 2004, when the trees were approximately 19-m tall. The trees were planted at the same time, and in the same manner as those in the FACE experiment, and the stand encompasses historical FACE rings 6 and 7. Thus, both experimental plantations consist of closed-canopy forests, in a linear growth phase. Most of the site parameters were similar, though total soil N content was initially greater at the fertilization site. The plantation was fertilized with 200 kg ha -1 of N as urea in March of 2004 before leaf out, in a replicated, randomized complete block design (Figure 2b). Several productivity parameters within the fertilization experiment were assessed, including tree annual basal area increment (BAI, cm 2 m -2 ), fine root productivity, and leaf litterfall. Leaf [N] and canopy N distribution were also measured by sampling from several heights within the canopy, and felling trees from within each experimental plot. Resin access tubes were used to assess fertilization effects on N availability (Figure 3). Summary: Increases in stem and leaf productivity and leaf N concentration support the conclusion that the sweetgum trees on the ORNL research plantation are limited by the availability of N, and it is likely that the trees in the FACE experiment are also limited by N. According to Luo et al. (2004), limited soil N availability will eventually lead to progressive nitrogen limitation of increased productivity responses to elevated CO 2. However, PNL is dependent on the effects of CO 2 on several biomass and soil N pools: whole-plant N amount, canopy N amount, annual N uptake, and N mineralization (Luo et al. 2004). Many of these parameters in FACE do not currently show signs of PNL. For example, canopy N was not affected by elevated [CO 2 ], and while whole plant N amount increased with CO 2 enrichment, the increase in N was mainly to supply fine root productivity. Thus, N was not sequestered in the woody biomass, which is one of the major mechanisms underlying PNL (see Figure 1). Also, N mineralization as measured previously did not change significantly with CO 2 enrichment. Nevertheless, N uptake has increased with increased productivity, and in an N-limited ecosystem, this may eventually lead to PNL. Nitrogen-limitation and the potential for long-term effects on production and carbon storage in a CO 2 -enriched forest Colleen M. Iversen 1, Richard J. Norby 2 1. University of Tennessee, Knoxville 2. Oak Ridge National Laboratory Preliminary results: Stem production: The basal area increment of the trees at the fertilization site increased significantly in response to fertilization with N, both on a daily basis and as an overall increment at the end of the growing season (Figure 4a, b). There is not a significant difference between the annual growth increment in the control plots in the fertilization experiment and FACE, which lends confidence to the conclusion that the trees in the FACE experiment would respond in a similar manner to N fertilization. Leaf [N]: Leaf N concentration in the fertilization experiment increased an average of 18% in response to N fertilization, but did not differ with canopy height (Figure 5). In a fertilization study, Scott et al. (2004) found that on average, 18 mg g -1 was the [N] sufficient for 90% of maximum stem growth. Foliar [N] in all plots in the FACE experiment and in the control plots of the N fertilization experiment are less than the critical [N] (Figure 5). Thus, increased BAI in the fertilized plots may have been possible because of the increase in foliar N, and it is possible that the stem production in FACE would have shown a positive response to elevated CO2 if foliar N concentrations were greater. Leaf litter/LAI: N addition increased the total amount of leaf litter produced (and thus, LAI, Figure 6) but did not alter the distribution of leaves within the canopy (data not shown). Fine-root production and biomass: We are also processing samples of fine root production and peak root biomass. In addition, fine root and litter N concentration are being measured. Future research: One of the main premises of PNL theory is that increases in whole plant N are due to N sequestration in plant biomass (pathway 1) However, previous research has found that the extra carbon fixed and N taken up is not being stored in plant long-lived plant biomass, such as wood. Instead, much of the N in the CO 2 -enriched plots is invested in fine root productivity, a pool which has a fast turnover time. Future research involving 15 N-enrichment and decomposition techniques will concentrate on the unanswered questions regarding interactions between C and N cycling belowground in ORNL FACE: 1.Will CO 2 -mediated increases in fine-root litter stimulate microbial activity and increase decomposition rates? 2.Will CO 2 -mediated increases in labile C substrate for microbial growth increase microbial N demand and immobilization? 3.Will CO 2 -mediated increases in microbial N demand decrease long-term net N mineralization and plant available N? Figure 1: Adapted from Luo et al. 2004. Elevated [CO 2 ] may lead to PNL via two pathways. Both pathways lead to decreased plant N availability and uptake, which in turn will provide a negative feedback to sustained increases in NPP, which may affect long- term C storage. Progressive Nitrogen Limitation (PNL): According to Luo et al. (2004), progressive limitation is dependent on three things: 1. A positive growth response to elevated [CO 2 ] 2. Increased N uptake 3. Limitation of N supply within the soil Thus, PNL depends both on a CO 2 -mediated stimulation of net primary productivity (NPP) and the related increased N demands, both of which have been observed in the CO 2 - enriched plots at ORNL FACE (see below). PNL also depends on the potential for the ecosystem to meet productivity and carbon storage demands (Luo et al. 2004). If soil N is limiting, PNL may be inevitable, though plants can compensate for N deficiency for a short time through nutrient stress alleviation mechanisms, including changes in plant and soil C:N ratios and NUE, and N mining via increased fine root growth. Block ThreeBlock TwoBlock One 12 m 16 m Control N Ring 7 NN N Control N N Ring 6 Figure 2 Figure 3: NO 3 - availability as assessed by WECSA ion- exchange resins throughout the growing season. There were no treatment effects on NH 4 + availability. * Research supported by US Department of Energy Office of Science, Biological and Environmental Research. civersen@utk.edu a) b) Figure 6: Cumulative litterfall weight as measured in litterfall baskets beneath the canopy in each plot. Figure 5: N concentration of leaves in differing canopy positions in the fertilization and FACE plots measured in late July, and late August, respectively. Figure 4: (a) Basal area increment (BAI) by tree in August 2004. Stem productivity in each treatment is significantly related to initial stem basal area. (b) Total basal area increment (cm 2 /m 2 ) of sweetgum trees during the 2004 growing season in the FACE experiment and in the adjacent N fertilization experiment. **** * *