A highly controversial issue in global biogeochemistry is the regulation of terrestrial carbon (C) sequestration by soil nitrogen (N) availability. This controversy translates into great uncertainty in predicting future global terrestrial C sequestration. We propose a new framework that centers on the concept of progressive N limitation (PNL) for studying the interactions between C and N in terrestrial ecosystems. In PNL, available soil N becomes increasingly limiting as C and N are sequestered in long-lived plant biomass and soil organic matter. Our analysis focuses on the role of PNL in regulating ecosystem responses to rising atmospheric carbon dioxide concentration, but the concept applies to any perturbation that initially causes C and N to accumulate in organic forms. This article examines conditions under which PNL may or may not constrain net primary production and C sequestration in terrestrial ecosystems. While the PNL-centered framework has the potential to explain diverse experimental results and to help researchers integrate models and data, direct tests of the PNL hypothesis remain a great challenge to the research community.

Trifolium repens L. and Lolium perenne L. were grown in monocultures and bi‐species mixture in a Free Air Carbon Dioxide Enrichment (FACE) experiment at elevated (60 Pa) and ambient (35 Pa) CO2 partial pressure (pCO2) for three years. The effects of defoliation frequencies (4 and 7 cuts in 1993; 4 and 8 cuts in 1994/95) and nitrogen fertilization (10 and 42 g m–2 y–1 N in 1993; 14 and 56 g m–2 y–1 N in 1994/95) on the growth response to pCO2 were investigated. There were significant interspecific differences in the CO2 responses during the first two years, while in the third growing season, these interspecific differences disappeared. Yield of T. repens in monocultures increased in the first two years by 20% when grown at elevated pCO2. This CO2 response was independent of defoliation frequency and nitrogen fertilization. In the third year, the CO2 response of T. repens declined to 11%. In contrast, yield of L. perenne monocultures increased by only 7% on average over three years at elevated pCO2. The yield response of L. perenne to CO2 changed according to defoliation frequency and nitrogen fertilization, mainly in the second and third year. The ratio of root/yield of L. perenne increased under elevated pCO2, low N fertilizer rate, and frequent defoliation, but it remained unchanged in T. repens. We suggest that the more abundant root growth of L. perenne was related to increased N limitation under elevated pCO2. The consequence of these interspecific differences in the CO2 response was a higher proportion of T. repens in the mixed swards at elevated pCO2. This was evident in all combinations of defoliation and nitrogen treatments. However, the proportion of the species was more strongly affected by N fertilization and defoliation frequency than by elevated pCO2. Based on these results, we conclude that the species proportion in managed grassland may change as the CO2 concentration increases. However, an adapted management could, at least partially, counteract such CO2 induced changes in the proportion of the species. Since the availability of mineral N in the soil may be important for the species’ responses to elevated pCO2, more long‐term studies, particularly of processes in the soil, are required to predict the entire ecosystem response.

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~Symbiotic N, fixation is one of the main processes that introduces N into terrestrial ecosystems. As such, it may be crucial for the sequestration of the extra C available in a world of continuously increasing atmospheric CO, partial pressure (pC0,). The effect of elevated pC0, (60 Pa) on symbiotic N, fixation ('5N-isotope dilution method) was investigated using Free-Air-C0,-Enrichment technology over a period of 3 years. Trifolium repens was cultivated either alone or together with Lolium perenne (a nonfixing reference crop) in mixed swards. Two different N fertilization levels and defoliation frequencies were applied. The total N yield increased consistently and the percentage of plant N derived from symbiotic N, fixation increased significantly in T. repens under elevated pC0,. All additionally assimilated N was derived from symbiotic N, fixation, not from the soil. In the mixtures exposed to elevated pCO,, an increased amount of symbiotically fixed N (+7.8,8.2, and 6.2 g m-' a-' in 1993, 1994, and 1995, respectively) was introduced into the system. lncreased N, fixation is a competitive advantage for T. repens in mixed swards with pasture grasses and may be a crucial factor in maintaining the C:N ratio in the ecosystem as a whole.

Flavonoid signals from alfalfa (Medicago sativa L.) induce transcription of nodulation (nod) genes in Rhizobium melioti. Previous investigations identified the flavone luteolin as an active inducer in alfalfa seed extracts, but the nature of nod inducers released from roots has not been reported. Root exudate from 3-day-old alfalfa seedlings was purified and then assayed for biological activity with a nodABC-IacZ fusion in R. meliloti. Indentities of major nod inducers were established by spectroscopic analyses (ultraviolet/visible, proton nuclear magnetic resonance, and mass spectroscopy) and comparison with authentic standards. Major nod inducers, which were identified as 4',7-dihydroxyflavone, 4'-7-dihydroxyflavanone, and 4,4'-dihydroxy-2'-methoxychalcone, were released from seedling roots at 54, 22, and 20 picomole.plantr'. day-1, respectively. Luteolin was not found in these root exudates. The 4,4'-dihydroxy-2'-methoxychalcone induced nod genes at a concentration one order of magnitude lower than luteolin and is the first naturally released chalcone reported to have this function. Moderate and weak nod-inducing activity was associated, respectively, with 4',7-dihydroxyflavone and 4',7-dihydroxyflavanone.Alfalfa (Medicago sativa L.), an important leguminous forage crop throughout the world, forms N2-fixing root nodules in association with the soil bacterium Rhizobium meliloti. The earliest events of alfalfa nodule formation require expression ofthe nodulation (nod) DABC genes on the megaplasmid (pSym) of R. meliloti (8,16). Transcription of nodABC is induced through the cooperative action of the constitutive nodD product and components of root and seed exudates (22). Luteolin, 3',4',5,7-tetrahydroxyflavone, was isolated from alfalfa seed extracts and shown to participate in nod induction (23) evidence that some N2-dependent alfalfa seedlings are initially N-limited due to insufficient root nodule formation (10) and suggests that more nodules might be formed if more rhizobial cells are induced to initiate the infection process.The presence of active nod inducers in plants does not guarantee release into the rhizosphere. Yelton et al. (30) observed that extracts from some plants induced nod transcription in R. meliloti even when exudates ofthe same plants did not. In contrast, both extracts and exudates of alfalfa induced nod genes, but it is unclear if luteolin was solely responsible for nod induction.In order to better understand the process of nod-inducer release, it is necessary to identify active compounds actually exuded into the rhizosphere. The purpose ofthis study was to identify, quantify, and characterize the major nod inducers exuded by roots of young, unnodulated alfalfa seedlings. MATERIALS AND METHODS Plant CultureOne g of alfalfa (Medicago sativa L.) seed (cv 'Moapa 69') containing about 400 seeds (94% viable) was scarified, surfacesterilized 3 min with 70% ethanol, rinsed with sterile water, and imbibed in sterile, aerated water. Imbibing solutions were changed after 4 and 8 h to rem...