Elevated CO(2) modifies N acquisition of Medicago truncatula by enhancing N fixation and reducing nitrate uptake from soil

Abstract

The effects of elevated CO2 (750 ppm vs. 390 ppm) were evaluated on nitrogen (N) acquisition and assimilation by three Medicago truncatula genotypes, including two N-fixing-deficient mutants (dnf1-1 and dnf1-2) and their wild-type (Jemalong). The proportion of N acquisition from atmosphere and soil were quantified by (15)N stable isotope, and N transportation and assimilation-related genes and enzymes were determined by qPCR and biochemical analysis. Elevated CO2 decreased nitrate uptake from soil in all three plant genotypes by down-regulating nitrate reductase (NR), nitrate transporter NRT1.1 and NR activity. Jemalong plant, however, produced more nodules, up-regulated N-fixation-related genes and enhanced percentage of N derived from fixation (%Ndf) to increase foliar N concentration and N content in whole plant (Ntotal Yield) to satisfy the requirement of larger biomass under elevated CO2. In contrast, both dnf1 mutants deficient in N fixation consequently decreased activity of glutamine synthetase/glutamate synthase (GS/GOGAT) and N concentration under elevated CO2. Our results suggest that elevated CO2 is likely to modify N acquisition of M. truncatula by simultaneously increasing N fixation and reducing nitrate uptake from soil. We propose that elevated CO2 causes legumes to rely more on N fixation than on N uptake from soil to satisfy N requirements.

Figure 1. Above- and below-ground biomass of M. truncatula plants as affected by CO₂ level and plant genotype: dnf1-1 and dnf1-2 are deficient in N fixation, and Jemalong is their wild type.

Each value represents the average (±SE) of four replicates. Different lowercase letters indicate significant differences between ambient CO₂ and elevated CO₂ within the same genotype. Different uppercase letters indicate significant differences among genotypes within the same CO₂ treatment as determined by Tukey’s multiple range test at P<0.05.

Figure 2. Nodule number per root of M. truncatula plant as affected by CO₂ level and plant genotype: dnf1-1 and dnf1-2 are deficient in N fixation, and Jemalong is their wild type.

Each value represents the average (±SE) of four replications. Different lowercase letters indicate significant differences between ambient CO₂ and elevated CO₂ within the same genotype. Different uppercase letters indicate significant differences among M. genotypes within the same CO₂ treatment as determined by Tukey’s multiple range test at P<0.05.

Figure 3. Total non-structural carbohydrate (TNC) content in leaves and roots of M. truncatula plants as affected by CO₂ level and plant genotype: dnf1-1 and dnf1-2 are deficient in N fixation, and Jemalong is their wild type.

Each value represents the average (±SE) of four replicates. Different lowercase letters indicate significant differences between ambient CO₂ and elevated CO₂ within the same genotype. Different uppercase letters indicate significant differences among genotypes with the same CO₂ treatment as determined by Tukey’s multiple range test at P<0.05.

Figure 4. N concentrations in leaves and roots as well as Ntotal Yield (N content in per plant) of M. truncatula plants as affected by CO₂ level and plant genotype: dnf1-1 and dnf1-2 are deficient in N fixation, and Jemalong is their wild type.

Each value represents the average (±SE) of four replicates. Different lowercase letters indicate significant differences between ambient CO₂ and elevated CO₂ within the same genotype. Different uppercase letters indicate significant differences among genotypes within the same CO₂ treatment as determined by Tukey’s multiple range test at P<0.05.

Figure 5. Activities of the enzymes involved in N reduction (NR) and in N assimilation (GS and GOGAT) in the leaves and roots of M. truncatula plants as affected by CO₂ level and plant genotype: dnf1-1 and dnf1-2 are deficient in N fixation, and Jemalong is their wild type.

Each value represents the average (±SE) of four replicates. Different lowercase letters indicate significant differences between ambient CO₂ and elevated CO₂ within the same genotype. Different uppercase letters indicate significant differences among genotypes within the same CO₂ treatment as determined by Tukey’s multiple range test at P<0.05.

Figure 6. Expression of genes involved in N fixation (ENOD, nodF, and nifH), nitrate transportation and reduction (NT and NR), ammonium uptake (AMT), and N assimilation (GS and GOGAT) in leaves of M. truncatula plants as affected by CO₂ level and plant genotype: dnf1-1 and dnf1-2 are deficient in N fixation, and Jemalong is their wild type.

Values indicate fold-change in expression based on qPCR determination, and each value represents the average (±SE) of four replicates. An asterisk above a column indicates a significant difference in gene expression under ambient vs. elevated CO₂ (P<0.05).