Elevated CO2 concentration around alfalfa nodules increases N2 fixation

Abstract

Nodule CO2 fixation via PEPC provides malate for bacteroids and oxaloacetate for N assimilation. The process is therefore of central importance for efficient nitrogen fixation. Nodule CO2 fixation is known to depend on external CO2 concentration. The hypothesis of the present paper was that nitrogen fixation in alfalfa plants is enhanced when the nodules are exposed to elevated CO2 concentrations. Therefore nodulated plants of alfalfa were grown in a hydroponic system that allowed separate aeration of the root/nodule compartment that avoided any gas leakage to the shoots. The root/nodule compartments were aerated either with a 2500 microl l(-1) (+CO2) or zero microl l(-1) (-CO2) CO2-containing N2/O2 gas flow (80/20, v/v). Nodule CO2 fixation, nitrogen fixation, and growth were strongly increased in the +CO2 treatment in a 3-week experimental period. More intensive CO2 and nitrogen fixation coincided with higher per plant amounts of amino acids and organic acids in the nodules. Moreover, the concentration of asparagine was increased in both the nodules and the xylem sap. Plants in the +CO2 treatment tended to develop nodules with higher %N concentration and individual activity. In a parallel experiment on plants with inefficient nodules (fix-) the +CO2 treatment remained without effect. Our data support the thesis that nodule CO2 fixation is pivotal for efficient nitrogen fixation. It is concluded that strategies which enhance nodule CO2 fixation will improve nitrogen fixation and nodule formation. Moreover, sufficient CO2 application to roots and nodules is necessary for growth and efficient nitrogen fixation in hydroponic and aeroponic growth systems.

Fig. 1.

Experimental set-up for the long-term application of an N₂/O₂ mixture (80/20; v/v) with different CO₂ concentrations to the root/nodule compartments. The parts enclosed in dotted lines are repeated eight times each. The gas input comes from pressurized gas bottles. The N₂ and O₂ gas was free of any CO₂ or H₂ contamination. FC, flow controller.

Fig. 2.

Nodulated alfalfa plants ‘Saranac’ grown for 3 weeks with either −CO₂ (left) or +CO₂ (right) application to a separated root/nodule compartment. Nodules of plants from both treatments are shown below the plants.

Fig. 3.

Development of leaf number per plant (‘Saranac’) during the course of a 3-week experimental period with the application of an N₂/O₂ mixture (80/20; v/v) with either zero (–CO₂) or 2500 μl l⁻¹ CO₂ (+CO₂) to the root/nodule compartment. Data are means of four replicates. Bars represent the standard deviation. An asterisk indicates a statistically significant difference from the +CO₂ treatment (t test, P ≤0.05).

Fig. 4.

Development of branch number per plant (‘Saranac’) during the course of a 3-week experimental period with the application of an N₂/O₂ mixture (80/20; v/v) with either zero (–CO₂) or 2500 μl l⁻¹ CO₂ (+CO₂) to the root/nodule compartment. Data are means of four replicates. Bars represent standard deviation. An asterisk indicates a statistically significant difference from the +CO₂ treatment (t test, P ≤0.05).

Fig. 5.

Amino acids in nodules and in the xylem sap 3 weeks after beginning the application of an N₂/O₂ mixture (80/20; v/v) with either zero (–CO₂) or 2500 μl l⁻¹ CO₂ (+CO₂) to the root/nodule compartment. Data are the means of four replicates. Bars represent standard deviation. An asterisk indicates a statistically significant difference from the +CO₂ treatment (t test, P ≤0.05). (A) Concentrations of the five most abundant amino acids in the nodules. (B) Total amount in nodules per plant of the five most abundant nodule amino acids. In addition to the amino acids shown, Ala, Gaba, Tyr, Arg, Try, Lys, Val, Thr, Leu, His, Ile, Gly, and Prol. were detected in concentrations below 0.5 μmol g⁻¹ nodule fresh weight. (C) Concentrations of amino acids found in the xylem sap in concentrations above 0.1 μM.

Fig. 6.

Nodule organic acid compostition. Data are the means of four replicates. Bars represent standard deviation. An asterisk indicates a statistically significant difference from the +CO₂ treatment (t test, P ≤0.05). (A) Total amount of organic acids in nodules per plant. (B) Concentration of detected organic acids in nodules.