Concerted changes in N and C primary metabolism in alfalfa (Medicago sativa) under water restriction

Abstract

Although the mechanisms of nodule N(2) fixation in legumes are now well documented, some uncertainty remains on the metabolic consequences of water deficit. In most cases, little consideration is given to other organs and, therefore, the coordinated changes in metabolism in leaves, roots, and nodules are not well known. Here, the effect of water restriction on exclusively N(2)-fixing alfalfa (Medicago sativa L.) plants was investigated, and proteomic, metabolomic, and physiological analyses were carried out. It is shown that the inhibition of nitrogenase activity caused by water restriction was accompanied by concerted alterations in metabolic pathways in nodules, leaves, and roots. The data suggest that nodule metabolism and metabolic exchange between plant organs nearly reached homeostasis in asparagine synthesis and partitioning, as well as the N demand from leaves. Typically, there was (i) a stimulation of the anaplerotic pathway to sustain the provision of C skeletons for amino acid (e.g. glutamate and proline) synthesis; (ii) re-allocation of glycolytic products to alanine and serine/glycine; and (iii) subtle changes in redox metabolites suggesting the implication of a slight oxidative stress. Furthermore, water restriction caused little change in both photosynthetic efficiency and respiratory cost of N(2) fixation by nodules. In other words, the results suggest that under water stress, nodule metabolism follows a compromise between physiological imperatives (N demand, oxidative stress) and the lower input to sustain catabolism.

Fig. 1.

Metabolomic analysis of leaf, root, and nodules under drought and control conditions. The figure shows only metabolites that vary significantly (P < 0.05) under drought conditions using a two-factor ANOVA [treatment (drought versus control) and nature of the organ (leaf, root, nodule), resulting in six groups]. The list of metabolites that vary significantly in each organ, using organ-specific statistics is shown in Table 2.

Fig. 2.

Nitrogenase (N_ase, nmol N₂ g DW^–1 s^–1) activity in nodules (A), N_ase photosynthetic yield N_ase/A (B; mol N₂ fixed g nodule DW^–1 s^–1 per mol CO₂ fixed g leaf DW^–1 s^–1, i.e. mol N₂ mol^–1 CO₂) and respiratory cost of N_ase activity R _nodule/N_ase (C; mol CO₂ mol^–1 NH₃). R _nodule used for calculations is from Table 1. N_ase activity was measured using the acetylene assay. In A, the difference between control and drought is significant (*P < 0.05). In B and C, there is no significant difference between treatments.

Fig. 3.

Total soluble proteins (A) and metabolic ratios (B) in leaves, roots, and nodules under control and drought conditions. In A, significant differences under drought are indicated with an asterisk (P < 0.05). In B, the drought-to-control quotient of the metabolite ratios is indicated on the x-axis. To facilitate reading, the steady line (no changes) is indicated with a broken line. Most visible changes in nodules are indicated with arrows. Source data are from metabolomic analyses (see also Fig. 1).

Fig. 4.

Silver-stained two-dimensional gel electrophoresis of proteins extracted from alfalfa (Medivago sativa L.) nodules exposed to control versus drought conditions. In the first dimension, 125 µg of total protein was loaded on a 18cm IEF strip with a linear gradient of pH 4–7. The second dimension was conducted in 10% polyacrylamide (w/v) gels (20×20cm) (for details see the Materials and methods). The gel image analyses was conducted with Progenesis SameSpots software v3.0 and the subsequent mass spectrometry analyses identified up to 17 proteins (marked by arrows) with significantly different expression depending on the water treatment.

Fig. 5.

Most visible changes in carbon primary metabolism of leaves, roots, and nodules of Medicago sativa under drought. This figure is a tentative summary of the present results. Thick and broken arrows represent enhanced and repressed pathways, respectively. GABA, γ-aminobutyrate; G3P, 3-phosphoglyceraldehyde; Mal, malate; PEP, phosphoenolpyruvate; PEPc, PEP carboxylase; OA, organic acids; OAA, oxaloacetate; SSA, succinic semialdehyde; TCAP, tricarboxylic acid pathway; 2OG, 2-oxoglutarate.