The response of carbon metabolism and antioxidant defenses of alfalfa nodules to drought stress and to the subsequent recovery of plants

Abstract

Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N(2)ase) activity. Exposure of plants to a moderate drought (leaf water potential of -1.3 MPa) had no effect on sucrose (Suc) synthase (SS) activity, but caused inhibition of N(2)ase activity (-43%), accumulation of succinate (+36%) and Suc (+58%), and up-regulation of genes encoding cytosolic CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic glutathione reductase, and bacterial MnSOD and catalases B and C. Intensification of stress (-2.1 MPa) decreased N(2)ase (-82%) and SS (-30%) activities and increased malate (+40%), succinate (+68%), and Suc (+435%). There was also up-regulation (mRNA) of cytosolic ascorbate peroxidase and down-regulation (mRNA) of SS, homoglutathione synthetase, and bacterial catalase A. Drought stress did not affect nifH mRNA level or leghemoglobin expression, but decreased MoFe- and Fe-proteins. Rewatering of plants led to a partial recovery of the activity (75%) and proteins (>64%) of N(2)ase, a complete recovery of Suc, and a decrease of malate (-48%) relative to control. The increase in O(2) diffusion resistance, the decrease in N(2)ase-linked respiration and N(2)ase proteins, the accumulation of respiratory substrates and oxidized lipids and proteins, and the up-regulation of antioxidant genes reveal that bacteroids have their respiratory activity impaired and that oxidative stress occurs in nodules under drought conditions prior to any detectable effect on SS or leghemoglobin. We conclude that a limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of N(2)ase activity in alfalfa nodules.

Figure 1.

Simplified representations of some metabolic routes examined in this work. A, Carbon and nitrogen metabolism in nodules of an amide-producing legume, such as alfalfa. Adapted from Arrese-Igor et al. (1999). B, Production of ROS and some major antioxidant systems in legume nodules. Adapted from Matamoros et al. (2003). AAT, Asp aminotransferase; ASC, ascorbate; ETC, electron transfer chain; GS, Gln synthetase; OAA, oxaloacetate; ox met, oxidative metabolism; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; MDH, malate dehydrogenase; TCA, tricarboxylic acid cycle.

Figure 2.

Expression of N₂ase in alfalfa plants exposed to drought stress and following recovery from drought. A, Steady-state mRNA levels of the nifH gene, encoding the Fe-protein (component 2), were quantified by qRT-PCR. Values are means ± se of six biological replicates, each corresponding to RNA extracts from different plants. B, Protein levels of the MoFe-protein (component 1) and Fe-protein (component 2) of N₂ase. Values are means ± se of four western blots that were analyzed densitometrically. C, Apparent N₂ase activity of intact plants measured as H₂ evolution with an open flow-through system. Values are means ± se of five or six replicates. For all panels, treatments are designated as C (control), D1 (moderate drought stress), D2 (severe drought stress), and R (recovery). For A, means of D1, D2, and R are indicated with an asterisk when >2 (up-regulation) or <0.5 (down-regulation). For B and C, means of D1, D2, and R marked with an asterisk are significantly different from C, as determined by the Dunnett's t test (P < 0.05).

Figure 3.

Contents of carbon substrates and associated enzyme activities in nodules of alfalfa plants exposed to drought stress and following recovery from drought. A, Contents of dicarboxylic acids and Suc in nodules. B, Specific activities of ICDH, SS, AI, and GOGAT in nodules. Plant treatments and statistical analysis are as described in Figure 2. Values are means ± se of six to 10 replicates.

Figure 4.

Steady-state levels of mRNAs encoding antioxidant enzymes (A) and other important proteins (B) in the nodule host cells of alfalfa plants exposed to drought stress and following recovery from drought. Plant treatments are designated as in Figure 2. Values are means ± se of four to six biological replicates, each corresponding to RNA extracts from different plants. Means of D1, D2, and R are indicated with an asterisk when >2 (up-regulation) or <0.5 (down-regulation).

Figure 5.

Steady-state levels of mRNAs encoding antioxidant enzymes in the bacteroids of alfalfa plants exposed to drought stress and following recovery from drought. Plant treatments are designated as in Figure 2 and statistical analysis is as described in Figure 4. Values are means ± se of four to six biological replicates, each corresponding to RNA extracts from different plants.

Figure 6.

Contents of antioxidant metabolites in nodules of alfalfa plants exposed to drought stress and following recovery from drought. Plant treatments and statistical analysis are as described in Figure 2. Values are means ± se of five to seven replicates.

Figure 7.

Oxidative damage of lipids and proteins in nodules of alfalfa plants exposed to drought stress and following recovery from drought. For both sections, treatments are designated as C (control), D1 (moderate drought stress), D2 (severe drought stress), and R (recovery). A, Content of MDA in nodules. Values are means ± se of four replicates and are statistically compared as described in Figure 2. B, Immunoblot analysis of carbonyl derivatives of proteins from nodules. Lanes labeled C′, D1′, D2′, and R′ were loaded with aliquots of the corresponding C, D1, D2, and R extracts, in which the derivatization step was omitted (negative controls). All lanes of the 10% SDS gel contain 15 μg of nodule protein. Approximate molecular masses in kilodaltons are given on the right and bars mark immunoreactive proteins with an enhanced signal in D1, D2, or R as compared to C. The immunoblot shown is representative of four of them, each loaded with nodule extracts from plants grown in different series.