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

Loreto Naya,Frank R Minchin,Cesar Arrese-Igor,Esther M. González,Manuel Becana,Ruben Ladrera,Javier Ramos

doi:10.1104/pp.107.099648

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.

Highlights

Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N2ase) activity
Following the same criteria for significant gene up-regulation or downregulation in the quantitative reverse transcription (qRT)-PCR analysis as those generally used in cDNA array studies (El Yahyaoui et al, 2004), our results show that the nifH mRNA level in alfalfa nodule bacteroids did not appreciably change under moderate or severe stress but decreased after the recovery period
This may be caused by alterations in the O2 availability and respiratory capacity of bacteroids and by the oxidative damage of nodule cell components

Summary

Introduction

Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N2ase) activity. The increase in O2 diffusion resistance, the decrease in N2ase-linked respiration and N2ase 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. Studies on soybean (Glycine max), common bean Another mechanism that could play a role in the drought-induced inhibition of N2 fixation, but has received much less attention, is oxidative stress. The application of low concentrations of paraquat, a ROS generator, to the rooting medium of pea plants inhibits SS activity of nodules prior to N2ase activity, suggesting that SS is an early target of oxidative stress in nodules (Marino et al, 2006). Physiological, and biochemical studies strongly suggest that alfalfa is more drought tolerant than pea (Moran et al, 1994) and ureideproducing grain legumes (Sinclair and Serraj, 1995)

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Conclusion