Nitrate reductases and hemoglobins contribute to nitric oxide (NO) balance in the Medicago truncatula – Sinorhizobium meliloti symbiosis

Abstract

The symbiosis between legumes and bacteria of rhizobium type leads to the formation of nitrogen-fixing nodules. In the symbiotic model Medicago truncatula/Sinorhizobium meliloti, nitric oxide (NO) is produced throughout the whole symbiotic process, from early interaction between the plant and the bacteria to senescence of nitrogen fixing nodules. Toxic, signalling or metabolic effects of NO depend on its concentration at the site of action. Therefore, NO steady-state concentration inside the cells should be tightly controlled to limit toxic effects and allow the signalling and metabolic functions to occur. In plants, several sources of NO have been described including nitrate reductase (NR), mitochondrial electron transfer chain (ETC) and NOS-like activity. Hemoglobins (Hbs) are known to metabolise NO. Based on their sequence homology and affinity for oxygen, three classes of Hbs have been essentially described in plants: non-symbiotic hemoglobins (ns-Hbs, Class 1), leghemoglobins (Lbs, Class 2) and truncated hemoglobins (tr-Hbs, Class 3). The three types of Hbs were reported to be expressed in legumes.     

To precisely assess the occurrence of NO during the whole symbiotic process, and the respective role of the different NRs and Hbs in its balance, we analysed the production of NO, the expression of 3 NRs and 6 Hb genes, and the global NR activity during short term (0 to 14 days post-inoculation and long term (0 to 8 weeks post-inoculation) symbiosis experiments.

Four peaks of NO production, which levels depend on nitrate concentration in culture medium, were observed 10 hours, 4 days, 3-4 weeks and 6 weeks post-inoculation. NO production was correlated with NR1, NR2, ns-HBs and tr-Hbs genes expression at the different stages of nodule formation. The use of various NO source inhibitors showed that NO production mainly depends on NR activity during early interaction steps as well as in mature and senescent nodules.

The different roles of NO, as a signalling and/or metabolic molecule, and its regulation by NRs and Hbs, in young, mature and senescent nodules are discussed.