Abscisic acid-induced nitric oxide and proline accumulation in independent pathways under water-deficit stress during seedling establishment in Medicago truncatula.

Nitric oxide (NO) production and amino acid metabolism modulation, in particular abscisic acid (ABA)-dependent proline accumulation, are stimulated in planta by most abiotic stresses. However, the relationship between NO production and proline accumulation under abiotic stress is still poorly understood, especially in the early phases of plant development. To unravel this question, this work investigated the tight relationship between NO production and proline metabolism under water-deficit stress during seedling establishment. Endogenous nitrate reductase-dependent NO production in Medicago truncatula seedlings increased in a time-dependent manner after short-term water-deficit stress. This water-deficit-induced endogenous NO accumulation was mediated through a ABA-dependent pathway and accompanied by an inhibition of seed germination, a loss of water content, and a decrease in elongation of embryo axes. Interestingly, a treatment with a specific NO scavenger (cPTIO) alleviated these water-deficit detrimental effects. However, the content of total amino acids, in particular glutamate and proline, as well as the expression of genes encoding enzymes of synthesis and degradation of proline were not affected by cPTIO treatment under water-deficit stress. Under normal conditions, exogenous NO donor stimulated neither the expression of P5CS2 nor the proline content, as observed after PEG treatment. These results strongly suggest that the modulation of proline metabolism is independent of NO production under short-term water-deficit stress during seedling establishment.

Overexpression of a Medicago truncatula stress-associated protein gene (MtSAP1) leads to nitric oxide accumulation and confers osmotic and salt stress tolerance in transgenic tobacco.

The impact of Medicago truncatula stress-associated protein gene (MtSAP1) overexpression has been investigated in Nicotiana tabacum transgenic seedlings. Under optimal conditions, transgenic lines overexpressing MtSAP1 revealed better plant development and higher chlorophyll content as compared to wild type seedlings. Interestingly, transgenic lines showed a stronger accumulation of nitric oxide (NO), a signaling molecule involved in growth and development processes. This NO production seemed to be partially nitrate reductase dependent. Due to the fact that NO has been also reported to play a role in tolerance acquisition of plants to abiotic stresses, the responses of MtSAP1 overexpressors to osmotic and salt stress have been studied. Compared to the wild type, transgenic lines were less affected in their growth and development. Moreover, NO content in MtSAP1 overexpressors was always higher than that detected in wild seedlings under stress conditions. It seems that this better tolerance induced by MtSAP1 overexpression could be associated with this higher NO production that would enable seedlings to reach a high protection level to prepare them to cope with abiotic stresses.

Involvement of Medicago truncatula glutamate receptor-like channels in nitric oxide production under short-term water deficit stress.

Early stages of plant development are highly susceptible to environmental cues, and seedlings have to develop sophisticated mechanisms to sense and respond to abiotic stresses. We have previously identified that abscisic acid (ABA), nitric oxide (NO) and modulation of nitrogen metabolism are involved in adaptive responses in Medicago truncatula seedlings under water deficit stress. Here, we investigated whether glutamate receptor-like channels (GLRs) played a role in the developmental physiological processes of Medicago seedlings during post-germination after a short-term water deficit stress. Twenty-nine independent MtGLR genes have been identified and then divided into four clades following a phylogenetic analysis; seventeen of them exhibited specific domains which are characteristic of animal ionotropic glutamate receptors. Under drought stress, ABA-induced NO accumulation was significantly reduced in presence of a GLR competitive antagonist, suggesting that this water deficit-induced endogenous NO production was mediated through a MtGLR-dependent pathway. Water deficit-induced inhibition of embryo axis elongation was strongly reduced whereas loss of water content was alleviated when MtGLRs were inhibited. These results suggest that glutamate receptors-like channels are required, through their involvement in NO production, in adaptive responses under short-term water-deficit stress during Medicago seedling establishment.