In Vivo Metabolic Regulation of Alternative Oxidase under Nutrient Deficiency-Interaction with Arbuscular Mycorrhizal Fungi and Rhizobium Bacteria.

The interaction of the alternative oxidase (AOX) pathway with nutrient metabolism is important for understanding how respiration modulates ATP synthesis and carbon economy in plants under nutrient deficiency. Although AOX activity reduces the energy yield of respiration, this enzymatic activity is upregulated under stress conditions to maintain the functioning of primary metabolism. The in vivo metabolic regulation of AOX activity by phosphorus (P) and nitrogen (N) and during plant symbioses with Arbuscular mycorrhizal fungi (AMF) and Rhizobium bacteria is still not fully understood. We highlight several findings and open questions concerning the in vivo regulation of AOX activity and its impact on plant metabolism during P deficiency and symbiosis with AMF. We also highlight the need for the identification of which metabolic regulatory factors of AOX activity are related to N availability and nitrogen-fixing legume-rhizobia symbiosis in order to improve our understanding of N assimilation and biological nitrogen fixation.

Overexpression of the arginine decarboxylase gene promotes the symbiotic interaction Medicago truncatula-Sinorhizobium meliloti and induces the accumulation of proline and spermine in nodules under salt stress conditions.

Legumes have the capacity to fix nitrogen in symbiosis with soil bacteria known as rhizobia by the formation of root nodules. However, nitrogen fixation is highly sensitive to soil salinity with a concomitant reduction of the plant yield and soil fertilization. Polycationic aliphatic amines known as polyamines (PAs) have been shown to be involved in the response to a variety of stresses in plants including soil salinity. Therefore, the generation of transgenic plants overexpressing genes involved in PA biosynthesis have been proposed as a promising tool to improve salt stress tolerance in plants. In this work we tested whether the modulation of PAs in transgenic Medicago truncatula plants was advantageous for the symbiotic interaction with Sinorhizobium meliloti under salt stress conditions, when compared to wild type plants. Consequently, we characterized the symbiotic response to salt stress of the homozygous M. truncatula plant line L-108, constitutively expressing the oat adc gene, coding for the PA biosynthetic enzyme arginine decarboxylase, involved in PAs biosynthesis. In a nodulation kinetic assay, nodule number incremented in L-108 plants under salt stress. In addition, these plants at vegetative stage showed higher nitrogenase and nodule biomass and, under salt stress, accumulated proline (Pro) and spermine (Spm) in nodules, while in wt plants, the accumulation of glutamic acid (Glu), γ-amino butyric acid (GABA) and 1-aminocyclopropane carboxylic acid (ACC) (the ethylene (ET) precursor) were the metabolites involved in the salt stress response. Therefore, overexpression of oat adc gene favours the symbiotic interaction between plants of M. truncatula L-108 and S. meliloti under salt stress and the accumulation of Pro and Spm, seems to be the molecules involved in salt stress tolerance.

Restrictive water condition modifies the root exudates composition during peanut-PGPR interaction and conditions early events, reversing the negative effects on plant growth.

Water deficit is one of the most serious environmental factors that affect the productivity of crops in the world. Arachis hypogaea is a legume with a high nutritional value and 70% is cultivated in semi-arid regions. This research aimed to study the effect of water deficit on peanut root exudates composition, analyzing the importance of exudates on peanut-PGPR interaction under restrictive water condition. Peanut seedlings were subjected to six treatments: 0 and 15 mM PEG, in combination with non-inoculated, Bradyrhizobium sp. and Bradyrhizobium-Azospirillum brasilense inoculated treatments. We analyzed the 7-day peanut root exudate in response to a water restrictive condition and the presence of bacterial inocula. Molecular analysis was performed by HPLC, UPLC and GC. Bacteria motility, chemotaxis, bacterial adhesion to peanut roots and peanut growth parameters were analyzed. Restrictive water condition modified the pattern of molecules exuded by roots, increasing the exudation of Naringenin, oleic FA, citric and lactic acid, and stimulation the release of terpenes of known antioxidant and antimicrobial activity. The presence of microorganisms modified the composition of root exudates. Water deficit affected the first events of peanut-PGPR interaction and the root exudates favored bacterial mobility, the chemotaxis and attachment of bacteria to peanut roots. Changes in the profile of molecules exuded by roots allowed A. hypogaea-Bradyrhizobium and A.hypogaea-Bradyrhizobium-Azospirillum interaction thus reversing the negative effects of restrictive water condition on peanut growth. These findings have a future potential application to improve plant-PGPR interactions under water deficit by formulating inoculants containing key molecules exuded during stress.

Polyamines contribute to salinity tolerance in the symbiosis Medicago truncatula-Sinorhizobium meliloti by preventing oxidative damage.

Polyamines (PAs) such as spermidine (Spd) and spermine (Spm) are small ubiquitous polycationic compounds that contribute to plant adaptation to salt stress. The positive effect of PAs has been associated to a cross-talk with other anti-stress hormones such as brassinosteroids (BRs). In this work we have studied the effects of exogenous Spd and Spm pre-treatments in the response to salt stress of the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti by analyzing parameters related to nitrogen fixation, oxidative damage and cross-talk with BRs in the response to salinity. Exogenous PAs treatments incremented the foliar and nodular Spd and Spm content which correlated with an increment of the nodule biomass and nitrogenase activity. Exogenous Spm treatment partially prevented proline accumulation which suggests that this polyamine could replace the role of this amino acid in the salt stress response. Additionally, Spd and Spm pre-treatments reduced the levels of H2O2 and lipid peroxidation under salt stress. PAs induced the expression of genes involved in BRs biosynthesis which support a cross-talk between PAs and BRs in the salt stress response of M. truncatula-S. meliloti symbiosis. In conclusion, exogenous PAs improved the response to salinity of the M. truncatula-S. meliloti symbiosis by reducing the oxidative damage induced under salt stress conditions. In addition, in this work we provide evidences of the cross-talk between PAs and BRs in the adaptive responses to salinity.

24-Epibrassinolide ameliorates salt stress effects in the symbiosis Medicago truncatula-Sinorhizobium meliloti and regulates the nodulation in cross-talk with polyamines.

Brassinosteroids (BRs) are steroid plant hormones that have been shown to be involved in the response to salt stress in cross-talk with other plant growth regulators such as polyamines (PAs). In addition, BRs are involved in the regulation of the nodulation in the rhizobium-legume symbiosis through the alteration of the PAs content in leaves. In this work, we have studied the effect of exogenous 24-epibrassinolide (EBL) in the response to salinity of nitrogen fixation in the symbiosis Medicago truncatula-Sinorhizobium meliloti. Foliar spraying of EBL restored the growth of plants subjected to salt stress and provoked an increment of the nitrogenase activity. In general, PAs levels in leaves and nodules decreased by the salt and EBL treatments, however, the co-treatment with NaCl and EBL augmented the foliar spermine (Spm) concentration. This increment of the Spm levels was followed by a reduction of the membrane oxidative damage and a diminution of the proline accumulation. The effect of BRs on the symbiotic interaction was evaluated by the addition of 0.01, 0.1 and 0.5 µM EBL to the growing solution, which provoked a reduction of the nodule number and an increment of the PAs levels in shoot. In conclusion, foliar treatment with EBL had a protective effect against salt stress in the M. truncatula-S. meliloti symbiosis mediated by an increment of the Spm levels. Treatment of roots with EBL incremented PAs levels in shoot and reduced the nodule number which suggests a cross-talk between PAs and BRs in the nodule suppression and the protection against salt stress.

Occurrence of polyamines in root nodules of Phaseolus vulgaris in symbiosis with Rhizobium tropici in response to salt stress.

Polyamines (PAs) are low molecular weight aliphatic compounds that have been shown to be an important part of plant responses to salt stress. For that reason in this work we have investigated the involvement of PAs in the response to salt stress in root nodules of Phaseolus vulgaris in symbiosis with Rhizobium tropici. The level and variety of PAs was higher in nodules, compared to leaves and roots, and in addition to the common PAs (putrescine, spermidine and spermine) we found homospermidine (Homspd) as the most abundant polyamine in nodules. UPLC-mass spectrometry analysis revealed the presence of 4-aminobutylcadaverine (4-ABcad), only described in nodules of Vigna angularis before. Indeed, the analysis of different nodular fractions revealed higher level of 4-ABcad, as well as Homspd, in bacteroids which indicate the production of these PAs by the bacteria in symbiosis. The genes involved in PAs biosynthesis in nodules displayed an induction under salt stress conditions which was not consistent with the decline of free PAs levels, probably due to the nitrogen limitations provoked by the nitrogenase activity depletion and/or the conversion of free PAs to theirs soluble conjugated forms, that seems to be one of the mechanisms involved in the regulation of PAs levels. On the contrary, cadaverine (Cad) and 4-ABcad concentrations augmented by the salinity, which might be due to their involvement in the response of bacteroids to hyper-osmotic conditions. In conclusion, the results shown in this work suggest the alteration of the bacteroidal metabolism towards the production of uncommon PAs such as 4-ABcad in the response to salt stress in legume root nodules.