Application of emerging technologies to obtain legume protein isolates with improved techno-functional properties and health effects.

Current demand of consumers for healthy and sustainable food products has led the industry to search for different sources of plant protein isolates and concentrates. Legumes represent an excellent nonanimal protein source with high-protein content. Legume species are distributed in a wide range of ecological conditions, including regions with drought conditions, making them a sustainable crop in a context of global warming. However, their use as human food is limited by the presence of antinutritional factors, such as protease inhibitors, lectins, phytates, and alkaloids, which have adverse nutritional effects. Antitechnological factors, such as fiber, tannins, and lipids, can affect the purity and protein extraction yield. Although most are removed or reduced during alkaline solubilization and isoelectric precipitation processes, some remain in the resulting protein isolates. Selection of appropriate legume genotypes and different emerging and sustainable facilitating technologies, such as high-power ultrasound, pulsed electric fields, high hydrostatic pressure, microwave, and supercritical fluids, can be applied to increase the removal of unwanted compounds. Some technologies can be used to increase protein yield. The technologies can also modify protein structure to improve digestibility, reduce allergenicity, and tune technological properties. This review summarizes recent findings regarding the use of emerging technologies to obtain high-purity protein isolates and the effects on techno-functional properties and health.

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.

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.

Trehalose and trehalase in root nodules of Medicago truncatula and Phaseolus vulgaris in response to salt stress.

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranoside), a non-reducing disaccharide, has been found in a wide variety of organisms playing an important role as an abiotic stress protectant. Plants may come into contact with trehalose from exogenous sources, such as in plant-rhizobia symbiosis in which the rhizobia have the capacity to produce trehalose. The aim of this work is to analyse how trehalose and trehalase respond to salt stress in root nodules of legumes. For this purpose, tissue expression of Medicago truncatula trehalase gene (MTTRE1) and the expression of MTTRE1 under salt stress were analysed by real-time quantitative reverse transcription-PCR method. Trehalase activity was determined and trehalose was also measured by gas chromatography. In addition, trehalase protein occurrence in different organs and at different developmental stages in Phaseolus vulgaris plants has been studied. MTTRE1 expression is induced in nodules compared with leaves and roots, indicating a transcriptional regulation of trehalase in the presence of the microsymbiont. Under salt stress conditions, trehalase activity is downregulated at the transcriptional level, allowing trehalose accumulation. The results found in this study led us to conclude that trehalase activity is induced in root nodules of legumes by the microsymbiont and that under salt stress conditions; trehalase activity is downregulated at the transcriptional level in M. truncatula nodules. This allows trehalose accumulation and supports the possible role of this disaccharide as a stabilizer against salt stress conditions.

Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism.

Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.

The relaxase of the Rhizobium etli symbiotic plasmid shows nic site cis-acting preference.

Genetic and biochemical characterization of TraA, the relaxase of symbiotic plasmid pRetCFN42d from Rhizobium etli, is described. After purifying the relaxase domain (N265TraA), we demonstrated nic binding and cleavage activity in vitro and thus characterized for the first time the nick site (nic) of a plasmid in the family Rhizobiaceae. We studied the range of N265TraA relaxase specificity in vitro by testing different oligonucleotides in binding and nicking assays. In addition, the ability of pRetCFN42d to mobilize different Rhizobiaceae plasmid origins of transfer (oriT) was examined. Data obtained with these approaches allowed us to establish functional and phylogenetic relationships between different plasmids of this family. Our results suggest novel characteristics of the R. etli pSym relaxase for previously described conjugative systems, with emphasis on the oriT cis-acting preference of this enzyme and its possible biological relevance.

Identification of the rctA gene, which is required for repression of conjugative transfer of rhizobial symbiotic megaplasmids.

An analysis of the conjugative transfer of pRetCFN42d, the symbiotic plasmid (pSym) of Rhizobium etli, has revealed a novel gene, rctA, as an essential element of a regulatory system for silencing the conjugative transfer of R. etli pSym by repressing the transcription of conjugal transfer genes in standard laboratory media. The rctA gene product lacks sequence conservation with other proteins of known function but may belong to the winged-helix DNA-binding subfamily of transcriptional regulators. Similar to that of many transcriptional repressors, rctA transcription seems to be positively autoregulated. rctA expression is greatly reduced upon overexpression of another gene, rctB, previously identified as a putative activator of R. etli pSym conjugal transfer. Thus, rctB seems to counteract the repressive action of rctA. rctA homologs are present in at least three other bacterial genomes within the order Rhizobiales, where they are invariably located adjacent to and divergently transcribed from putative virB-like operons. We show that similar to that of R. etli pSym, conjugative transfer of the 1.35-Mb symbiotic megaplasmid A of Sinorhizobium meliloti is also subjected to the inhibitory action of rctA. Our data provide strong evidence that the R. etli and S. meliloti pSym plasmids are indeed self-conjugative plasmids and that this property would only be expressed under optimal, as yet unknown conditions that entail inactivation of the rctA function. The rctA gene seems to represent novel but probably widespread regulatory systems controlling the transfer of conjugative elements within the order Rhizobiales.

Physiological implications of trehalase from Phaseolus vulgaris root nodules: partial purification and characterization.

The purification and characterization of trehalase from common bean nodules as well as the role of this enzyme on growth, nodulation nitrogen fixation by examining the effects of the trehalase inhibitor validamycin A, was studied. Validamycin A did not affect plant and nodule mass, neither root trehalase and nitrogenase activity; however this treatment applied at the time of sowing increased nodule number about 16% and decreased nodule trehalase activity (16-fold) and the size of nodules. These results suggest that nodule trehalase activity of Phaseolus vulgaris could be involved in nodule formation and development. In addition, acid trehalase (EC 3.2.1.28) was purified from root nodules by fractionating ammonium sulfate, column chromatography on DEAE-sepharose and sephacryl S-300, and finally on native polyacrylamide gel electrophoresis. The purified homogeneous preparation of native acid trehalase exhibited a molecular mass of 42 and 45 kDa on SDS-PAGE. The enzyme has the optimum pH 3.9, Km of 0.109 mM, Vmax of 3630 nkat mg-1 protein and is relatively heat stable. Besides trehalose, it shows maximal activity with sucrose and maltose and, to a lesser degree melibiose, cellobiose and raffinose, and it does not hydrolyze on lactose and turanose. Acid trehalase was activated by Na+, Mn2+, Mg2+, Li+, Co2+, K+ and inhibited by Fe3+, Hg+ and EDTA.

Identification of functional mob regions in Rhizobium etli: evidence for self-transmissibility of the symbiotic plasmid pRetCFN42d.

An approach originally designed to identify functional origins of conjugative transfer (oriT or mob) in a bacterial genome (J. A. Herrera-Cervera, J. M. Sanjuán-Pinilla, J. Olivares, and J. Sanjuán, J. Bacteriol. 180:4583-4590, 1998) was modified to improve its reliability and prevent selection of undesired false mob clones. By following this modified approach, we were able to identify two functional mob regions in the genome of Rhizobium etli CFN42. One corresponds to the recently characterized transfer region of the nonsymbiotic, self-transmissible plasmid pRetCFN42a (C. Tun-Garrido, P. Bustos, V. González, and S. Brom, J. Bacteriol. 185:1681-1692, 2003), whereas the second mob region belongs to the symbiotic plasmid pRetCFN42d. The new transfer region identified contains a putative oriT and a typical conjugative (tra) gene cluster organization. Although pRetCFN42d had not previously been shown to be self-transmissible, mobilization of cosmids containing this tra region required the presence of a wild-type pRetCFN42d in the donor cell; the presence of multiple copies of this mob region in CFN42 also promoted conjugal transfer of the Sym plasmid pRetCFN42d. The overexpression of a small open reading frame, named yp028, located downstream of the putative relaxase gene traA, appeared to be responsible for promoting the conjugal transfer of the R. etli pSym under laboratory conditions. This yp028-dependent conjugal transfer required a wild-type pRetCFN42d traA gene. Our results suggest for the first time that the R. etli symbiotic plasmid is self-transmissible and that its transfer is subject to regulation. In wild-type CFN42, pRetCFN42d tra gene expression appears to be insufficient to promote plasmid transfer under standard laboratory conditions; gene yp028 may play some role in the activation of conjugal transfer in response to as-yet-unknown environmental conditions.