Oxylipin dynamics in Medicago truncatula in response to salt and wounding stresses

Multiple stresses are becoming common challenges in modern agriculture due to environmental changes. A large set of phytochemicals collectively known as oxylipins play a key role in responses to several stresses. Understanding the fine‐tuned plant responses to multiple and simultaneous stresses could open new perspectives for developing more tolerant varieties. We carried out the molecular and biochemical profiling of genes, proteins and active compounds involved in oxylipin metabolism in response to single/combined salt and wounding stresses on Medicago truncatula. Two new members belonging to the CYP74 gene family were identified. Gene expression profiling of each of the six CYP74 members indicated a tissue‐ and time‐specific expression pattern for each member in response to single/combined salt and wounding stresses. Notably, hormonal profiling pointed to an attenuated systemic response upon combined salt and leaf wounding stresses. Combined, these results confirm the important role of jasmonates in legume adaptation to abiotic stresses and point to the existence of a complex molecular cross‐talk among signals generated by multiple stresses.     

Effect of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula

In the present study, photosynthetic parameters including gas exchanges, pigment contents, and chlorophyll fluorescence, were compared in two contrasting local Medicago truncatula lines TN6.18 and TN8.20, in response to salt added to the nutrient solution. Plants were cultivated under symbiotic nitrogen fixation (SNF) after inoculation with a reference strain Sinorhizobium meliloti 2011, a very tolerant strain to salinity (700 mM NaCl), and grown in a controlled glasshouse. On one month old plants (with active SNF), salt treatment (75 mM NaCl) was gradually applied. Photosynthesis, assimilating pigments and chlorophyll fluorescence were monitored throughout the experiment during both short and long terms, compared to control (non-saline) conditions. A genotypic variation in salt tolerance was found; TN6.18 was the more sensitive to salinity. The relative tolerance of TN8.20 was concomitant with the highest photochemical quenching coefficient (q_P) affecting the maximum quantum yield of PSII (Y); the real quantum yield (?_exc) was the most affected in the sensitive line. Moreover, stomatal and PSII reaction centers activities differed clearly between the studied lines. We found that the effect of salinity on photosynthesis of M. truncatula was related to PSII activity reduction rather than to stomatal conductance limitation. Photosynthesis was reduced by the inhibition of CO₂ assimilation caused by PSII damage. This was clearly estimated by the Y, ?_exc and especially by the quantum yield of electron transport of PSII (Φ_PSII). Thus, on the basis of our results on the two local M. truncatula lines, we recommend the use of chlorophyll fluorescence as non-destructive screening method to discriminate susceptible and resistant legumes to salt stress.     

Growth, nitrogen fixation and ion distribution in Medicago truncatula subjected to salt stress

This study compared the growth, nodulation, N₂ fixation, and ion distribution in three Medicago truncatula lines, in response to salt in nutrient solution. Two local lines (TN8.20 and TN6.18) and a reference line (Jemalong 6) were inoculated with a reference strain Sinorhizobium meliloti 2011, a very tolerant strain to salinity (700 mM NaCl) and grown in a controlled glasshouse with or without 75 mM NaCl. A genotypic variation in tolerance to salt was found: TN6.18 was the most sensitive line whereas TN8.20 was the most tolerant. The relative tolerance of TN8.20 was concomitant with the lowest leaf Na⁺ concentration and the highest nodule biomass production. However, nodule efficiency (amount of nitrogen fixed per g dry weight nodule) decreased in all lines. Results suggest that the tolerance to salt seems to depend on the host plant ability to protect its leaves against an excessive Na⁺ (and Cl^?) accumulation, and its ability to maintain the development of an abundant nodular system, which in turn determines an important rate of nitrogen fixation and allows the plants to conserve their growth potentialities. The loss of the nodular efficiency under salt stress seems to be compensated by a large nodule biomass.     

Biodiversity within Medicago truncatula genotypes toward response to iron deficiency: Investigation of main tolerance mechanisms

Iron (Fe) deficiency is one of the major environmental stresses affecting plant production in the world. The selection of tolerant genotypes is considered an effective remediation strategy for this stress. The present study was carried out in order to investigate the biodiversity within Medicago truncatula plants in response to Fe deficiency, to identify tolerant genotypes and to assess the main tolerance mechanisms. To do this, a screening test was performed on 20 M. truncatula genotypes cultivated in minimal medium. Biometric and physiological markers were analyzed, including plant biomass, chlorophyll and root architecture. Results showed a biodiversity among the 20 genotypes. Interestingly, Fe deficiency tolerance was highest in TN8.20 and A17 genotypes. However, the lowest tolerance behavior was observed in TN1.11 and TN6.18. In order to investigate the main tolerance mechanisms, an experiment was conducted in the hydroponic system on already selected genotypes. Assessment of Fe deficiency tolerance was performed mainly on plant growth parameters, Fe (III)-chelate-reductase activity, rhizosphere acidification and antioxidant system defense. The relative better tolerance of A17 and TN8.20 to Fe deficiency was positively correlated with their capacity to maintain higher Fe-acquisition efficiency in roots via rhizosphere acidification and the stimulation of Fe (III)-chelate-reductase activity. Moreover, tolerant genotypes showed the lowest decreases in chlorophyll content and photosynthetic activity (CO₂ assimilation) compared to the sensitive ones. The efficiency of antioxidant capacity of the tolerant genotypes was revealed in stimulation of catalase (CAT) and peroxidase (POX) activities as well as accumulation of polyphenols, leading to the maintenance of cell integrity under Fe deficiency.     

Crosstalk between the nodulation signaling pathway and the autoregulation of nodulation in Medicago truncatula

A subset of CLAVATA3/endosperm-surrounding region-related (CLE) peptides are involved in autoregulation of nodulation (AON) in Medicago truncatula (e.g. MtCLE12 and MtCLE13). However, their linkage to other components of the AON pathways downstream of the shoot-derived inhibitor (SDI) is not understood. We have ectopically expressed the putative peptide ligand encoding genes MtCLE12 and MtCLE13 in M. truncatula which abolished nodulation completely in wild-type roots but not in the supernodulating null mutant sunn-4. Further, root growth inhibition was detected when MtCLE12 was ectopically expressed in wild-type roots or synthetic CLE12 peptide was applied exogenously. To identify downstream genes, roots of wild-type and sunn-4 mutant overexpressing MtCLE12 were used for quantitative gene expression analysis. We found that, in 35S:MtCLE12 roots, NODULE INCEPTION (NIN, a central regulator of nodulation) was down-regulated, whereas MtEFD (ethylene response factor required for nodule differentiation) and MtRR8 (a type-A response regulator thought to be involved in the negative regulation of cytokinin signaling), were up-regulated. Moreover, we found that the up-regulation of MtEFD and MtRR8 caused by overexpressing MtCLE12 is SUNN-dependent. Hence, our data link for the first time the pathways for Nod factor signaling, cytokinin perception and AON.     

Variability of interactions between barrel medic (Medicago truncatula) genotypes and Orobanche species

A number of broomrapes ( Orobanche spp.) are major threats for grain legumes in the Mediterranean area. Previous studies have shown very high levels of resistance to Orobanche crenata in Medicago truncatula with little variation among accessions hampering, therefore, its use for genetic analysis. In order to identify alternative systems for ulterior genetic and genomic analysis, we studied early stages of the interaction between M. truncatula accessions and a range of Orobanche species. We found significant differences in the induction of germination on Orobanche aegyptiaca , Orobanche foetida var. broteri , Orobanche minor , Orobanche nana and Orobanche ramosa seeds. Accessions also varied in the number of O. aegyptiaca , O. crenata , O. foetida var. broteri , O. nana and O. ramosa attachments supported. Applications of the synthetic germination stimulant GR24 increased the germination level of Orobanche cumana and O. minor . No attachments were observed for any of these species because of physical barriers typical of host resistance. On the contrary, increase of O. nana germination by GR24 led to an increase in number of attachments albeit the normal development of the nodules was stopped in a later stage. The genetic variation observed for induction of germination and subsequent attachment will be useful for isolating and characterising genes involved in early stages of Orobanche –host plant interaction and for the study of the biosynthetic pathways of production of germination stimulants.     

Differential expression within the glutamine synthetase gene family of the model legume Medicago truncatula.

The glutamine synthetase (GS) gene family of Medicago truncatula Gaertn. contains three genes related to cytosolic GS (MtGSa, MtGSb, and MtGSc), although one of these (MtGSc) appears not to be expressed. Sequence analysis suggests that the genes are more highly conserved interspecifically rather than intraspecifically: MtGSa and MtGSb are more similar to their homologs in Medicago sativa and Pisum sativum than to each other. Studies in which gene-specific probes are used show that both MtGSa and MtGSb are induced during symbiotic root nodule development, although not coordinately. MtGSa is the most highly expressed GS gene in nodules but is also expressed to lower extents in a variety of other organs. MtGSb shows higher levels of expression in roots and the photosynthetic cotyledons of seedlings than in nodules or other organs. In roots, both genes are expressed in the absence of an exogenous nitrogen source. However the addition of nitrate leads to a short-term, 2- to 3-fold increase in the abundance of both mRNAs, and the addition of ammonium leads to a 2-fold increase in MtGSb mRNA. The nitrogen supply, therefore, influences the expression of the two genes in roots, but it is clearly not the major effector of their expression. In the discussion section, the expression of the GS gene family of the model legume M. truncatula is compared to those of other leguminous plants.     

Antioxidant gene-enzyme responses in Medicago truncatula genotypes with different degree of sensitivity to salinity

Antioxidant responses and nodule function of Medicago truncatula genotypes differing in salt tolerance were studied. Salinity effects on nodules were analysed on key nitrogen fixation proteins such as nitrogenase and leghaemoglobin as well as estimating lipid peroxidation levels, and were found more dramatic in the salt-sensitive genotype. Antioxidant enzyme assays for catalase (CAT, EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and guaiacol peroxidase (EC 1.11.1.7) were analysed in nodules, roots and leaves treated with increasing concentrations of NaCl for 24 and 48 h. Symbiosis tolerance level, depending essentially on plant genotype, was closely correlated with differences of enzyme activities, which increased in response to salt stress in nodules (except CAT) and roots, whereas a complex pattern was observed in leaves. Gene expression responses were generally correlated with enzymatic activities in 24-h treated roots in all genotypes. This correlation was lost after 48 h of treatment for the sensitive and the reference genotypes, but it remained positively significant for the tolerant one that manifested a high induction for all tested genes after 48 h of treatment. Indeed, tolerance behaviour could be related to the induction of antioxidant genes in plant roots, leading to more efficient enzyme stimulation and protection. High induction of CAT gene was also distinct in roots of the tolerant genotype and merits further consideration. Thus, part of the salinity tolerance in M. truncatula is related to induction and sustained expression of highly regulated antioxidant mechanisms.     

Nodule carbohydrate metabolism and polyols involvement in the response of Medicago sativa to salt stress

Alterations of plant growth, chlorophyll fluorescence parameters, nodule carbon metabolism and polyols concentration as result of salt stress were examined in alfalfa (Medicago sativa). Plants, in symbiosis with Sinorhizobium meliloti GR4 strain, were grown under controlled conditions for 35 days (DAS) and subjected to 150 mM of NaCl stress. Plant biomass (PDW) and nitrogen fixation rate (NFR) were markedly affected by salt stress conditions; the highest reductions of PDW (50%) and NFR (40%) were registered at 84 DAS and 56 DAS, respectively. In addition, salinity affected the chlorophyll fluorescence parameters, decreased initial chlorophyll fluorescence (F₀) and increased the optimum quantum yield of PSII (F_v/F_m ratio). The enzyme activities sucrose synthase activity and phosphoenolpyruvate carboxylase, responsible for the carbon supply to the bacteroids by the formation of dicarboxylates, were drastically inhibited by salinity, mainly at 56 DAS with the beginning of flowering. The content of total soluble sugars and proline increased under salt stress, and these concentrations were higher in nodule than in leaf. This last result suggests that the nodule is an organ specially protected in order to maintain its functioning, even under stress conditions. Besides, the content of myoinositol and pinitol in leaves and nodules changed with the plant growth stage and the saline treatment. Under salinity stress, the concentrations of pinitol in nodule were higher than in leaf, which supports the central function of this molecule in the adaptive response of nodules to salt stress. The increase of pinitol synthesis in nodule of M. sativa under salt stress could be one of the adaptive features used by the plant.     

Nitrogen fixation persists under conditions of salt stress in transgenic Medicago truncatula plants expressing a cyanobacterial flavodoxin

Several recent studies have demonstrated that the expression of a cyanobacterial flavodoxin in plants can provide tolerance to a wide range of environmental stresses. Indeed, this strategy has been proposed as a potentially powerful biotechnological tool to generate multiple‐tolerant crops. To determine whether flavodoxin expression specifically increased tolerance to salt stress and whether it might also preserve legume nitrogen fixation under saline conditions, the flavodoxin gene was introduced into the model legume Medicago truncatula. Expression of flavodoxin did not confer saline tolerance to the whole plant, although the sensitive nitrogen‐fixing activity was maintained under salt stress in flavodoxin‐expressing plants. Our results indicate that flavodoxin induced small but significant changes in the enzymatic activities involved in the nodule redox balance that might be responsible for the positive effect on nitrogen fixation. Expression of flavodoxin can be regarded as a potential tool to improve legume symbiotic performance under salt stress, and possibly other environmental stresses.     

Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress

NaCl stress causes the accumulation of several mRNAs in tomato seedlings. An upregulated cDNA clone, SAM1, was found to encode a S-adenosyl-L-methionine synthetase enzyme (AdoMet synthetase). Expression of the cDNA SAM1 in a yeast mutant lacking functional SAM genes resulted in high AdoMet synthetase activity and AdoMet accumulation. We show that tomato plants contain at least four SAM isogenes. Clones corresponding to isogenes SAM2 and SAM3 have also been isolated and sequenced. they encode predicted polypeptides 95% and 92% identical, respectively, to the SAM1-encoded AdoMet Synthetase. RNA hybridization analysis showed a differential response of SAM genes to salt and other stress treatments. SAM1 and SAM3 mRNAs accumulated in the root in response to NaCl, mannitol or ABA treatments. SAM1 mRNA accumulated also in leaf tissue. These increases of mRNA level were apparent as soon as 8 h after the initiation of the salt treatment and were maintained for at least 3 days. A possible role for AdoMet synthetases in the adaptation to salt stress is discussed.     

Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula

Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.     

Nitrogen fixation is synchronized with carbon metabolism in Lotus japonicus and Medicago truncatula nodules under salt stress

Abstract

In the present work, the response to NaCl applied at the vegetative stage to Medicago truncatula and Lotus japonicus has been evaluated in order to ascertain whether the effect of salt stress on nitrogen fixation is due to a limitation on nodular carbon metabolism. Results show maximum sucrose synthase (SS) and alkaline invertase (AI) activities were obtained at the vegetative stage, when maximum nitrogenase activity was detected in both species. SS activity decreased with the salt treatment, providing evidence of the regulatory role of this enzyme for the carbon supply to the bacteroids. Phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activities could account for higher nitrogen fixation efficiency detected in L. japonicus nodules and isocitrate dehydrogenase (ICDH) activity compensated for the carbon limitations that occur under salt stress. These results support that nitrogenase inhibition in nodules experiencing salt stress is doubt to a carbon flux shortage, as result of carbon metabolism enzymes activities down-regulation.