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.     

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.     

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.     

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.     

The tyrosyl-DNA phosphodiesterase gene family in Medicago truncatula Gaertn.: bioinformatic investigation and expression profiles in response to copper- and PEG-mediated stress

The Tdp1 gene encoding tyrosyl-DNA phosphodiesterase has been extensively investigated in animal cells, due to the role of this enzyme in the repair of topoisomerase I-DNA covalent lesions. In contrast, information in this regard is totally missing in plants. We report for the first time in plants on the Tdp1 gene family from barrel medic (Medicago truncatula Gaertn.), composed of two members, hereby named MtTdp1α and MtTdp1β. The expression profiles of MtTdp1α and MtTdp1β genes were evaluated in plantlets grown in vitro using copper and polyethylene glycol (PEG 6000) as stress agents. In situ detection of reactive oxygen species (ROS) was carried out by histochemical staining, while the level of oxidative DNA damage, quantified in terms of 7,8-dihydro-8-oxoguanine (8-oxo-dG), increased up to 7.4- and 6.7-fold in response to copper and PEG 6000 treatments, respectively. Quantitative real-time polymerase chain reaction revealed that both Tdp1 genes were significantly up-regulated in response to copper and PEG. The Tdp1 genes were also significantly up-regulated during seed rehydration, an aspect of seed physiology in which DNA repair is a key component. Thus, the Tdp1 genes might be used as novel tools for improving stress tolerance in crops. The expression patterns of the barrel medic top1α and top1β genes, encoding distinct isoforms of DNA topoisomerase I, were also analyzed and discussed to acquire additional information on their specific functions, closely related to that of the Tdp1 gene in animal cells.     

Asymmetric somatic hybridisation between the annual legumes Medicago truncatula and Medicago scutellata

 Interspecific asymmetric somatic hybrids have been produced between the sexually incompatible annual Medicago species Medicago truncatula and M. scutellata using only one regenerable parent and improved protoplast techniques. Regenerable M. truncatula leaf protoplasts were inactivated by lethal doses of iodoacetamide. Cotyledon protoplasts of non-regenerable M. scutellata were produced from 9 to 13 day old γ-irradiated cotyledons. Polyethylene glycol was used for protoplast fusion, and its successful use depended on a strategy to eliminate toxic effects. More than 50 mature regenerated plants were obtained from the fusion products and amplified fragment length polymorphism and chromosome analysis of a sample of 20 plants allowed the identification of two asymmetric hybrid plants. This technique is suitable for transferring characters from incompatible annual Medicago species to Medicago truncatula. Received: 23 July 1998 / Revision received: 21 December 1998 / Accepted: 15 January 1999     

蒺藜苜蓿花器官特异基因的表达分析

蒺藜苜蓿(Medicago truncatula G)花器官特异表达基因是参与其花器官形成与发育的重要基因。筛选蒺藜苜蓿的花器官特异表达基因,寻找这类基因在其他模式植物中的直系同源基因,并将其表达模式在不同植物间进行比较,有利于深入的理解这类基因在蒺藜苜蓿花器官发育中的功能。根据蒺藜苜蓿表达谱,并以其PISTILLAZA(PI)基因为模板,文章筛选了97个蒺藜苜蓿花器官特异表达基因(Ratio≥10,且Z≥7.9).通过同源比对,确定了这类基因在拟南芥(Arabidopsis thaliana L.)、大豆(Glycinemax L.)、百脉根(Lotusjaponicus L.)和水稻(Oryzasativa L.)中的直系同源基因。对这类基因在5种植物中的表达量、表达部位和功能进行比较,发现进化关系较近的植物,直系同源基因的表达变异较小,而进化关系较远的植物,直系同源基因的表达变异较大。进一步对表达分化较大的直系同源基因进行启动子分析,发现不同植物中直系同源基因表达模式的变化与启动子中调控元件的特性有关。     

Physiological and biochemical responses to acute ozone-induced oxidative stress in Medicago truncatula.

Oxidative signaling mediated by reactive oxygen species (ROS) is a central component of biotic and abiotic stresses in plants. Acute ozone (O(3)) fumigation is a useful non-invasive treatment for eliciting endogenous ROS in planta. In this study, 38 different accessions of the model legume, Medicago truncatula, from various geographical regions were fumigated with 300 nmol mol(-1) of O(3) for a period of six hours. Phenotypic symptoms were evaluated 24 and 48 h after the end of treatment. A majority of the accessions showed distinct visible damage. Eight accessions showing varying sensitivities to ozone were subjected to biochemical analysis to evaluate correlations between ozone damage and levels of ROS, antioxidants, and lipid peroxidation. Two-way analysis of variance indicated highly significant interactions between O(3) damage and levels of ROS, ascorbate, glutathione and lipid peroxidation. There were significant differences among the accessions for these traits before and after the end of O(3) fumigation, as indicated by equal variance Student's t-test. This study suggests that multiple physiological and biochemical mechanisms may govern O(3) tolerance or sensitivity. Surveying a large collection of germplasm led to identification of multiple resistant and sensitive lines for investigating molecular basis of O(3) phytotoxicity. The most resistant JE154 accession also showed enhanced tolerance to chronic O(3) and dehydration stress, suggesting germplasm with increased tolerance to acute O(3) can be a useful resource for improving resistance to multiple abiotic stressors.     

Overlap of proteome changes in Medicago truncatula in response to auxin and Sinorhizobium meliloti

We used proteome analysis to identify proteins induced during nodule initiation and in response to auxin in Medicago truncatula. From previous experiments, which found a positive correlation between auxin levels and nodule numbers in the M. truncatula supernodulation mutant sunn (supernumerary nodules), we hypothesized (1) that auxin mediates protein changes during nodulation and (2) that auxin responses might differ between the wild type and the supernodulating sunn mutant during nodule initiation. Increased expression of the auxin response gene GH3:beta-glucuronidase was found during nodule initiation in M. truncatula, similar to treatment of roots with auxin. We then used difference gel electrophoresis and tandem mass spectrometry to compare proteomes of wild-type and sunn mutant roots after 24 h of treatment with Sinorhizobium meliloti, auxin, or a control. We identified 131 of 270 proteins responding to treatment with S. meliloti and/or auxin, and 39 of 89 proteins differentially displayed between the wild type and sunn. The majority of proteins changed similarly in response to auxin and S. meliloti after 24 h in both genotypes, supporting hypothesis 1. Proteins differentially accumulated between untreated wild-type and sunn roots also showed changes in auxin response, consistent with altered auxin levels in sunn. However, differences between the genotypes after S. meliloti inoculation were largely not due to differential auxin responses. The role of the identified candidate proteins in nodule initiation and the requirement for their induction by auxin could be tested in future functional studies.