Genome-wide characterization, transcriptome profiling, and functional analysis of the ALMT gene family in Medicago for aluminum resistance.

Aluminum (Al) is the major limiting factor affecting plant productivity in acidic soils. Al3+ ions exhibit increased solubility at a pH below 5, leading to plant root tip toxicity. Alternatively, plants can perceive very low concentrations of Al3+, and Al triggers downstream signaling even at pH 5.7 without causing Al toxicity. The ALUMINUM-ACTIVATED-MALATE-TRANSPORTER (ALMT) family members act as anion channels, with some regulating the secretion of malate from root apices to chelate Al, which is a crucial mechanism for plant Al resistance. To date, the role of the ALMT gene family within the legume Medicago species has not been fully characterized. In this study, we investigated the ALMT gene family in M. sativa and M. truncatula and identified 68 MsALMTs and 18 MtALMTs, respectively. Phylogenetic analysis classified these genes into five clades, and synteny analysis uncovered genuine paralogs and orthologs. The real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that MtALMT8, MtALMT9, and MtALMT15 in clade 2-2b are expressed in both roots and root nodules, and MtALMT8 and MtALMT9 are significantly upregulated by Al in root tips. We also observed that MtALMT8 and MtALMT9 can partially restore the Al sensitivity of Atalmt1 in Arabidopsis. Moreover, transcriptome analysis examined the expression patterns of these genes in M. sativa in response to Al at both pH 5.7 and pH 4.6, as well as to protons, and found that Al and protons can independently induce some Al-resistance genes. Overall, our findings indicate that MtALMT8 and MtALMT9 may play a role in Al resistance, and highlight the resemblance between the ALMT genes in Medicago species and those in Arabidopsis.

The peptide GOLVEN10 alters root development and noduletaxis in Medicago truncatula.

The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.

Gibberellins Inhibit Flavonoid Biosynthesis and Promote Nitrogen Metabolism in Medicago truncatula.

Bioactive gibberellic acids (GAs) are diterpenoid plant hormones that are biosynthesized through complex pathways and control various aspects of growth and development. Although GA biosynthesis has been intensively studied, the downstream metabolic pathways regulated by GAs have remained largely unexplored. We investigated Tnt1 retrotransposon insertion mutant lines of Medicago truncatula with a dwarf phenotype by forward and reverse genetics screening and phylogenetic, molecular, biochemical, proteomic and metabolomic analyses. Three Tnt1 retrotransposon insertion mutant lines of the gibberellin 3-beta-dioxygenase 1 gene (GA3ox1) with a dwarf phenotype were identified, in which the synthesis of GAs (GA3 and GA4) was inhibited. Phenotypic analysis revealed that plant height, root and petiole length of ga3ox1 mutants were shorter than those of the wild type (Medicago truncatula ecotype R108). Leaf size was also much smaller in ga3ox1 mutants than that in wild-type R108, which is probably due to cell-size diminution instead of a decrease in cell number. Proteomic and metabolomic analyses of ga3ox1/R108 leaves revealed that in the ga3ox1 mutant, flavonoid isoflavonoid biosynthesis was significantly up-regulated, while nitrogen metabolism was down-regulated. Additionally, we further demonstrated that flavonoid and isoflavonoid biosynthesis was induced by prohexadione calcium, an inhibitor of GA3ox enzyme, and inhibited by exogenous GA3. In contrast, nitrogen metabolism was promoted by exogenous GA3 but inhibited by prohexadione calcium. The results of this study further demonstrated that GAs play critical roles in positively regulating nitrogen metabolism and transport and negatively regulating flavonoid biosynthesis through GA-mediated signaling pathways in leaves.

Nitrate-induced CLE35 signaling peptides inhibit nodulation through the SUNN receptor and miR2111 repression.

Legume plants form nitrogen (N)-fixing symbiotic nodules when mineral N is limiting in soils. As N fixation is energetically costly compared to mineral N acquisition, these N sources, and in particular nitrate, inhibit nodule formation and N fixation. Here, in the model legume Medicago truncatula, we characterized a CLAVATA3-like (CLE) signaling peptide, MtCLE35, the expression of which is upregulated locally by high-N environments and relies on the Nodule Inception-Like Protein (NLP) MtNLP1. MtCLE35 inhibits nodule formation by affecting rhizobial infections, depending on the Super Numeric Nodules (MtSUNN) receptor. In addition, high N or the ectopic expression of MtCLE35 represses the expression and accumulation of the miR2111 shoot-to-root systemic effector, thus inhibiting its positive effect on nodulation. Conversely, ectopic expression of miR2111 or downregulation of MtCLE35 by RNA interference increased miR2111 accumulation independently of the N environment, and thus partially bypasses the nodulation inhibitory action of nitrate. Overall, these results demonstrate that the MtNLP1-dependent, N-induced MtCLE35 signaling peptide acts through the MtSUNN receptor and the miR2111 systemic effector to inhibit nodulation.

Genome-Wide Identification and Expression Profiling Analysis of the Trihelix Gene Family Under Abiotic Stresses in Medicago truncatula.

The trihelix transcription factor (GT) family is widely involved in regulating plant growth and development, and most importantly, responding to various abiotic stresses. Our study first reported the genome-wide identification and analysis of GT family genes in Medicago truncatula. Overall, 38 trihelix genes were identified in the M. truncatula genome and were classified into five subfamilies (GT-1, GT-2, SH4, GTγ and SIP1). We systematically analyzed the phylogenetic relationship, chromosomal distribution, tandem and segmental duplication events, gene structures and conserved motifs of MtGTs. Syntenic analysis revealed that trihelix family genes in M. truncatula had the most collinearity relationship with those in soybean followed by alfalfa, but very little collinearity with those in the maize and rice. Additionally, tissue-specific expression analysis of trihelix family genes suggested that they played various roles in the growth and development of specific tissues in M. truncatula. Moreover, the expression of some MtGT genes, such as MtGT19, MtGT20, MtGT22, and MtGT33, was dramatically induced by drought, salt, and ABA treatments, illustrating their vital roles in response to abiotic stresses. These findings are helpful for improving the comprehensive understanding of trihelix family; additionally, the study provides candidate genes for achieving the genetic improvement of stress resistance in legumes.

Short chain chito-oligosaccharides promote arbuscular mycorrhizal colonization in Medicago truncatula.

During the establishment of arbuscular mycorrhizal (AM) symbiosis, the fungus and the host plant exchange chemical signals that are crucial to reciprocal recognition. Short-chain chitin oligomers (CO) released by AM fungi are known to trigger symbiotic signaling in all host plant species tested. Here we applied exogenous CO, derived from crustacean exoskeleton, to pot-grown Medicago truncatula inoculated with the AM fungus Funneliformis mosseae and investigated root colonization, plant gene regulation and biomass production. CO treatment strongly promoted AM colonization with significant increases in arbuscule development, biomass production and photosynthetic surface compared to untreated mycorrhizal plants. Gene expression analyses indicated that CO treatment anticipated the expression of MtBCP and MtPT4 plant symbiotic markers, during the first two weeks post inoculation. Altogether, our results provide evidence that plant treatment with symbiotic fungal elicitors, anticipated and enhanced AM development, encouraging the use of CO to promote AM establishment in sustainable agricultural practices.

A combination of chitooligosaccharide and lipochitooligosaccharide recognition promotes arbuscular mycorrhizal associations in Medicago truncatula.

Plants associate with beneficial arbuscular mycorrhizal fungi facilitating nutrient acquisition. Arbuscular mycorrhizal fungi produce chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs), that promote symbiosis signalling with resultant oscillations in nuclear-associated calcium. The activation of symbiosis signalling must be balanced with activation of immunity signalling, which in fungal interactions is promoted by COs resulting from the chitinaceous fungal cell wall. Here we demonstrate that COs ranging from CO4-CO8 can induce symbiosis signalling in Medicago truncatula. CO perception is a function of the receptor-like kinases MtCERK1 and LYR4, that activate both immunity and symbiosis signalling. A combination of LCOs and COs act synergistically to enhance symbiosis signalling and suppress immunity signalling and receptors involved in both CO and LCO perception are necessary for mycorrhizal establishment. We conclude that LCOs, when present in a mix with COs, drive a symbiotic outcome and this mix of signals is essential for arbuscular mycorrhizal establishment.

The effects of soil phosphorus and zinc availability on plant responses to mycorrhizal fungi: a physiological and molecular assessment.

The positive effects of arbuscular mycorrhizal fungi (AMF) have been demonstrated for plant biomass, and zinc (Zn) and phosphorus (P) uptake, under soil nutrient deficiency. Additionally, a number of Zn and P transporter genes are affected by mycorrhizal colonisation or implicated in the mycorrhizal pathway of uptake. However, a comprehensive study of plant physiology and gene expression simultaneously, remains to be undertaken. Medicago truncatula was grown at different soil P and Zn availabilities, with or without inoculation of Rhizophagus irregularis. Measures of biomass, shoot elemental concentrations, mycorrhizal colonisation, and expression of Zn transporter (ZIP) and phosphate transporter (PT) genes in the roots, were taken. Mycorrhizal plants had a greater tolerance of both P and Zn soil deficiency; there was also evidence of AMF protecting plants against excessive Zn accumulation at high soil Zn. The expression of all PT genes was interactive with both P availability and mycorrhizal colonisation. MtZIP5 expression was induced both by AMF and soil Zn deficiency, while MtZIP2 was down-regulated in mycorrhizal plants, and up-regulated with increasing soil Zn concentration. These findings provide the first comprehensive physiological and molecular picture of plant-mycorrhizal fungal symbiosis with regard to soil P and Zn availability. Mycorrhizal fungi conferred tolerance to soil Zn and P deficiency and this could be linked to the induction of the ZIP transporter gene MtZIP5, and the PT gene MtPT4.

IAOx induces the SUR phenotype and differential signalling from IAA under different types of nitrogen nutrition in Medicago truncatula roots.

Indole-3-acetaldoxime (IAOx) is a particularly relevant molecule as an intermediate in the pathway for tryptophan-dependent auxin biosynthesis. The role of IAOx in growth-signalling and root phenotype is poorly studied in cruciferous plants and mostly unknown in non-cruciferous plants. We synthesized IAOx and applied it to M. truncatula plants grown axenically with NO3-, NH4+ or urea as the sole nitrogen source. During 14 days of growth, we demonstrated that IAOx induced an increase in the number of lateral roots, especially under NH4+ nutrition, while elongation of the main root was inhibited. This phenotype is similar to the phenotype known as "superroot" previously described in SUR1- and SUR2-defective Arabidopsis mutants. The effect of IAOx, IAA or the combination of both on the root phenotype was different and dependent on the type of N-nutrition. Our results also showed the endogenous importance of IAOx in a legume plant in relation to IAA metabolism, and suggested IAOx long-distance transport depending on the nitrogen source provided. Finally, our results point out to CYP71A as the major responsible enzymes for IAA synthesis from IAOx, while they exclude indole-3-acetaldehyde oxidases.

A Mass Spectrometry-Based Study Shows that Volatiles Emitted by Arthrobacter agilis UMCV2 Increase the Content of Brassinosteroids in Medicago truncatula in Response to Iron Deficiency Stress.

Iron is an essential plant micronutrient. It is a component of numerous proteins and participates in cell redox reactions; iron deficiency results in a reduction in nutritional quality and crop yields. Volatiles from the rhizobacterium Arthrobacter agilis UMCV2 induce iron acquisition mechanisms in plants. However, it is not known whether microbial volatiles modulate other metabolic plant stress responses to reduce the negative effect of iron deficiency. Mass spectrometry has great potential to analyze metabolite alterations in plants exposed to biotic and abiotic factors. Direct liquid introduction-electrospray-mass spectrometry was used to study the metabolite profile in Medicago truncatula due to iron deficiency, and in response to microbial volatiles. The putatively identified compounds belonged to different classes, including pigments, terpenes, flavonoids, and brassinosteroids, which have been associated with defense responses against abiotic stress. Notably, the levels of these compounds increased in the presence of the rhizobacterium. In particular, the analysis of brassinolide by gas chromatography in tandem with mass spectrometry showed that the phytohormone increased ten times in plants grown under iron-deficient growth conditions and exposed to microbial volatiles. In this mass spectrometry-based study, we provide new evidence on the role of A. agilis UMCV2 in the modulation of certain compounds involved in stress tolerance in M. truncatula.

Metabolic signatures of germination triggered by kinetin in Medicago truncatula.

In the present work, non-targeted metabolomics was used to investigate the seed response to kinetin, a phytohormone with potential roles in seed germination, still poorly explored. The aim of this study was to elucidate the metabolic signatures of germination triggered by kinetin and explore changes in metabolome to identify novel vigor/stress hallmarks in Medicago truncatula. Exposure to 0.5 mM kinetin accelerated seed germination but impaired seedling growth. Metabolite composition was investigated in seeds imbibed with water or with 0.5 mM kinetin collected at 2 h and 8 h of imbibition, and at the radicle protrusion stage. According to Principal Component Analysis, inositol pentakisphosphate, agmatine, digalactosylglycerol, inositol hexakisphosphate, and oleoylcholine were the metabolites that mostly contributed to the separation between 2 h, 8 h and radicle protrusion stage, irrespective of the treatment applied. Overall, only 27 metabolites showed significant changes in mean relative contents triggered by kinetin, exclusively at the radicle protrusion stage. The observed metabolite depletion might associate with faster germination or regarded as a stress signature. Results from alkaline comet assay, highlighting the occurrence of DNA damage at this stage of germination, are consistent with the hypothesis that prolonged exposure to kinetin induces stress conditions leading to genotoxic injury.

Comparative Physiological Analysis Reveals the Role of NR-Derived Nitric Oxide in the Cold Tolerance of Forage Legumes.

The role of nitric oxide (NO) signaling in the cold acclimation of forage legumes was investigated in this study. Medicago sativa subsp. falcata (L.) Arcang. (hereafter M. falcata) is a forage legume with a higher cold tolerance than Medicago truncatula, a model legume. Cold acclimation treatment resulted in increased cold tolerance in both M. falcata and M. truncatula, which was suppressed by pretreatment with tungstate, an inhibitor of nitrate reductase (NR), and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of NO. Likely, NITRATE REDUCTASE 1 (NIA1), but not NIA2 transcript, NR activity, and NO production were increased after cold treatment. Treatments with exogenous NO donors resulted in increased cold tolerance in both species. Superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX) activities and Cu,Zn-SOD2, Cu,Zn-SOD3, cytosolic APX1 (cAPX1), cAPX3 and chloroplastic APX1 (cpAPX1) transcript levels were induced in both species after cold treatment, which was suppressed by tungstate and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Treatment with exogenous NO resulted in enhanced activities of SOD, CAT, and APX. Moreover, higher levels of NIA1 transcript, NR activity, NO production, and antioxidant enzyme activities and transcripts were observed in M. falcata as compared with M. truncatula after cold treatment. The results suggest that NR-derived NO production and upregulated antioxidant defense are involved in cold acclimation in both species, while the higher levels of NO production and its derived antioxidant enzymes are associated with the higher cold tolerance in M. falcata as compared with M. truncatula.

Gibberellins negatively regulate the development of Medicago truncatula root system.

The root system displays a remarkable plasticity that enables plants to adapt to changing environmental conditions. This plasticity is tightly linked to the activity of root apical meristems (RAMs) and to the formation of lateral roots, both controlled by related hormonal crosstalks. In Arabidopsis thaliana, gibberellins (GAs) were shown to positively control RAM growth and the formation of lateral roots. However, we showed in Medicago truncatula that GAs negatively regulate root growth and RAM size as well as the number of lateral roots depending at least on the MtDELLA1 protein. By using confocal microscopy and molecular analyses, we showed that GAs primarily regulate RAM size by affecting cortical cell expansion and additionally negatively regulate a subset of cytokinin-induced root expansin encoding genes. Moreover, GAs reduce the number of cortical cell layers, resulting in the formation of both shorter and thinner roots. These results suggest contrasting effects of GA regulations on the root system architecture depending on plant species.

Hydrogen Peroxide and Nitric Oxide Crosstalk Mediates Brassinosteroids Induced Cold Stress Tolerance in Medicago truncatula.

Brassinosteroids (BRs) play pivotal roles in modulating plant growth, development, and stress responses. In this study, a Medicago truncatula plant pretreated with brassinolide (BL, the most active BR), enhanced cold stress tolerance by regulating the expression of several cold-related genes and antioxidant enzymes activities. Previous studies reported that hydrogen peroxide (H2O2) and nitric oxide (NO) are involved during environmental stress conditions. However, how these two signaling molecules interact with each other in BRs-induced abiotic stress tolerance remain largely unclear. BL-pretreatment induced, while brassinazole (BRZ, a specific inhibitor of BRs biosynthesis) reduced H2O2 and NO production. Further, application of dimethylthiourea (DMTU, a H2O2 and OH- scavenger) blocked BRs-induced NO production, but BRs-induced H2O2 generation was not sensitive to 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, a scavenger of NO). Moreover, pretreatment with DMTU and PTIO decreased BL-induced mitochondrial alternative oxidase (AOX) and the photosystem capacity. However, pretreatment with PTIO was found to be more effective than DMTU in reducing BRs-induced increases in Valt, Vt, and MtAOX1 gene expression. Similarly, BRs-induced photosystem II efficiency was found in NO dependent manner than H2O2. Finally, we conclude that H2O2 was involved in NO generation, whereas NO was found to be crucial in BRs-induced AOX capacity, which further contributed to the protection of the photosystem under cold stress conditions in Medicago truncatula.

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.

Metabolic and gene expression hallmarks of seed germination uncovered by sodium butyrate in Medicago truncatula.

Because high-quality seeds are essential for successful crop production in challenging environments, understanding the molecular bases of seed vigour will lead to advances in seed technology. Histone deacetylase inhibitors, promoting histone hyperacetylation, are used as tools to explore aspects still uncovered of the abiotic stress response in plants. The aim of this work was to investigate novel signatures of seed germination in Medicago truncatula, using the histone deacetylase inhibitor sodium butyrate (NaB) as stress agent. NaB-treated and untreated seeds collected at 2 and 8 hr of imbibition and at the radicle protrusion stage underwent molecular phenotyping and nontargeted metabolome profiling. Quantitative enrichment analysis revealed the influence of NaB on seed nucleotide, amino acid, lipid, and carbohydrate metabolism. Up-regulation of antioxidant and polyamine biosynthesis genes occurred in response to NaB. DNA damage evidenced in NaB-treated seeds correlated with up-regulation of base-excision repair genes. Changes in N1 -methyladenosine and N1 -methylguanine were associated with up-regulation of MtALKBH1 (alkylation repair homolog) gene. N2 ,N2 -dimethylguanosine and 5-methylcytidine, tRNA modifications involved in the post-transcriptional regulation of DNA damage response, were also accumulated in NaB-treated seeds at the radicle protrusion stage. The observed changes in seed metabolism can provide novel potential metabolic hallmarks of germination.

Arbuscular mycorrhizal fungi possess a CLAVATA3/embryo surrounding region-related gene that positively regulates symbiosis.

The arbuscular mycorrhizal (AM) symbiosis is a beneficial association established between land plants and the members of a subphylum of fungi, the Glomeromycotina. How the two symbiotic partners regulate their association is still enigmatic. Secreted fungal peptides are candidates for regulating this interaction. We searched for fungal peptides with similarities with known plant signalling peptides. We identified CLAVATA (CLV)/EMBRYO SURROUNDING REGION (ESR)-RELATED PROTEIN (CLE) genes in phylogenetically distant AM fungi: four Rhizophagus species and one Gigaspora species. These CLE genes encode a signal peptide for secretion and the conserved CLE C-terminal motif. They seem to be absent in the other fungal clades. Rhizophagus irregularis and Gigaspora rosea CLE genes (RiCLE1 and GrCLE1) are transcriptionally induced in symbiotic vs asymbiotic conditions. Exogenous application of synthetic RiCLE1 peptide on Medicago truncatula affects root architecture, by slowing the apical growth of primary roots and stimulating the formation of lateral roots. In addition, pretreatment of seedlings with RiCLE1 peptide stimulates mycorrhization. Our findings demonstrate for the first time that in addition to plants and nematodes, AM fungi also possess CLE genes. These results pave the way for deciphering new mechanisms by which AM fungi modulate plant cellular responses during the establishment of AM symbiosis.

Targeting Induced Local Lesions IN Genomes (TILLING) in Medicago truncatula.

TILLING is a reverse genetics strategy that combines the high density of point mutations provided by traditional chemical mutagenesis with rapid screening of DNA pools from a mutagenized population for induced mutations (McCallum et al., Nat Biotechnol 18:455-457, 2000). This high-throughput technique allows the identification of point mutations in any gene of interest.

Localization of calreticulin and calcium ions in mycorrhizal roots of Medicago truncatula in response to aluminum stress.

Aluminum (Al) toxicity limits growth and symbiotic interactions of plants. Calcium plays essential roles in abiotic stresses and legume-Rhizobium symbiosis, but the sites and mechanism of Ca2+ mobilization during mycorrhizae have not been analyzed. In this study, the changes of cytoplasmic Ca2+ and calreticulin (CRT) in Medicago truncatula mycorrhizal (MR) and non-mycorrizal (NM) roots under short Al stress [50 µM AlCl3 pH 4.3 for 3 h] were analyzed. Free Ca2+ ions were detected cytochemically by their reaction with potassium pyroantimonate and anti-CRT antibody was used to locate this protein in Medicago roots by immunocytochemical methods. In MR and NM roots, Al induced accumulation of CRT and free Ca2+. Similar calcium and CRT distribution in the MR were found at the surface of fungal structures (arbuscules and intercellular hyphae), cell wall and in plasmodesmata, and in plant and fungal intracellular compartments. Additionally, degenerated arbuscules were associated with intense Ca2+ and CRT accumulation. In NM roots, Ca2+ and CRT epitopes were observed in the stele, near wall of cortex and endodermis. The present study provides new insight into Ca2+ storage and mobilization in mycorrhizae symbiosis. The colocalization of CRT and Ca2+ suggests that CRT is essential for calcium mobilization for normal mycorrhiza development and response to Al stress.

Quantitative proteomic analysis using iTRAQ to identify salt-responsive proteins during the germination stage of two Medicago species.

Salt stress is one of the primary abiotic stresses responsible for decreasing crop yields worldwide. Germinating seeds can be greatly influenced by saline conditions. In this study, the physiological and phenotypic changes induced by salt treatments (10-50 mM NaCl and Na2SO4 mixtures) were analysed for Zhongmu-3 (Medicago sativa) and R108 (Medicago truncatula) seedlings. Our observations indicated that Zhongmu-3 was more salt-tolerant than R108. To characterize the protein expression profiles of these two Medicago species in response to salt stress, an iTRAQ-based quantitative proteomic analysis was applied to examine salt-responsive proteins. We identified 254 differentially changed salt-responsive proteins. Compared with control levels, the abundance of 121 proteins increased and 44 proteins decreased in salt-treated Zhongmu-3 seedlings, while 119 proteins increased and 18 proteins decreased in R108 seedlings. Moreover, 48 differentially changed proteins were common to Zhongmu-3 and R108 seedlings. A subsequent functional annotation indicated these proteins influenced diverse processes, such as catalytic activity, binding, and antioxidant activity. Furthermore, the corresponding transcript levels of 15 differentially changed proteins were quantified by qRT-PCR. The data presented herein provide new insights into salt-responsive proteins, with potential implications for enhancing the salt tolerance of Medicago species.