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.

AIR12 confers cold tolerance through regulation of the CBF cold response pathway and ascorbate homeostasis.

Auxin induced in root culture (AIR12) is a single gene in Arabidopsis and codes for a mono-heme cytochrome b, but it is unknown whether plant AIR12 is involved in abiotic stress responses. MfAIR12 was identified from Medicago falcata that is legume germplasm with great cold tolerance. Transcript levels of MfAIR12 and its homolog MtAIR12 from Medicago truncatula was induced under low temperature. Overexpression of MfAIR12 led to the accumulation of H2 O2 in apoplast and enhanced cold tolerance, which was blocked by H2 O2 scavengers, indicating that the increased cold tolerance was dependent upon the accumulated H2 O2 . In addition, declined cold tolerance was observed in Arabidopsis mutant air12, which could be restored by expressing MfAIR12. Compared to the wild type, higher levels of ascorbic acid and ascorbate redox state, as well as transcripts of the C repeat/dehydration responsive element-binding factor (CBF) transcription factors and their downstream cold-responsive genes, were observed in MfAIR12 transgenic lines, but lower levels of those in air12 mutant. It is suggested AIR12 confers cold tolerance as a result of the altered H2 O2 in the apoplast that is signaling in the regulation of CBF cold response pathway and ascorbate homeostasis.

An eukaryotic elongation factor 2 from Medicago falcata (MfEF2) confers cold tolerance.

BACKGROUND: An eukaryotic translation elongation factor-2 (eEF-2) plays an important role in protein synthesis, however, investigation on its role in abiotic stress responses is limited. A cold responsive eEF2 named as MfEF2 was isolated from yellow-flowered alfalfa [Medicago sativa subsp. falcata (L.) Arcang, thereafter M. falcata], a forage legume with great cold tolerance, and transgenic tobacco (Nicotiana tabacum L.) plants overexpressing MfEF2 were analyzed in cold tolerance and proteomic profiling was conducted under low temperature in this study. RESULTS: MfEF2 transcript was induced and peaked at 24 h and remained at the high level during cold treatment up to 96 h. Overexpression of MfEF2 in trasngenic tobacco plants resulted in enhanced cold tolerance. Compared to the wild type, transgenic plants showed higher survival rate after freezing treatment, higher levels of net photosynthetic rate (A), maximum photochemical efciency of photosystem (PS) II (Fv/Fm) and nonphotochemical quenching (NPQ) and lower levels of ion leakage and reactive oxygen species (ROS) production after chilling treatment. iTRAQ-based quantitative proteomic analysis identified 336 differentially expressed proteins (DEPs) from leaves of one transgenic line versus the wild type after chilling treatment for 48 h. GO and KEGG enrichment were conducted for analysis of the major biological process, cellular component, molecular function, and pathways of the DEPs involving in. It is interesting that many down-regulated DEPs were grouped into "photosynthesis" and "photosynthesis-antenna", such as subunits of PSI and PSII as well as light harvesting chlorophyll protein complex (LHC), while many up-regulated DEPs were grouped into "spliceosome". CONCLUSIONS: The results suggest that MfEF2 confers cold tolerance through regulating hundreds of proteins synthesis under low temperature conditions. The elevated cold tolerance in MfEF2 transgenic plants was associated with downregulation of the subunits of PSI and PSII as well as LHC, which leads to reduced capacity for capturing sunlight and ROS production for protection of plants, and upregulation of proteins involving in splicesome, which promotes alternative splicing of pre-mRNA under low temperature.

Comprehensive analysis of phenotype, microstructure and global transcriptional profiling to unravel the effect of excess copper on the symbiosis between nitrogen-fixing bacteria and Medicago lupulina.

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. However, knowledge of the molecular mechanisms, especially the microstructure and global transcriptional profiling, of the symbiosis process under heavy metal contamination is limited. In this study, a heavy metal-tolerant legume, Medicago lupulina, was treated with different concentrations of copper (Cu). The results showed that the early infection process was inhibited and the nodule ultrastructure was changed under 200 mg kg-1 Cu stress. Most infection threads (ITs) were prevented from entering the nodule cells, and few rhizobia were released into the host cells, in which thickening of the plant cell wall and IT wall was observed, demonstrating that rhizobial invasion was inhibited under Cu stress. RNA-seq analysis indicated that a strong shift in gene expression occurred (3257 differentially expressed genes, DEGs). The most pronounced effect was the upregulation of a set of 71 of 73 DEGs for nodule-specific cysteine-rich peptides, which have been shown to control the terminal differentiation of rhizobia in the nodules and to have antimicrobial activity. Various genes for metal transport, chelation binding and antioxidant defence were regulated. In particular, the DEGs for Cu trafficking and detoxification were induced during nodule formation. The DEGs for ethylene (ET) biosynthesis and signalling were also differentially expressed during nodulation, suggesting that the inhibition of nodulation by Cu occurred partially through ET signalling. Furthermore, the genes related to the cell wall were mostly upregulated and most likely involved in cell wall thickening. These findings provide an integrated understanding of the effects of Cu on legume nodule symbiosis at the molecular and phenotypic levels.

Medicago LINC Complexes Function in Nuclear Morphology, Nuclear Movement, and Root Nodule Symbiosis.

Nuclear movement is involved in cellular and developmental processes across eukaryotic life, often driven by Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, which bridge the nuclear envelope (NE) via the interaction of Klarsicht/ANC-1/Syne-1 Homology (KASH) and Sad1/UNC-84 (SUN) proteins. Arabidopsis (Arabidopsis thaliana) LINC complexes are involved in nuclear movement and positioning in several cell types. Observations since the 1950s have described targeted nuclear movement and positioning during symbiosis initiation between legumes and rhizobia, but it has not been established whether these movements are functional or incidental. Here, we identify and characterize LINC complexes in the model legume Medicago truncatula We show that LINC complex characteristics such as NE localization, dependence of KASH proteins on SUN protein binding for NE enrichment, and direct SUN-KASH binding are conserved between plant species. Using a SUN dominant-negative strategy, we demonstrate that LINC complexes are necessary for proper nuclear shaping and movement in Medicago root hairs, and are important for infection thread initiation and nodulation.

A comparison of lateral root patterning among dicot and monocot plants.

Lateral root branching along the primary root involves complex gene regulatory networks in model plant Arabidopsis. However, it is largely unclarified whether different plant species share a common mechanism to pattern the lateral root along the primary axis. In this study, we assessed the development pattern of lateral root among several dicot and monocot plants, including Arabidopsis, tomato, Medicago, Nicotiana, rice, and ryegrass by using an agar-gel culture system. Our results reveal a regular-spaced distribution pattern of lateral roots along the primary root axis of both dicot and monocot plants. Meanwhile, the root patterning is tightly controlled by root bending and the plant hormone auxin. However, nitrogen and phosphate starvations trigger distinguished root growth patterns among different plant species. Our studies strongly suggest a partially shared signaling pathway underlying root patterning of various plant species, and also provide a foundation for further identification of genes associated with root development.

A cold responsive ethylene responsive factor from Medicago falcata confers cold tolerance by up-regulation of polyamine turnover, antioxidant protection, and proline accumulation.

Ethylene responsive factor (ERF) subfamily transcription factors play an important role in plant abiotic and biotic stress tolerance. A cold responsive ERF, MfERF1, was isolated from Medicago falcata, an important forage legume that has great cold tolerance. Overexpression of MfERF1 resulted in an increased tolerance to freezing and chilling in transgenic tobacco plants, whereas down-regulation of the ortholog of MfERF1 in Medicago truncatula resulted in reduced freezing tolerance in RNAi plants. Higher transcript levels of some stress responsive genes (CHN50, OSM, ERD10C, and SAMS) and those involved in spermidine (Spd) and spermine (Spm) synthesis (SAMDC1, SAMDC2, SPDS1, SPDS2, and SPMS) and catabolism (PAO) were observed in transgenic plants than in wild type. However, neither Spd nor Spm level was accumulated in transgenic plants as a result of promoted polyamine oxidase activity. Transgenic plants had higher activities of antioxidants associated with the induced encoding genes including Cu, Zn-SOD, CAT1, CAT2, CAT3, and cpAPX and accumulated more proline associated with induced P5CS and reduced PROX2 transcription as compared with wild type. The results suggest that MfERF1 confers cold tolerance through promoted polyamine turnover, antioxidant protection, and proline accumulation.

A Lipid-Anchored NAC Transcription Factor Is Translocated into the Nucleus and Activates Glyoxalase I Expression during Drought Stress.

The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play a vital role in the response to drought stress. Here, we report a lipid-anchored NACsa TF in Medicago falcata MfNACsa is an essential regulator of plant tolerance to drought stress, resulting in the differential expression of genes involved in oxidation reduction and lipid transport and localization. MfNACsa is associated with membranes under unstressed conditions and, more specifically, is targeted to the plasma membrane through S-palmitoylation. However, a Cys26-to-Ser mutation or inhibition of S-palmitoylation results in MfNACsa retention in the endoplasmic reticulum/Golgi. Under drought stress, MfNACsa translocates to the nucleus through de-S-palmitoylation mediated by the thioesterase MtAPT1, as coexpression of APT1 results in the nuclear translocation of MfNACsa, whereas mutation of the catalytic site of APT1 results in colocalization with MfNACsa and membrane retention of MfNACsa. Specifically, the nuclear MfNACsa binds the glyoxalase I (MtGlyl) promoter under drought stress, resulting in drought tolerance by maintaining the glutathione pool in a reduced state, and the process is dependent on the APT1-NACsa regulatory module. Our findings reveal a novel mechanism for the nuclear translocation of an S-palmitoylated NAC in response to stress.

A temperature induced lipocalin gene from Medicago falcata (MfTIL1) confers tolerance to cold and oxidative stress.

Temperature-induced lipocalins (TIL) are plasmalemma-localized proteins and responsive to environmental stresses. Physiological functions of MfTIL1 from Medicago sativa subsp. falcata (L.) Arcang. (hereafter falcata), a forage legume with cold and drought tolerance, were investigated in this study. MfTIL1 expression was greatly induced by 4-96 h of cold treatment, while transcript levels of the orthologs in Medicago truncatula, a model legume plant with lower cold tolerance than falcata, were reduced or not altered within 48-96 h. MfTIL1 expression was not responsive to dehydration and salinity. Compared to the wild type, transgenic tobacco plants overexpressing MfTIL1 had lower temperature (LT50) that resulted in 50 % lethal and elevated survival rate in response to freezing, elevated F v/F m and decreased ion leakage after treatments with chilling, high light and methyl viologen (MV). H2O2 and O2 (-) were less accumulated in transgenic plants than in the wild type after treatments with chilling, high light and MV, while antioxidant enzyme activities showed no difference between the two types of plants prior to or following treatments. Higher transcript levels of NtDREB3 and NtDREB4 genes were observed in transgenic plants than in the wild type under non-stressed conditions, but higher transcript levels of NtDREB1, NtDREB2, NtDREB4 and NtCOR15a genes under chilling conditions. It is suggested that MfTIL1 plays an important role in plant tolerance to cold and oxidative stress through promoted scavenging of reactive oxygen species and up-regulating expression of multiple cold responsive genes.

Assessing genotypic diversity and symbiotic efficiency of five rhizobial legume interactions under cadmium stress for soil phytoremediation.

In the framework of soil phytoremediation using local legume plants coupled with their native root-nodulating bacteria to increase forage yields and preserve contaminated soils in arid regions of Tunisia, we investigated the diversity of bacteria from root nodules of Lathyrus sativus, Lens culinaris, Medicago marina, M. truncatula, and M. minima and the symbiotic efficiency of these five legume symbiosis under Cadmium stress. Fifty bacterial strains were characterized using physiological and biochemical features such heavy metals resistant, and PCR-RFLP of 16S rDNA. Taxonomically, the isolates nodulating L. sativus, and L. culinaris are species within the genera Rhizobium and the ones associated to Medicago sp, within the genera Sinorhizobium. The results revealed also that the cadmium tolerance of the different legumes-rhizobia interaction was as follows: M. minima < M. truncatula < M. marina < L. sativus < L. culinaris indicating that the effect of Cadmium on root nodulation and biomass production is more deleterious on M. minima-S. meliloti and M. truncatula-S. meliloti than in other symbiosis. Knowledge on genetic and functional diversity of M. marina, L. sativus and L. culinaris microsymbiotes is very useful for inoculant strain selection and can be selected to develop inoculants for soil phytoremediation.

STENOFOLIA acts as a repressor in regulating leaf blade outgrowth.

We recently reported that the Medicago WOX gene, STENOFOLIA (STF), acts as a transcriptional repressor in regulating leaf blade outgrowth. By using the Nicotiana sylvestris bladeless lam1 mutant as a genetic tool, we showed that the WUS-box, which is conserved among WUS clade WOX genes, is partly responsible for the repressive activity of STF. All members of the modern/WUS clade genes (WUS, WOX1-WOX7) in Arabidopsis that contain intact WUS-box can substitute for STF/LAM1 function while the intermediate and ancient clade members including WOX9,WOX11 and WOX13 cannot, due to lack of the intact WUS-box. Taken together, our results reveal a conserved repression mechanism playing a central role in cell proliferation conferred to the evolutionarily dynamic WOX gene family with acquisition of a repressor domain.

A cold responsive galactinol synthase gene from Medicago falcata (MfGolS1) is induced by myo-inositol and confers multiple tolerances to abiotic stresses.

Galactinol synthase (GolS, EC 2.4.1.123) catalyzes formation of galactinol and the subsequent synthesis of raffinose family oligosaccharides. The relationship of GolS to drought and salt tolerance has been well documented, however, little information is available about the role of GolS gene in cold tolerance. A coding sequence of MfGolS1 cDNA was cloned from Medicago sativa spp falcata (i.e. M. falcata), a species that exhibits greater cold tolerance than alfalfa (M. sativa). MfGolS1 transcript was not detected in untreated vegetative tissues using RNA blot hybridization; however, it was greatly induced in leaves, but not in stem and petiole, after cold treatment. Higher levels of MfGolS1 transcript were induced and maintained in M. falcata than in M. sativa during cold acclimation. Accordingly, more sugars including sucrose, galactinol, raffinose and stachyose were accumulated in M. falcata than in M. sativa. The data indicated that MfGolS1 transcript and its resultant sugar accumulation were associated with the differential cold tolerance between M. falcata and M. sativa. MfGolS1 transcript was weakly induced by dehydration and salt stresses, but not responsive to abscisic acid. MfGolS1 could be induced by myo-inositol, which is proposed to participate in cold-induced MfGolS1 expression. Overexpression of MfGolS1 in tobacco resulted in elevated tolerance to freezing and chilling in transgenic plants as a result of enhanced levels of galactinol, raffinose and stachyose. Tolerance to drought and salt stresses was also increased in the transgenic tobacco plants. It is suggested that MfGolS1 plays an important role in plant tolerance to abiotic stresses.

Interactive effects of salinity and iron deficiency in Medicago ciliaris.

In calcareous salt-affected soils, iron availability to plants is subjected to the effects of both sodium and bicarbonate ions. Our aim was to study interactive effects of salinity and iron deficiency on iron acquisition and root acidification induced by iron deficiency in Medicago ciliaris L., a species commonly found in saline ecosystems. Four treatments were used: C, control treatment, complete medium (CM) containing 30 microM Fe; S, salt treatment, CM with 75 mM NaCl; D, deficient treatment, CM containing only 1 microM Fe; DS, interactive treatment, CM containing 1 microM Fe with 75 mM NaCl. Our study showed that plant growth and chlorophyll content were much more affected by the interactive treatment than by iron deficiency or by the salt treatment, indicating an additive effect of these constraints in DS plants. These results could be partially explained by Na accumulation in shoots as well as a limitation of nutrient uptake such as Fe and K under salt stress, under iron deficiency, and especially under their combined effect. The study also showed that root acidification was deeply diminished when iron deficiency was associated with salinity. This probably explained the decrease of Fe uptake and suggested that root proton pump activity would be inhibited by salinity.

Influence of organic and inorganic soil amendments on plant growth in crude oil-contaminated soil.

Phytoremediation can be a viable alternative to traditional, more costly remediation techniques. Three greenhouse studies were conducted to evaluate plant growth with different soil amendments in crude oil-contaminated soil. Growth of alfalfa (Medicago sativa L., cultivar: Riley), bermudagrass (Cynodon dactylon L., cultivar: Common), crabgrass (Digitaria sanguinalis, cultivar: Large), fescue (Lolium arundinaceum Schreb., cultivar: Kentucky 31), and ryegrass (Lolium multiflorum Lam., cultivar: Marshall) was determined in crude oil-contaminated soil amended with either inorganic fertilizer, hardwood sawdust, papermill sludge, broiler litter or unamended (control). In the first study, the addition of broiler litter reduced seed germination for ryegrass, fescue, and alfalfa. In the second study, bermudagrass grown in broiler litter-amended soil produced the most shoot biomass, bermudagrass produced the most root biomass, and crabgrass and bermudagrass produced the most root length. In the third study, soil amended with broiler litter resulted in the greatest reduction in gravimetric total petroleum hydrocarbon (TPH) levels across the six plant treatments following the 14-wk study. Ryegrass produced more root biomass than any other species when grown in inorganic fertilizer- or hardwood sawdust + inorganic fertilizer-amended soil. The studies demonstrated that soil amendments and plant species selection were important considerations for phytoremediation of crude oil-contaminated soil.

[Bioremediation of mineral oil and polycyclic aromatic hydrocarbons (PAHs) in soils with two plant species].

With alfalfa (Medicago sativa) and paddy rice as test plants, and with pollutant level, specific bacteria, fungi and organic fertilizer as control factors, the bioremediation of mineral oil and polycyclic aromatic hydrocarbons (PAHs) in soils with two plant species was conducted. The results showed that the degradation rate of mineral oil was promoted by fertilization in alfalfa soil, but not in paddy rice soil. The degradation rate of total PAHs (11 PAH listed in USEPA) was increased by fertilization both in alfalfa and in paddy rice soil, and the promotion effect of fertilizer was better in paddy rice soil than in alfalfa soil. The fertilization rate was positively related to the rhizospheric indigenous fungi and bacteria's CFU in alfalfa soil, but only to the indigenous bacteria's CFU in paddy rice soil. The fungi and bacteria's CFU in both test soils had no positive relation with the amount of specific bacteria and fungi spiked. The degradation rate of 3-ring PAHs was enhanced in both alfalfa and paddy rice soil by fertilization, and the effect was stronger in paddy rice soil than in alfalfa soil. However, fertilization had no positive effect on the degradation of 4-ring PAHs.

Losses of diesel oil by volatilisation and effects of diesel oil on seed germination and seedling growth.

For phytoremediation to be effective, seeds must germinate and subsequently grow, or seedlings must become established, in contaminated soil. In this study, the effect of diesel oil on the viability of seeds of white clover and ryegrass together with long term abiotic diesel oil loss were investigated. Losses of diesel by volatilisation were found to be as high as 58% over 360 days suggesting that volatilisation (abiotic loss) may be a significant method of contaminant removal that may have been previously underestimated or overlooked in short term studies. White clover and ryegrass seeds were able to germinate in the presence of volatile diesel components and following 24 weeks immersion in diesel oil, which suggested that properties of their seed coats prevented diesel oil causing damage to the seeds.