Genome-Wide Analysis of BBX Gene Family in Three Medicago Species Provides Insights into Expression Patterns under Hormonal and Salt Stresses.

BBX protein is a class of zinc finger transcription factors that have B-box domains at the N-terminus, and some of these proteins contain a CCT domain at the C-terminus. It plays an important role in plant growth, development, and metabolism. However, the expression pattern of BBX genes in alfalfa under hormonal and salt stresses is still unclear. In this study, we identified a total of 125 BBX gene family members by the available Medicago reference genome in diploid alfalfa (Medicago sativa spp. Caerulea), a model plant (M. truncatula), and tetraploid alfalfa (M. sativa), and divided these members into five subfamilies. We found that the conserved motifs of BBXs of the same subfamily reveal similarities. We analyzed the collinearity relationship and duplication mode of these BBX genes and found that the expression pattern of BBX genes is specific in different tissues. Analysis of the available transcriptome data suggests that some members of the BBX gene family are involved in multiple abiotic stress responses, and the highly expressed genes are often clustered together. Furthermore, we identified different expression patterns of some BBX genes under salt, ethylene, salt and ethylene, salicylic acid, and salt and salicylic acid treatments, verified by qRT-PCR, and analyzed the subcellular localization of MsBBX2, MsBBX17, and MsBBX32 using transient expression in tobacco. The results showed that BBX genes were localized in the nucleus. This study systematically analyzed the BBX gene family in Medicago plants, which provides a basis for the study of BBX gene family tolerance to abiotic stresses.

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.

Over-expression of Medicago Acyl-CoA-binding 2 genes enhance salt and drought tolerance in Arabidopsis.

Acyl-CoA-binding proteins (ACBPs) are mainly involved in acyl-CoA ester binding and trafficking in eukaryotic cells, and they function in lipid metabolism, membrane biosynthesis, cellular signaling, stress response, disease resistance, and other biological activities in plants. However, the roles of ACBP family members in Medicago remain unclear. In this study, a total of eight ACBP genes were identified in the genome of Medicago truncatula and Medicago sativa, and they were clustered into four sub-families (Class I-IV). Many cis-acting elements related to abiotic response were identified in the promoter region of these ACBP genes, in particular light-responsive elements. These ACBP genes exhibited distinct expression pattern in various tissues, and the expression level of MtACBP1/MsACBP1 and MtACBP2/MsACBP2 gene pairs were significantly increased under NaCl treatment. Subcellular localization analysis showed that MtACBP1/MsACBP1 and MtACBP2/MsACBP2 were localized in the endoplasmic reticulum of tobacco epidermal cells. Arabidopsis seedlings over-expressing MtACBP2/MsACBP2 displayed increased root length than the wild type under short light, Cu2+, ABA, PEG, and NaCl treatments. Over-expression of MtACBP2/MsACBP2 also significantly enhanced Arabidopsis tolerance under NaCl and PEG treatments in mature plants. Collectively, our study identified salt and drought responsive ACBP genes in Medicago and verified their functions in increasing resistance against salt and drought stresses.

Haplotype-Resolved, Chromosome-Level Assembly of White Clover (Trifolium repens L., Fabaceae).

White clover (Trifolium repens L.; Fabaceae) is an important forage and cover crop in agricultural pastures around the world and is increasingly used in evolutionary ecology and genetics to understand the genetic basis of adaptation. Historically, improvements in white clover breeding practices and assessments of genetic variation in nature have been hampered by a lack of high-quality genomic resources for this species, owing in part to its high heterozygosity and allotetraploid hybrid origin. Here, we use PacBio HiFi and chromosome conformation capture (Omni-C) technologies to generate a chromosome-level, haplotype-resolved genome assembly for white clover totaling 998 Mbp (scaffold N50 = 59.3 Mbp) and 1 Gbp (scaffold N50 = 58.6 Mbp) for haplotypes 1 and 2, respectively, with each haplotype arranged into 16 chromosomes (8 per subgenome). We additionally provide a functionally annotated haploid mapping assembly (968 Mbp, scaffold N50 = 59.9 Mbp), which drastically improves on the existing reference assembly in both contiguity and assembly accuracy. We annotated 78,174 protein-coding genes, resulting in protein BUSCO completeness scores of 99.6% and 99.3% against the embryophyta_odb10 and fabales_odb10 lineage datasets, respectively.

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.

Constitutive expression of a group 3 LEA protein from Medicago falcata (MfLEA3) increases cold and drought tolerance in transgenic tobacco.

KEY MESSAGE: MfLEA3 is involved in protection of catalase activity and confers multiple abiotic stress tolerance. Late embryogenesis abundant (LEA) proteins are involved in plant growth, development and abiotic stress tolerance. A member of group 3 LEA proteins from Medicago sativa subsp. falcata (L.) Arcang, MfLEA3, was investigated in the study. MfLEA3 transcript was induced in response to cold, dehydration, and abscisic acid (ABA), while the cold-induced transcript of MfLEA3 was blocked by pretreatment with inhibitor of ABA synthesis. Constitutive expression of MfLEA3 led to enhanced tolerance to cold, drought, and high-light stress in transgenic tobacco plants. Compared to accumulated reactive oxygen species (ROS) in the wild-type in response to treatments with low temperature, drought, and high light, ROS were not accumulated in transgenic plants. Superoxide dismutase, catalase (CAT), and ascorbate-peroxidase activities were increased in all plants after treatments with the above stresses, while higher CAT activity was maintained in transgenic plants compared with wild-type. However, transcript level of CAT-encoding genes including CAT1, CAT2, and CAT3 showed no significant difference between transgenic plants and wild-type, indicating that the higher CAT activity was not associated with its gene expression. ABA sensitivity and transcripts of several ABA and stress-responsive genes showed no difference between transgenic plant and wild-type, indicating that ABA signaling was not affected by constitutive expression of MfLEA3. The results suggest that MfLEA3 may be involved in the protection of CAT activity and confers multiple abiotic stress tolerance.

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.

A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis.

Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells' area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 (CTL1) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (Arabidopsis, cucumber or Medicago). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions.

Allele phasing is critical to revealing a shared allopolyploid origin of Medicago arborea and M. strasseri (Fabaceae).

BACKGROUND: Whole genome duplication plays a central role in plant evolution. There are two main classes of polyploid formation: autopolyploids which arise within one species by doubling of similar homologous genomes; in contrast, allopolyploidy (hybrid polyploidy) arise via hybridization and subsequent doubling of nonhomologous (homoeologous) genomes. The distinction between polyploid origins can be made using gene phylogenies, if alleles from each genome can be correctly retrieved. We examined whether two closely related tetraploid Mediterranean shrubs (Medicago arborea and M. strasseri) have an allopolyploid origin - a question that has remained unsolved despite substantial previous research. We sequenced and analyzed ten low-copy nuclear genes from these and related species, phasing all alleles. To test the efficacy of allele phasing on the ability to recover the evolutionary origin of polyploids, we compared these results to analyses using unphased sequences. RESULTS: In eight of the gene trees the alleles inferred from the tetraploids formed two clades, in a non-sister relationship. Each of these clades was more closely related to alleles sampled from other species of Medicago, a pattern typical of allopolyploids. However, we also observed that alleles from one of the remaining genes formed two clades that were sister to one another, as is expected for autopolyploids. Trees inferred from unphased sequences were very different, with the tetraploids often placed in poorly supported and different positions compared to results obtained using phased alleles. CONCLUSIONS: The complex phylogenetic history of M. arborea and M. strasseri is explained predominantly by shared allotetraploidy. We also observed that an increase in woodiness is correlated with polyploidy in this group of species and present a new possibility that woodiness could be a transgressive phenotype. Correctly phased homoeologues are likely to be critical for inferring the hybrid origin of allopolyploid species, when most genes retain more than one homoeologue. Ignoring homoeologous variation by merging the homoeologues can obscure the signal of hybrid polyploid origins and produce inaccurate results.

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 cryptic species produced by autopolyploidy and subsequent introgression involving Medicago prostrata (Fabaceae).

Although hybridisation through genome duplication is well known, hybridisation without genome duplication (homoploid hybrid speciation, HHS) is not. Few well-documented cases have been reported. A possible instance of HHS in Medicago prostrata Jacq. was suggested previously, based on only two genes and one individual. We tested whether this species was formed through HHS by sampling eight nuclear loci and 22 individuals, with additional individuals from related species, using gene capture and Illumina sequencing. Phylogenetic inference and coalescent simulations were performed to infer the causes of gene tree incongruence. We found no evidence that phylogenetic differences among M. prostrata individuals were the result of HHS. Instead, an autopolyploid origin of tetraploids with introgression from tetraploids of the M. sativa complex is likely. We argue that tetraploid M. prostrata individuals constitute a new species, characterised by a partially non-overlapping distribution and distinctive alleles (from the M. sativa complex). No gene flow from tetraploid to diploid M. prostrata is apparent, suggesting partial reproductive isolation. Thus, speciation via autopolyploidy appears to have been reinforced by introgression. This raises the intriguing possibility that introgressed alleles may be responsible for the increased range exploited by tetraploid M. prostrata with respect to that of the diploids.

Overexpression of MfPIP2-7 from Medicago falcata promotes cold tolerance and growth under NO3 (-) deficiency in transgenic tobacco plants.

BACKGROUND: Plasma membrane intrinsic proteins (PIPs), which belong to aquaporins (AQPs) superfamily, are subdivided into two groups, PIP1 and PIP2, based on sequence similarity. Several PIP2s function as water channels, while PIP1s have low or no water channel activity, but have a role in water permeability through interacting with PIP2. A cold responsive PIP2 named as MfPIP2-7 was isolated from Medicago falcata (hereafter falcata), a forage legume with great cold tolerance, and transgenic tobacco plants overexpressing MfPIP2-7 were analyzed in tolerance to multiple stresses including freezing, chilling, and nitrate reduction in this study. RESULTS: MfPIP2-7 transcript was induced by 4 to 12 h of cold treatment and 2 h of abscisic acid (ABA) treatment. Pretreatment with inhibitor of ABA synthesis blocked the cold induced MfPIP2-7 transcript, indicating that ABA was involved in cold induced transcription of MfPIP2-7 in falcata. Overexpression of MfPIP2-7 resulted in enhanced tolerance to freezing, chilling and NO3 (-) deficiency in transgenic tobacco (Nicotiana tabacum L.) plants as compared with the wild type. Moreover, MfPIP2-7 was demonstrated to facilitate H2O2 diffusion in yeast. Higher transcript levels of several stress responsive genes, such as NtERD10B, NtERD10C, NtDREB1, and 2, and nitrate reductase (NR) encoding genes (NtNIA1, and NtNIA2) were observed in transgenic plants as compared with the wild type with dependence upon H2O2. In addition, NR activity was increased in transgenic plants, which led to alterations in free amino acid components and concentrations. CONCLUSIONS: The results suggest that MfPIP2-7 plays an important role in plant tolerance to freezing, chilling, and NO3 (-) deficiency by promoted H2O2 diffusion that in turn up-regulates expression of NIAs and multiple stress responsive genes.

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.

Incomplete sequence homogenization in 45S rDNA multigene families: intermixed IGS heterogeneity within the single NOR locus of the polyploid species Medicago arborea (Fabaceae).

BACKGROUND AND AIMS: Ribosomal sequences have become the classical example of the genomic homogenization of nuclear multigene families. Despite theoretical advantages and modelling predictions that support concerted evolution of the 45S rDNA, several reports have found intragenomic polymorphisms. However, the origins and causes of these rDNA polymorphisms are difficult to assess because seed plants show a wide range of 45S rDNA loci number variation, especially in polyploids. Medicago arborea is a tetraploid species that has a single 45S rDNA locus. This feature makes this species a suitable case study to assess the fate of ribosomal IGS homogenization in polyploid species showing nucleolus organizer region (NOR) reduction. METHODS: The intergenic spacer (IGS) region was amplified by long PCR and the fragments were cloned and sequenced by a primer-walking strategy. The physical mapping of the whole and partial IGS variants was assessed by fluorescent in situ hybridization (FISH) and fibre-FISH methods on mitotic chromosomes and extended DNA fibres, respectively. KEY RESULTS: Two IGS fragments of 4·8 and 3·5 kb were obtained showing structural features of functional sequences. The shorter variant appears to be a truncated copy of the 4·8 kb fragment that lacks the duplication of the transcription initiation site region and the entire D region. The physical localization of the two IGS variants on metaphase chromosomes and extended DNA fibres using FISH corroborated their joint presence within the same locus. In addition, no spatial structure of the two variants was detected within the NOR. CONCLUSIONS: The results suggest that full sequence homogenization is not operating within the NOR locus of M. arborea. The structure of the NOR locus reported here departs from the models of IGS heterogeneity present in plants and caution against assuming the widespread belief that intragenomic ribosomal heterogeneity is mainly due to sequence variation between paralogous loci.

Early transcriptional responses to mercury: a role for ethylene in mercury-induced stress.

Understanding the cellular mechanisms of plant tolerance to mercury (Hg) is important for developing phytoremediation strategies of Hg-contaminated soils. The early responses of alfalfa (Medicago sativa) seedlings to Hg were studied using transcriptomics analysis. A Medicago truncatula microarray was hybridized with high-quality root RNA from M. sativa treated with 3 µM Hg for 3, 6 and 24 h. The transcriptional pattern data were complementary to the measurements of root growth inhibition, lipid peroxidation, hydrogen peroxide (H2 O2 ) accumulation and NADPH-oxidase activity as stress indexes. Of 559 differentially expressed genes (DEGs), 91% were up-regulated. The majority of DEGs were shared between the 3 and 6 h (60%) time points, including the 'stress', 'secondary metabolism' and 'hormone metabolism' functional categories. Genes from ethylene metabolism and signalling were highly represented, suggesting that this phytohormone may be relevant for metal perception and homeostasis. Ethylene-insensitive alfalfa seedlings preincubated with the ethylene signalling inhibitor 1-methylcyclopronene and Arabidopsis thaliana ein2-5 mutants confirmed that ethylene participates in the early perception of Hg stress. It modulates root growth inhibition, NADPH-oxidase activity and Hg-induced apoplastic H2 O2 accumulation. Therefore, ethylene signalling attenuation could be useful in future phytotechnological applications to ameliorate stress symptoms in Hg-polluted plants.

Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus, Medicago, and Phaseolus.

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.

Identification of duplicated and stress-inducible Aox2b gene co-expressed with Aox1 in species of the Medicago genus reveals a regulation linked to gene rearrangement in leguminous genomes.

In flowering plants, alternative oxidase (Aox) is encoded by 3-5 genes distributed in 2 subfamilies (Aox1 and Aox2). In several species only Aox1 is reported as a stress-responsive gene, but in the leguminous Vigna unguiculata Aox2b is also induced by stress. In this work we investigated the Aox genes from two leguminous species of the Medicago genus (Medicago sativa and Medicago truncatula) which present one Aox1, one Aox2a and an Aox2b duplication (named here Aox2b1 and Aox2b2). Expression analyses by semi-quantitative RT-PCR in M. sativa revealed that Aox1, Aox2b1 and Aox2b2 transcripts increased during seed germination. Similar analyses in leaves and roots under different treatments (SA, PEG, H2O2 and cysteine) revealed that these genes are also induced by stress, but with peculiar spatio-temporal differences. Aox1 and Aox2b1 showed basal levels of expression under control conditions and were induced by stress in leaves and roots. Aox2b2 presented a dual behavior, i.e., it was expressed only under stress conditions in leaves, and showed basal expression levels in roots that were induced by stress. Moreover, Aox2a was expressed at higher levels in leaves and during seed germination than in roots and appeared to be not responsive to stress. The Aox expression profiles obtained from a M. truncatula microarray dataset also revealed a stress-induced co-expression of Aox1, Aox2b1 and Aox2b2 in leaves and roots. These results reinforce the stress-inducible co-expression of Aox1/Aox2b in some leguminous plants. Comparative genomic analysis indicates that this regulation is linked to Aox1/Aox2b proximity in the genome as a result of the gene rearrangement that occurred in some leguminous plants during evolution. The differential expression of Aox2b1/2b2 suggests that a second gene has been originated by recent gene duplication with neofunctionalization.

Nitric oxide mediates cold- and dehydration-induced expression of a novel MfHyPRP that confers tolerance to abiotic stress.

Hybrid proline-rich proteins (HyPRPs) are cell wall-localized proteins, and are frequently responsive to environmental stresses. The coding sequence of a HyPRP cDNA was isolated from Medicago falcata, a forage crop that shows cold and drought tolerance. The predicted MfHyPRP contains a proline-rich domain at N-terminus after the signal peptide and a conserved eight-cysteine motif at the C-terminus. Higher level of MfHyPRP transcript was observed in leaves than in stems and roots under control conditions, while more MfHyPRP transcript was induced in leaves and stems than in roots after cold treatment. Levels of MfHyPRP transcript and MfHyPRP protein in leaves were induced by cold, dehydration, abscisic acid (ABA), hydrogen peroxide (H2 O2) and nitric oxide (NO), but not responsive to salt stress. The cold- or dehydration-induced expression of MfHyPRP was blocked by scavenger of NO, but not affected by inhibitor of ABA biosynthesis or scavenger of H2 O2. The results indicated that NO, but not ABA and H2 O2, was essential in the cold- and dehydration-induced expression of MfHyPRP. Overexpression of MfHyPRP in tobacco led to increased tolerance to freezing, chilling and osmotic stress as well as methyl viologen-induced oxidative stress. The increased cold and osmotic stress tolerance was proposed to be associated with improved protection against oxidative damages. It is suggested that NO mediates cold- and dehydration-induced expression of MfHyPRP that confers tolerance to abiotic stress.

Hydrogen peroxide and nitric oxide mediated cold- and dehydration-induced myo-inositol phosphate synthase that confers multiple resistances to abiotic stresses.

myo-Inositol phosphate synthase (MIPS) is the key enzyme of myo-inositol synthesis, which is a central molecule required for cell metabolism and plant growth as a precursor to a large variety of compounds. A full-length fragment of MfMIPS1 cDNA was cloned from Medicago falcata that is more cold-tolerant than Medicago sativa. While MfMIPS1 transcript was induced in response to cold, dehydration and salt stress, MIPS transcript and myo-inositol were maintained longer and at a higher level in M. falcata than in M. sativa during cold acclimation at 5 °C. MfMIPS1 transcript was induced by hydrogen peroxide (H(2) O(2)) and nitric oxide (NO), but was not responsive to abscisic acid (ABA). Pharmacological experiments revealed that H(2) O(2) and NO are involved in the regulation of MfMIPS1 expression by cold and dehydration, but not by salt. Overexpression of MfMIPS1 in tobacco increased the MIPS activity and levels of myo-inositol, galactinol and raffinose, resulting in enhanced resistance to chilling, drought and salt stresses in transgenic tobacco plants. It is suggested that MfMIPS1 is induced by diverse environmental factors and confers resistance to various abiotic stresses.