Heterotrimeric G proteins are crucial transducers of signaling from receptors,participating in growth and development,as well as in responses to biotic and abiotic stimuli.However,little is known about their roles in ...Heterotrimeric G proteins are crucial transducers of signaling from receptors,participating in growth and development,as well as in responses to biotic and abiotic stimuli.However,little is known about their roles in regulating various yield-related traits in legumes.In this study,we systematically analyzed the functions of two G-protein-encoding genes,MtGα1 and MtGβ1,along with Regulator of G-protein Signaling1(MtRGS1),in Medicago truncatula.All three genes were ubiquitously expressed in roots,stems,leaves,nodules,flowers,and pods.We generated the knockout mutants Mtgα1,Mtgβ1,and Mtrgs1 using CRISPR/Cas9 and assessed their growth and development.MtGα1 knockout resulted in slightly shorter plants with smaller pods and shorter spines,but larger seeds,without affecting overall biomass or other traits.MtGβ1 knockout led to dwarfism,weak root development,a severe drop in biomass production,smaller legume pods with shorter spines,and smaller seeds.However,the Mtrgs1 mutants were largely similar to wild-type plants,with few significant defects in growth and development.We also investigated the symbiotic nodulation-related phenotypes of these mutants,discovering that Mtgβ1 mutants produce lighter nodules,whereas Mtgα1 and Mtrgs1 mutants have normal nodulation phenotypes similar to those of wild-type plants.These observations suggest that MtGβ1 positively regulates nodulation,although the detailed mechanisms by which G proteins regulate symbiotic nitrogen fixation in M.truncatula remain to be explored.This work provides potentially valuable genetic resources for further functional analysis and elucidation of the molecular mechanisms of G proteins in this model legume.展开更多
Dof (DNA binding with one finger)基因家族是植物特有的一类转录因子,参与植物生长发育过程中种子的萌发、休眠及植物的开花等过程,在逆境响应中起到调控作用。为了研究截形苜蓿(Medicago truncatula) Dof 32启动子的组成及功能,以截...Dof (DNA binding with one finger)基因家族是植物特有的一类转录因子,参与植物生长发育过程中种子的萌发、休眠及植物的开花等过程,在逆境响应中起到调控作用。为了研究截形苜蓿(Medicago truncatula) Dof 32启动子的组成及功能,以截形苜蓿DNA为模板,进行启动子片段扩增,预测启动子相关转录因子的结合位点。据此对5’端序列进行缺失克隆,构建相应的表达载体并转化拟南芥,通过GUS染色和实时荧光定量分析各功能区缺失启动子在自然条件下和盐胁迫条件下的生物学活性,探索启动子核心区域及顺式作用元件的功能。结果表明:除DPS3F启动子缺失片段活性偏弱外,全长片段DP32及其余2个缺失片段DPS1F、DPS2F的活性均较强,能够驱动下游GUS基因的表达。在盐胁迫条件下DP32、DPS1F、DPS2F转基因拟南芥GUS基因的表达量较自然条件下显著上升。本研究为探索Dof基因在截形苜蓿抗逆过程中的调控机理提供资料。展开更多
The R2R3-MYB genes make up one of the largest transcription factor families in plants, and play regulatory roles in various biological processes such as development, metabolism and defense response. Although genome-wi...The R2R3-MYB genes make up one of the largest transcription factor families in plants, and play regulatory roles in various biological processes such as development, metabolism and defense response. Although genome-wide analyses of this gene family have been conducted in several species, R2R3-MYB genes have not been systematically analyzed in Medicago truncatula, a sequenced model legume plant. Here, we performed a comprehensive, genome-wide computational analysis of the structural characteristics, phylogeny, functions and expression patterns of M. truncatula R2R3-MYB genes. DNA binding domains are highly conserved among the 155 putative MtR2R3-MYB proteins that we identified. Chromosomal location analysis revealed that these genes were distributed across all eight chromosomes. Results showed that the expansion of the MtR2R3-MYB family was mainly attributable to segmental duplication and tandem duplication. A comprehensive classification was performed based on phylogenetic analysis of the R2R3-MYB gene families in M. truncatula, Arabidopsis thaliana and other plant species. Evolutionary relationships within clades were supported by clade-specific conserved motifs outside the MYB domain. Species-specific clades have been gained or lost during evolution, resulting in functional divergence. Also, tissue-specific expression patterns were investigated. The functions of stress response-related clades were further verified by the changes in transcript levels of representative R2R3-MYB genes upon treatment with abiotic and biotic stresses. This study is the first report on identification and characterization of R2R3-MYB gene family based on the genome of M. truncatula, and will facilitate functional analysis of this gene family in the future.展开更多
Synonymous codon usage pattern presumably reflects gene expression optimization as a result of molecular evolution. Though much attention has been paid to various model organisms ranging from prokaryotes to eukaryotes...Synonymous codon usage pattern presumably reflects gene expression optimization as a result of molecular evolution. Though much attention has been paid to various model organisms ranging from prokaryotes to eukaryotes, codon usage has yet been extensively investigated for model legume Medicago truncatula. In present study, 39 531 available coding sequences （CDSs） from M. truncatula were examined for codon usage bias （CUB）. Based on analyses including neutrality plots, effective number of codons plots, and correlations between optimal codons frequency and codon adaptation index, we conclude that natural selection is a major driving force in M. truncatula CUB. We have identified 30 optimal codons encoding 18 amino acids based on relative synonymous codon usage. These optimal codons characteristically end with A or T, except for AGG and TTG encoding arginine and leucine respectively. Optimal codon usage is positively correlated with the GC content at three nucleotide positions of codons and the GC content of CDSs. The abundance of expressed sequence tag is a proxy for gene expression intensity in the legume, but has no relatedness with either CDS length or GC content. Collectively, we unravel the synonymous codon usage pattern in M. truncatula, which may serve as the valuable information on genetic engineering of the model legume and forage crop.展开更多
Objective: To investigate the potential of anemonin and Clematis flammula(C.flammula) extracts against infective organisms.Methods: The molluscicidal activities of anemonin and C.flammula extracts against Galba trunca...Objective: To investigate the potential of anemonin and Clematis flammula(C.flammula) extracts against infective organisms.Methods: The molluscicidal activities of anemonin and C.flammula extracts against Galba truncatula Mull.(Lymnaeidae) and Fasciola hepatica larval stages contaminating this snail in Tunisia were assessed by testing six groups of snails in 250 mL of extracts and aqueous dechlorinated solutions with different concentrations(ranging from 2.5 to 20.0 mg/L) for 48 h.Besides, the antifungal potential of C.flammula leaves and flowers was evaluated by using the diffusion agar and broth dilution methods against four fungal strains: Aspergillus niger, Pythium catenulatum, Rhizoctonia solani and Fusarium phyllophilum.Results: As a result, hexane and ethyl acetate flower extracts exhibited significant molluscicidal activities with LC_(50) median lethal concentrations values of 11.87 and 11.65 mg/L, respectively while LC_(50) value of anemonin was 9.64 mg/L after 48 h exposure.The flower extracts showed a larvicidal effect with a deterioration rate exceeding 35.39% where flower ethyl acetate residue gave a deterioration rate of cercariae close to 97%.Moreover, C.flammula extracts were not noxious to the associated fauna survival.All extracts inhibited the growth of P.catenulatum, the leaves and flowers methanolic extracts had the more important fungicide action with minimum inhibitory concentrations of 1.56 and 3.12 mg/mL together with minimum fungistatic concentrations of 3.12 and 6.25 mg/mL respectively.Only flower extracts were active against Rhizoctonia solani with minimum inhibitory concentrations varying between 0.70 and 1.56 mg/mL and 6.25 mg/mL of minimum fungistatic concentration.Phytochemical tests showed that the antifungal activity may be attributed to the presence of the flavonoids/saponins in the methanolic extracts and the molluscicide effects could be due to the richness of hexane and ethyl acetate extracts on sterols and triterpenoids.Conclusions: This study emphasizes the important molluscicidal and antiparasitic effects of flower ethyl acetate extracts and anemonin compound as well as the considerable antifungal activities of methanolic extracts.These results improve the therapeutic virtues of C.flammula aerial part extracts.展开更多
Phytosulfokine-α(PSK-α),a sulfated pentapeptide with the sequence YIYTQ,is encoded by a small precursor gene family in Arabidopsis.PSK-αregulates multiple growth and developmental processes as a novel peptide hormo...Phytosulfokine-α(PSK-α),a sulfated pentapeptide with the sequence YIYTQ,is encoded by a small precursor gene family in Arabidopsis.PSK-αregulates multiple growth and developmental processes as a novel peptide hormone.Despite its importance,functions of PSK-αin M.truncatula growth remains unknown.In this study,we identified five genes to encode PSK-αprecursors in M.truncatula.All of these precursors possess conserved PSK-αsignature motif.Expression pattern analysis of these MtPSK genes revealed that each gene was expressed in a tissue-specific or ubiquitous pattern and three of them were remarkably expressed in root.Treatment of M.truncatula seedlings with synthetic PSK-αpeptide significantly promoted root elongation.In addition,expression analysis of downstream genes by RNA-seq and qRT-PCR assays suggested that PSK-αsignaling might regulate cell wall structure via PMEI-PME module to promote root cell growth.Taken together,our results shed light on the mechanism by which PSK-αpromotes root growth in M.truncatula,providing a new resource for improvement of root growth in agriculture.展开更多
Plants are capable of regulating their shoot architecture in response to diverse internal and external environments.The circadian clock is an adaptive mechanism that integrates information from internal and ambient co...Plants are capable of regulating their shoot architecture in response to diverse internal and external environments.The circadian clock is an adaptive mechanism that integrates information from internal and ambient conditions to help plants cope with recurring environmental fluctuations.Despite the current understanding of plant circadian clock and genetic framework underlying plant shoot architecture,the intricate connection between these two adaptive mechanisms remains largely unclear.In this study,we elucidated how the core clock gene LUX ARRHYTHMO(LUX)regulates shoot architecture in the model legume plant Medicago truncatula.We show that mtlux mutant displays increased main stem height,reduced lateral shoot length,and decreased the number of lateral branches and biomass yield.Gene expression analysis revealed that Mt LUX regulated shoot architecture by repressing the expression of strigolactone receptor MtD14 and MtTB1/MtTCP1A,a TCP gene that functions centrally in modulating shoot architecture.In vivo and in vitro experiments showed that Mt LUX directly binds to a cis-element in the promoter of MtTB1/MtTCP1A,suggesting that Mt LUX regulates branching by rhythmically suppressing MtTB1/MtTCP1A.This work demonstrates the regulatory effect of the circadian clock on shoot architecture,offering a new understanding underlying the genetic basis towards the flexibility of plant shoot architecture.展开更多
Background: It is important to understand the mechanisms of bio-effects in low-energy ion irradiated plant seeds. Recently, the increasing data have been accumulated concerning
Legumes can form a symbiotic relationship with rhizobia to fix atmospheric nitrogen, which is essential for their growth. This process is initiated by the recognition of molecular signals known as Nod factors(NFs), re...Legumes can form a symbiotic relationship with rhizobia to fix atmospheric nitrogen, which is essential for their growth. This process is initiated by the recognition of molecular signals known as Nod factors(NFs), released by rhizobia. These signals are perceived by NF receptors located on the plasma membrane, such as LYK3 and NFP in Medicago truncatula(Amor et al., 2003;Limpens et al., 2003).展开更多
Root nodule symbiosis(RNs)between legumes and rhizobia is a major source of nitrogen in agricultural systems.Effective symbiosis requires precise regulation of plant defense responses.The role of the defense hormone j...Root nodule symbiosis(RNs)between legumes and rhizobia is a major source of nitrogen in agricultural systems.Effective symbiosis requires precise regulation of plant defense responses.The role of the defense hormone jasmonic acid(JA)in the immune response has been extensively studied.Current research shows that JA can play either a positive or negative regulatory role in RNS depending on its concentration,but the molecular mechanisms remain to be elucidated.In this study,we found that inoculation with the rhizobia Sm1021 induces the JA pathway in Medicago truncatula,and blocking the JA pathway significantly reduces the number of infection threads.Mutations in the MtMYc2 gene,which encodes a JA signaling master transcription factor,significantly inhibited rhizobia infection,terminal differentiation,and symbiotic cell formation.Combining RNA sequencing and chromatin immunoprecipitation sequencing,we discovered that MtMYc2 regulates the expression of nodule-specific MtDNF2,MtNAD1,and MtSymCRK to suppress host defense,while it activates MtDNF1 expression to regulate the maturation of MtNCRs,which in turn promotes bacteroid formation.More importantly,MtMYC2 participates in symbiotic signal transduction by promoting the expression of MtiPD3.Notably,the MtMYC2-MtiPD3 transcriptional regulatory module is specifically present in legumes,and the Mtmyc2 mutants are susceptible to the infection by the pathogen Rhizoctonia solani.Collectively,these findings reveal the molecular mechanisms of how the JA pathway regulates RNS,broadening our understanding of the roles of JA in plant-microbe interactions.展开更多
In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmac...In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 （CYP72A67） involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethyl- methanesulfonate （EMS） mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s （CYP72A68） responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities （leaves/roots） of sapogenin synthesis.展开更多
Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid （ABA） but how this hormone controls seed germination is...Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid （ABA） but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.展开更多
We identified de novo sucrose transporter （SUT） genes involved in long-distance transport of sucrose from photosynthetic source leaves towards sink organs in the model leguminous species Medicago truncatula. The ide...We identified de novo sucrose transporter （SUT） genes involved in long-distance transport of sucrose from photosynthetic source leaves towards sink organs in the model leguminous species Medicago truncatula. The iden- tification and functional analysis of sugar transporters provide key information on mechanisms that underlie carbon partitioning in plant-microorganism interactions. In that way, full-length sequences of the M. truncatula SUT （MtSUT） family were retrieved and biochemical characterization of MtSUT members was performed by heterologous expression in yeast. The MtSUT family now comprises six genes which distribute among Dicotyledonous clades. MtSUTI-1 and MtSUT4-1 are key members in regard to their expression profiles in source leaves and sink roots and were characterized as functional H~/sucrose transporters. Physiological and molecular responses to phosphorus supply and inoculation by the arbuscular mycorrhizal fungus （AMF） Glomus intraradices was studied by gene expression and sugar quantification analyses. Sucrose represents the main sugar transport form in M. truncatula and the expression profiles of MtSUTI-1, MtSUT2, and MtSUT4-1 highlight a fine-tuning regulation for beneficial sugar fluxes towards the fungal symbiont. Taken together, these results suggest distinct functions for proteins from the SUT1, SUT2, and SUT4 clades in plant and in bio- trophic interactions.展开更多
Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides （LCOs）. Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation....Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides （LCOs）. Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accu- mulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizo- bium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and iso- pentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase （CCaMK）, a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein21 Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.展开更多
In the present study, the phosphorus-absorption capacity of transgenic Arabidopsis plants ectopically ex- pressing a novel phytase gene from Medicago truncatula Barrel Medic was evaluated. A full-length cDNA encoding ...In the present study, the phosphorus-absorption capacity of transgenic Arabidopsis plants ectopically ex- pressing a novel phytase gene from Medicago truncatula Barrel Medic was evaluated. A full-length cDNA encoding an extracellular form of phytase was isolated from the model legume M. truncatula. The phytase gene （MtPHY1） has an open reading frame of I 632 bp predicted to encode 543 amino acids, including an N- terminal signal peptide of 27 amino acids. The genomic sequence of the MtPHY1 gene is 5 151 bp, containing seven exons interrupted by six introns. Under high-Pi （2 mmol/L） growth conditions, higher levels of MtPHY1 transcripts accumulated in the leaf and stem than in the root. The transcript level was reduced in the stem and increased in the root, with no obvious changes in the hybridization signal detected in the leaf under IowPi （10 pmol/L） conditions. Chimeric transgenes were constructed by placing MtPHY1 under the control of the constitutive CaMV35S promoter and the root-specific MtPT1 promoter. Phytase activities in root apoplast of transgenic Arabidopsis were 12.3- to 16.2-fold of that in control plants. The phytase expressed was secreted into the rhizosphere, as demonstrated by HPLC analysis of phytate degradation by root exudates. Ectopic expression of MtPHY1 in Arabidopsis, leading to significant improvement in organic phosphorus absorption and plant growth, indicated that MtPHY1 has great potential for improving plant phosphorus absorption and phytoremediation.展开更多
基金supported by the Major Demonstration Project of the Open Competition for Seed Industry Science and Technology Innovation in Inner Mongolia(2022JBGS0016)the National Natural Science Foundation of China(32370253)+1 种基金the National Key Research and Development Program of China(2023YFF1001400,and 2022YFA0912100)a Faculty Resources Project of the College of Life Sciences,Inner Mongolia University(2022-101).
文摘Heterotrimeric G proteins are crucial transducers of signaling from receptors,participating in growth and development,as well as in responses to biotic and abiotic stimuli.However,little is known about their roles in regulating various yield-related traits in legumes.In this study,we systematically analyzed the functions of two G-protein-encoding genes,MtGα1 and MtGβ1,along with Regulator of G-protein Signaling1(MtRGS1),in Medicago truncatula.All three genes were ubiquitously expressed in roots,stems,leaves,nodules,flowers,and pods.We generated the knockout mutants Mtgα1,Mtgβ1,and Mtrgs1 using CRISPR/Cas9 and assessed their growth and development.MtGα1 knockout resulted in slightly shorter plants with smaller pods and shorter spines,but larger seeds,without affecting overall biomass or other traits.MtGβ1 knockout led to dwarfism,weak root development,a severe drop in biomass production,smaller legume pods with shorter spines,and smaller seeds.However,the Mtrgs1 mutants were largely similar to wild-type plants,with few significant defects in growth and development.We also investigated the symbiotic nodulation-related phenotypes of these mutants,discovering that Mtgβ1 mutants produce lighter nodules,whereas Mtgα1 and Mtrgs1 mutants have normal nodulation phenotypes similar to those of wild-type plants.These observations suggest that MtGβ1 positively regulates nodulation,although the detailed mechanisms by which G proteins regulate symbiotic nitrogen fixation in M.truncatula remain to be explored.This work provides potentially valuable genetic resources for further functional analysis and elucidation of the molecular mechanisms of G proteins in this model legume.
文摘Dof (DNA binding with one finger)基因家族是植物特有的一类转录因子,参与植物生长发育过程中种子的萌发、休眠及植物的开花等过程,在逆境响应中起到调控作用。为了研究截形苜蓿(Medicago truncatula) Dof 32启动子的组成及功能,以截形苜蓿DNA为模板,进行启动子片段扩增,预测启动子相关转录因子的结合位点。据此对5’端序列进行缺失克隆,构建相应的表达载体并转化拟南芥,通过GUS染色和实时荧光定量分析各功能区缺失启动子在自然条件下和盐胁迫条件下的生物学活性,探索启动子核心区域及顺式作用元件的功能。结果表明:除DPS3F启动子缺失片段活性偏弱外,全长片段DP32及其余2个缺失片段DPS1F、DPS2F的活性均较强,能够驱动下游GUS基因的表达。在盐胁迫条件下DP32、DPS1F、DPS2F转基因拟南芥GUS基因的表达量较自然条件下显著上升。本研究为探索Dof基因在截形苜蓿抗逆过程中的调控机理提供资料。
基金supported by the National Natural Science Foundation of China(31372362)
文摘The R2R3-MYB genes make up one of the largest transcription factor families in plants, and play regulatory roles in various biological processes such as development, metabolism and defense response. Although genome-wide analyses of this gene family have been conducted in several species, R2R3-MYB genes have not been systematically analyzed in Medicago truncatula, a sequenced model legume plant. Here, we performed a comprehensive, genome-wide computational analysis of the structural characteristics, phylogeny, functions and expression patterns of M. truncatula R2R3-MYB genes. DNA binding domains are highly conserved among the 155 putative MtR2R3-MYB proteins that we identified. Chromosomal location analysis revealed that these genes were distributed across all eight chromosomes. Results showed that the expansion of the MtR2R3-MYB family was mainly attributable to segmental duplication and tandem duplication. A comprehensive classification was performed based on phylogenetic analysis of the R2R3-MYB gene families in M. truncatula, Arabidopsis thaliana and other plant species. Evolutionary relationships within clades were supported by clade-specific conserved motifs outside the MYB domain. Species-specific clades have been gained or lost during evolution, resulting in functional divergence. Also, tissue-specific expression patterns were investigated. The functions of stress response-related clades were further verified by the changes in transcript levels of representative R2R3-MYB genes upon treatment with abiotic and biotic stresses. This study is the first report on identification and characterization of R2R3-MYB gene family based on the genome of M. truncatula, and will facilitate functional analysis of this gene family in the future.
基金supported by the National Basic Research Program of China (2014CB138702)the National Natural Science Foundation of China (31502001)
文摘Synonymous codon usage pattern presumably reflects gene expression optimization as a result of molecular evolution. Though much attention has been paid to various model organisms ranging from prokaryotes to eukaryotes, codon usage has yet been extensively investigated for model legume Medicago truncatula. In present study, 39 531 available coding sequences （CDSs） from M. truncatula were examined for codon usage bias （CUB）. Based on analyses including neutrality plots, effective number of codons plots, and correlations between optimal codons frequency and codon adaptation index, we conclude that natural selection is a major driving force in M. truncatula CUB. We have identified 30 optimal codons encoding 18 amino acids based on relative synonymous codon usage. These optimal codons characteristically end with A or T, except for AGG and TTG encoding arginine and leucine respectively. Optimal codon usage is positively correlated with the GC content at three nucleotide positions of codons and the GC content of CDSs. The abundance of expressed sequence tag is a proxy for gene expression intensity in the legume, but has no relatedness with either CDS length or GC content. Collectively, we unravel the synonymous codon usage pattern in M. truncatula, which may serve as the valuable information on genetic engineering of the model legume and forage crop.
基金supported by the Tunisian National Ministry of Higher Education and Scientific Research
文摘Objective: To investigate the potential of anemonin and Clematis flammula(C.flammula) extracts against infective organisms.Methods: The molluscicidal activities of anemonin and C.flammula extracts against Galba truncatula Mull.(Lymnaeidae) and Fasciola hepatica larval stages contaminating this snail in Tunisia were assessed by testing six groups of snails in 250 mL of extracts and aqueous dechlorinated solutions with different concentrations(ranging from 2.5 to 20.0 mg/L) for 48 h.Besides, the antifungal potential of C.flammula leaves and flowers was evaluated by using the diffusion agar and broth dilution methods against four fungal strains: Aspergillus niger, Pythium catenulatum, Rhizoctonia solani and Fusarium phyllophilum.Results: As a result, hexane and ethyl acetate flower extracts exhibited significant molluscicidal activities with LC_(50) median lethal concentrations values of 11.87 and 11.65 mg/L, respectively while LC_(50) value of anemonin was 9.64 mg/L after 48 h exposure.The flower extracts showed a larvicidal effect with a deterioration rate exceeding 35.39% where flower ethyl acetate residue gave a deterioration rate of cercariae close to 97%.Moreover, C.flammula extracts were not noxious to the associated fauna survival.All extracts inhibited the growth of P.catenulatum, the leaves and flowers methanolic extracts had the more important fungicide action with minimum inhibitory concentrations of 1.56 and 3.12 mg/mL together with minimum fungistatic concentrations of 3.12 and 6.25 mg/mL respectively.Only flower extracts were active against Rhizoctonia solani with minimum inhibitory concentrations varying between 0.70 and 1.56 mg/mL and 6.25 mg/mL of minimum fungistatic concentration.Phytochemical tests showed that the antifungal activity may be attributed to the presence of the flavonoids/saponins in the methanolic extracts and the molluscicide effects could be due to the richness of hexane and ethyl acetate extracts on sterols and triterpenoids.Conclusions: This study emphasizes the important molluscicidal and antiparasitic effects of flower ethyl acetate extracts and anemonin compound as well as the considerable antifungal activities of methanolic extracts.These results improve the therapeutic virtues of C.flammula aerial part extracts.
基金This work is supported by the National Natural Science Foundation of China(31500197).
文摘Phytosulfokine-α(PSK-α),a sulfated pentapeptide with the sequence YIYTQ,is encoded by a small precursor gene family in Arabidopsis.PSK-αregulates multiple growth and developmental processes as a novel peptide hormone.Despite its importance,functions of PSK-αin M.truncatula growth remains unknown.In this study,we identified five genes to encode PSK-αprecursors in M.truncatula.All of these precursors possess conserved PSK-αsignature motif.Expression pattern analysis of these MtPSK genes revealed that each gene was expressed in a tissue-specific or ubiquitous pattern and three of them were remarkably expressed in root.Treatment of M.truncatula seedlings with synthetic PSK-αpeptide significantly promoted root elongation.In addition,expression analysis of downstream genes by RNA-seq and qRT-PCR assays suggested that PSK-αsignaling might regulate cell wall structure via PMEI-PME module to promote root cell growth.Taken together,our results shed light on the mechanism by which PSK-αpromotes root growth in M.truncatula,providing a new resource for improvement of root growth in agriculture.
基金supported by Laboratory of Lingnan Modern Agriculture Project(NZ2021001)State Key Laboratory for Conservation and Utilization of Subtropical Agrobioresources(SKICUSA-a202007)Natural Science Foundation of Guangdong Province(2022A1515011027,2019A1515012009)。
文摘Plants are capable of regulating their shoot architecture in response to diverse internal and external environments.The circadian clock is an adaptive mechanism that integrates information from internal and ambient conditions to help plants cope with recurring environmental fluctuations.Despite the current understanding of plant circadian clock and genetic framework underlying plant shoot architecture,the intricate connection between these two adaptive mechanisms remains largely unclear.In this study,we elucidated how the core clock gene LUX ARRHYTHMO(LUX)regulates shoot architecture in the model legume plant Medicago truncatula.We show that mtlux mutant displays increased main stem height,reduced lateral shoot length,and decreased the number of lateral branches and biomass yield.Gene expression analysis revealed that Mt LUX regulated shoot architecture by repressing the expression of strigolactone receptor MtD14 and MtTB1/MtTCP1A,a TCP gene that functions centrally in modulating shoot architecture.In vivo and in vitro experiments showed that Mt LUX directly binds to a cis-element in the promoter of MtTB1/MtTCP1A,suggesting that Mt LUX regulates branching by rhythmically suppressing MtTB1/MtTCP1A.This work demonstrates the regulatory effect of the circadian clock on shoot architecture,offering a new understanding underlying the genetic basis towards the flexibility of plant shoot architecture.
文摘Background: It is important to understand the mechanisms of bio-effects in low-energy ion irradiated plant seeds. Recently, the increasing data have been accumulated concerning
基金supported by the National Key Research and Development Program of China (2023YFD1200600)the National Natural Science Foundation of China (301/ 590224002)。
文摘Legumes can form a symbiotic relationship with rhizobia to fix atmospheric nitrogen, which is essential for their growth. This process is initiated by the recognition of molecular signals known as Nod factors(NFs), released by rhizobia. These signals are perceived by NF receptors located on the plasma membrane, such as LYK3 and NFP in Medicago truncatula(Amor et al., 2003;Limpens et al., 2003).
基金National Natural Science Foundation of China(32370253,and 32070272)the National Key Research Development Program of China(2022YFA0912100,and 2023YFF1001400).
文摘Root nodule symbiosis(RNs)between legumes and rhizobia is a major source of nitrogen in agricultural systems.Effective symbiosis requires precise regulation of plant defense responses.The role of the defense hormone jasmonic acid(JA)in the immune response has been extensively studied.Current research shows that JA can play either a positive or negative regulatory role in RNS depending on its concentration,but the molecular mechanisms remain to be elucidated.In this study,we found that inoculation with the rhizobia Sm1021 induces the JA pathway in Medicago truncatula,and blocking the JA pathway significantly reduces the number of infection threads.Mutations in the MtMYc2 gene,which encodes a JA signaling master transcription factor,significantly inhibited rhizobia infection,terminal differentiation,and symbiotic cell formation.Combining RNA sequencing and chromatin immunoprecipitation sequencing,we discovered that MtMYc2 regulates the expression of nodule-specific MtDNF2,MtNAD1,and MtSymCRK to suppress host defense,while it activates MtDNF1 expression to regulate the maturation of MtNCRs,which in turn promotes bacteroid formation.More importantly,MtMYC2 participates in symbiotic signal transduction by promoting the expression of MtiPD3.Notably,the MtMYC2-MtiPD3 transcriptional regulatory module is specifically present in legumes,and the Mtmyc2 mutants are susceptible to the infection by the pathogen Rhizoctonia solani.Collectively,these findings reveal the molecular mechanisms of how the JA pathway regulates RNS,broadening our understanding of the roles of JA in plant-microbe interactions.
文摘In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 （CYP72A67） involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethyl- methanesulfonate （EMS） mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s （CYP72A68） responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities （leaves/roots） of sapogenin synthesis.
文摘Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid （ABA） but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 μM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of α-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.
文摘We identified de novo sucrose transporter （SUT） genes involved in long-distance transport of sucrose from photosynthetic source leaves towards sink organs in the model leguminous species Medicago truncatula. The iden- tification and functional analysis of sugar transporters provide key information on mechanisms that underlie carbon partitioning in plant-microorganism interactions. In that way, full-length sequences of the M. truncatula SUT （MtSUT） family were retrieved and biochemical characterization of MtSUT members was performed by heterologous expression in yeast. The MtSUT family now comprises six genes which distribute among Dicotyledonous clades. MtSUTI-1 and MtSUT4-1 are key members in regard to their expression profiles in source leaves and sink roots and were characterized as functional H~/sucrose transporters. Physiological and molecular responses to phosphorus supply and inoculation by the arbuscular mycorrhizal fungus （AMF） Glomus intraradices was studied by gene expression and sugar quantification analyses. Sucrose represents the main sugar transport form in M. truncatula and the expression profiles of MtSUTI-1, MtSUT2, and MtSUT4-1 highlight a fine-tuning regulation for beneficial sugar fluxes towards the fungal symbiont. Taken together, these results suggest distinct functions for proteins from the SUT1, SUT2, and SUT4 clades in plant and in bio- trophic interactions.
文摘Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides （LCOs）. Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accu- mulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizo- bium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and iso- pentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase （CCaMK）, a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein21 Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.
基金Supported by the Samuel Roberts Noble Foundation and the Hebei Provincial Natural Science Foundation of China （300112）.
文摘In the present study, the phosphorus-absorption capacity of transgenic Arabidopsis plants ectopically ex- pressing a novel phytase gene from Medicago truncatula Barrel Medic was evaluated. A full-length cDNA encoding an extracellular form of phytase was isolated from the model legume M. truncatula. The phytase gene （MtPHY1） has an open reading frame of I 632 bp predicted to encode 543 amino acids, including an N- terminal signal peptide of 27 amino acids. The genomic sequence of the MtPHY1 gene is 5 151 bp, containing seven exons interrupted by six introns. Under high-Pi （2 mmol/L） growth conditions, higher levels of MtPHY1 transcripts accumulated in the leaf and stem than in the root. The transcript level was reduced in the stem and increased in the root, with no obvious changes in the hybridization signal detected in the leaf under IowPi （10 pmol/L） conditions. Chimeric transgenes were constructed by placing MtPHY1 under the control of the constitutive CaMV35S promoter and the root-specific MtPT1 promoter. Phytase activities in root apoplast of transgenic Arabidopsis were 12.3- to 16.2-fold of that in control plants. The phytase expressed was secreted into the rhizosphere, as demonstrated by HPLC analysis of phytate degradation by root exudates. Ectopic expression of MtPHY1 in Arabidopsis, leading to significant improvement in organic phosphorus absorption and plant growth, indicated that MtPHY1 has great potential for improving plant phosphorus absorption and phytoremediation.
