Core clock component MtLUX controls shoot architecture through repression of MtTB1/MtTCP1A in Medicago truncatula

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.

In silico genome-wide identification,phylogeny and expression analysis of the R2R3-MYB gene family in Medicago truncatula

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.

Synonymous codon usage pattern in model legume Medicago truncatula

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.

Phytosulfokine-α Promotes Root Growth by Repressing Expression of Pectin Methylesterase Inhibitor (PMEI) Genes in Medicago truncatula

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.

蒺藜苜蓿(Medicago truncatula)全长转录组测序及分析

为了深入分析和探索豆科模式植物蒺藜苜蓿的mRNA完整结构,使用单分子长读数测序技术(single-molecule long-read sequencing technology,SMRT)对蒺藜苜蓿进行全长转录组测序及分析。共获得7 728 183个subread和509 014条全长非嵌合序列(full-length non-chimeric read,FLNC),通过比对分析发现,94.36%的序列与93.01%的序列分别与蒺藜苜蓿R108与A17参考基因组匹配。总计存在8 406种可变性剪接,其中主要的剪接方式为内含子保留(intron retention,RI)。共发现23 926个基因,其中12 049个基因存在295 545条转录本,在这些转录本中至少存在一个poly(A)位点。此外,共鉴定出3 223条转录因子,6 595条长非编码RNA(long non-coding RNA,lncRNA)和479条融合转录本。使用SMRT技术能够深入发掘蒺藜苜蓿转录数据,也为更好地利用蒺藜苜蓿基因组资源提供数据补充。

蒺藜苜蓿(Medicago truncatula)叶片衰老的转录组分析

为探究蒺藜苜蓿(Medicago truncatula)叶片衰老过程中相关基因的分子机制及代谢通路,本研究以其成熟叶片和衰老初期、中期、末期叶片为研究对象。通过转录组测序,比较4个发育阶段样品之间的转录组差异。结果表明,与成熟叶片相比,共筛选出2990个差异表达基因(DEGs),在所有衰老样本中表达水平均发生显著变化,其中有2684条(89.77%)可进行基因功能注释;同时利用实时荧光定量PCR(qRT-PCR)结果证实了转录组测序的可靠性。进一步分析发现,DEGs涉及高等植物转录因子33个家族,包括ERF、WRKY、bHLH、MYB、NAC和bZIP。此外,还发现了837个与叶片衰老相关的基因。本研究发现了蒺藜苜蓿叶片衰老过程中大量差异表达的基因,为进一步分析蒺藜苜蓿叶片发育和衰老的调控机制提供参考。

蒺藜苜蓿(Medicago truncatula)MtAP2转录因子的克隆及自激活检测

AP2转录因子在植物生长发育过程中具有重要作用,为深入研究蒺藜苜蓿MtAP2转录因子的功能,于蒺藜苜蓿中克隆MtAP2转录因子,进行生物信息学分析,构建诱饵表达载体PGBKT7-MtAP2并转入酵母菌中,进行自激活活性检测。结果显示,本研究成功克隆了蒺藜苜蓿MtAP2转录因子,其开放阅读框为624 bp,编码207个氨基酸。生物信息学分析显示,蒺藜苜蓿MtAP2蛋白与鹰嘴豆的亲缘关系最为接近。诱饵表达载体PGBKT7-AP2构建成功,转入到Y2H酵母菌中,且通过在营养缺陷型培养基培养证明其没有自激活活性。本研究成功克隆了蒺藜苜蓿MtAP2转录因子,构建了酵母诱饵表达载体PGBKT7-MtAP2,并证明其不存在自激活现象,后续可进行互作蛋白筛选。

Medicago truncatula resources to study legume biology and symbiotic nitrogen fixation

Medicago truncatula is a chosen model for legumes towards deciphering fundamental legume biology,especially symbiotic nitrogen fixation.Current genomic resources for M.truncatula include a completed whole genome sequence information for R108 and Jemalong A17 accessions along with the sparse draft genome sequences for other 226 M.truncatula accessions.These genomic resources are complemented by the availability of mutant resources such as retrotransposon(Tnt1)insertion mutants in R108 and fast neutron bombardment(FNB)mutants in A17.In addition,several M.truncatula databases such as small secreted peptides(SSPs)database,transporter protein database,gene expression atlas,proteomic atlas,and metabolite atlas are available to the research community.This review describes these resources and provide information regarding how to access these resources.

CYP72A67 Catalyzes a Key Oxidative Step in Medicago truncatula Hemolytic Saponin Biosynthesis

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.

The Medicago truncatula Sucrose Transporter Family： Characterization and Implication of Key Members in Carbon Partitioning towards Arbuscular Mycorrhizal Fungi

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.

ABA-Mediated Inhibition of Germination Is Related to the Inhibition of Genes Encoding Cell-Wall Biosynthetic and Architecture： Modifying Enzymes and Structural Proteins in Medicago truncatula Embryo Axis

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.

截形苜蓿(Medicago truncatula)Dof 32基因启动子克隆及功能区缺失分析

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。

Rhizobium Lipo-chitooligosaccharide Signaling Triggers Accumulation of Cytokinins in Medicago truncatula Roots

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.

Circadian rhythms driving a fast-paced root clock implicate species-specific regulation in Medicago truncatula

Plants have a hierarchical circadian structure comprising multiple tissue-specific oscillators that operate at different speeds and regulate the expression of distinct sets of genes in different organs.However,the identity of the genes differentially regulated by the circadian clock in different organs,such as roots,and how their oscillations create functional specialization remain unclear.Here,we profiled the diurnal and circadian landscapes of the shoots and roots of Medicago truncatula and identified the conserved regulatory sequences contributing to transcriptome oscillations in each organ.We found that the light-dark cycles strongly affect the global transcriptome oscillation in roots,and many clock genes oscillate only in shoots.Moreover,many key genes involved in nitrogen fixation are regulated by circadian rhythms.Surprisingly,the root clock runs faster than the shoot clock,which is contrary to the hierarchical circadian structure showing a slow-paced root clock in both detached and intact Arabidopsis thaliana(L.)Heynh.roots.Our result provides important clues about the species-specific circadian regulatory mechanism,which is often overlooked,and possibly coordinates the timing between shoots and roots independent of the current prevailing model.

Differential mRNA Translation in Medicago truncatula Accessions with Contrasting Responses to Ozone-Induced Oxidative Stress

Acute ozone is a model abiotic elicitor of oxidative stress and a useful tool for understanding biochemical and molecular events during oxidative signaling. Two Medicago truncatula accessions with contrasting responses to ozone were used to examine translational regulation during ozone stress. In ozone-resistant JE154, significant reduction in ri- bosome loading was observed within one hour of ozone treatment, suggesting energy homeostasis as a vital factor for oxidative stress management. Polysomal RNA-based expression profiling with Affymetrix arrays revealed extensive changes in the translatomes of both accessions. Messenger RNAs with low GC content in their 5＇ and 3＇-UTRs were pref- erentially associated with polysomes during oxidative stress. Genebins analysis revealed extensive changes in various gene ontologies in both accessions. Extensive changes in nicotinate and nicotinamide metabolism genes were corrobo- rated with increased levels of NAD＋ and NADH in JE154. The significantly lower NAD＋：NADH redox status in JE154, in conjunction with higher ATP amounts, provided a cellular milieu conducive for overcoming oxidative stress. Low levels of ATP, NADH, and suppression of antioxidant defense responses, abet build-up of ozone-derived ROS and ultimately lead to oxidative cell death in Jemalong.

Depletion of tyrosyl-DNA phosphodiesterase 1a(MtTdp1a)affects transposon expression in Medicago truncatula

The role of plant tyrosyl-DNA phosphodiesteritdase 1a in genome stability is studied using a Medicago Etruncatula Mt Tdp1a-depleted line. Lack of Mt Tdp1 a results e in a 39% reduction of methylated cytosines as compared thto control. RNA-Seq analyses revealed that 11 DNA transto posons and 22 retrotransposons were differentially exrpressed in the Tdp1a-2a line. Among them all, DNA etttransposons（Mu DR, h AT, DNA3-11_Mad） and seven retroetransposons（LTR（Long Terminal Repeat）/Gipsy, LTR/Copia,LLTR and Non LTR/L1） were down-regulated, while the15 retrotransposons were up regulated. Results suggest that the occurrence of stress-responsive cis-elements as well as changes in the methylation pattern at the LTR promoters might be responsible for the enhanced retrotransposon transcription.

Genome-wide analysis of nucleotide-binding site disease resistance genes in Medicago truncatula

The class of nucleotide-binding site(NBS)-Leucine-rich repeat(LRR) disease resistance genes play an important role in defending plants from a variety of pathogens and insect pests. Consequently, many NBS-LRR genes have been identified in various plant species. In this study, we identified 617 NBS-encoding genes in the Medicago truncatula genome(Mt3.5v5) and divided them into two groups, regular(490) and non-regular(127) NBSLRR genes. The regular NBS-LRR genes were characterized on the bases of structural diversity, chromosomal location, gene duplication, conserved protein motifs, and EST expression profiling. According to N-terminal motifs and LRR motifs, the 490 regular NBS-LRR genes were then classified into 10 types: CC-NBS(4), CC-NBS-LRR(212), TIR-NBS(20), TIR-NBS-LRR(160), TIR-NBS-TIR(1), TIR-NBS-TIR-LRR(2), NBS-TIR(7), NBS-TIR-LRR(1), NBS(10), and NBS-LRR(73). Analysis of the physical location and duplications of the regular NBS-LRR genes revealed that the M. truncatula genome is similar to rice. Interestingly, we found that TIR-type genes are more frequently expressed than non-TIR-type genes in M. truncatula, whereas the number of non-TIR-type regular NBSLRR genes was greater than TIR-type genes, suggesting the gene expression was not associated with the total number of NBS-LRR genes. Moreover, we found that the phylogenetic tree supported our division of the regular NBS-LRR genes into two distinct clades(TIR-type and non-TIR-type), but some of the non-TIR-type lineages contain TIR-type genes. These analyses provide a robust database of NBS-LRR genes in M. truncatula that will facilitate the isolation of new resistance genes and breeding strategies to engineer disease resistance in leguminous crop.

Molecular studies of the Medicago truncatula MtAnn3 gene involved in root hair deformation

The full-length annexin gene,MtAnn3,of Medicago truncatula was cloned by 5' RACE.Compared with typical annexins,which contain a head domain and four homologous repeats in the conserved core domain,the MtAnn3 protein has only one repeat in the core domain.MtAnn3 can bind cell membranes when transiently expressed in onion epidermal cells.Agrobacterium rhizogenes-mediated transformation of MtAnn3 into Medicago roots revealed that overexpression of the gene can change the polarity of root hair growth in Ca2+-free medium.The plant hormone cytokinin was able to upregulate the expression of MtAnn3.While MtAnn3 transcripts were detected in young nodules,expression was not nodule-specific,and could be detected at high levels in the roots,stems and leaves as well.

Reactive Oxygen Species Acted as a Bystander Signals to Generate Bio-effects in Low-Energy Ion Irradiated Medicago truncatula Seeds

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

蒺藜苜蓿MtMYB16基因克隆、表达及转录自激活分析

植物中MYB转录因子数量众多,功能多样,在植物生长发育、逆境响应等多方面发挥着重要作用。为研究MYB转录因子在蒺藜苜蓿中的功能,本研究采用PCR技术从蒺藜苜蓿中克隆MtMYB16基因,该基因开放阅读框1074 bp,共编码357个氨基酸。进化树分析结果显示,MtMYB16蛋白与白花草木樨中MYB蛋白同源性最高。亚细胞定位结果显示,MtMYB16蛋白定位在细胞核中。酵母自激活检测结果显示,MtMYB16蛋白具有自激活活性,自激活结构域位于C端。表达分析结果显示,MtMYB16在蒺藜苜蓿根、茎、叶、花、果实中均有表达,在根中表达水平最高;IAA、MeJA能够降低MtMYB16表达水平;盐胁迫和干旱胁迫下,MtMYB16表达量在1.0 h时迅速增加,说明MtMYB16可能具有响应盐及干旱胁迫的功能。