Morphological characterisation and genetic analysis of a bi-pistil mutant (bip) in Medicago truncatula Gaertn.

A floral organ mutant was observed in transgenic Medicago truncatula Gaertn. plants that had two separate stigmas borne on two separate styles that emerged from a single superior carpel primordium. We propose the name bi-pistil, bip for the mutation. We believe this is the first report of such a mutation in this species. Genetic and molecular analyses of the mutant were conducted. The mutant plant was crossed to a mtapetala plant with a wild-type pistil. Expression of the mutant trait in the F₁ and F₂ generations indicates that the bi-pistil trait is under the control of a single recessive gene. Other modifying genes may influence its expression. The mutation was associated with the presence of a T-DNA insert consisting of the Alfalfa mosaic virus (AMV) coat protein gene in antisense orientation and the nptII selectable marker gene. It is suggested that the mutation is due to gene disruption because multiple copies of the T-DNA were observed in the mutant. The bi-pistil gene was found to be independent of the male-sterile gene, tap. This novel mutant may assist in understanding pistil development in legumes.     

中国重要农业害虫韭菜迟眼蕈蚊多态性EST-SSR标记的开发

【背景】韭菜迟眼蕈蚊是我国重要的农业害虫,然而它的遗传资源有限。本研究旨在开发韭菜迟眼蕈蚊EST-SSR标记,为研究不同地区的韭菜迟眼蕈蚊种群结构和遗传多样性奠定基础。【方法】从韭菜迟眼蕈蚊的表达序列标签(EST序列)中设计16对简单重复序列(SSR)引物,进一步筛选出9对具有多态性的SSR引物。【结果】从42095条unigene中确定了3383个SSR位点。利用查找到的SSR位点共设计出16对引物,进一步检测筛选发现9对引物具有多态性,引物的每个位点平均有3.33个等位基因。利用9对引物对30头韭菜迟眼蕈蚊进行检测,共获得30个等位基因,观测杂合度和期望杂合度的范围分别为0.0000~0.6875和0.0370~0.6877;其中,9个位点中有5个位点显著偏离Hardy-Weinberg平衡。【结论与意义】本研究成功从迟眼蕈蚊EST序列中筛选出9个具有多态性的微卫星位点,这为进一步分析该害虫种群的遗传结构和遗传多样性奠定了基础。     

Integrative approaches to determining Csl function

While there is an ever-increasing amount of information regarding cellulose synthase catalytic subunits (CesA) and their role in the formation of the cell wall, the remainder of the enzymes that synthesize structural cell wall polysaccharides are unknown. The completion of the Arabidopsis genome and the wealth of the sequence information from other plant genome projects provide a rich resource for determining the identity of these enzymes. Arabidopsis contains six families of genes related to cellulose synthase, the cellulose synthase-like (Csl) genes. Our laboratory is taking a multidisciplinary approach to determine the function of the Csl genes, incorporating genomic, genetic and biochemical data. Information from expressed sequence tag (EST) projects has revealed the presence of Csl genes in all plant species with a significant number of ESTs. Certain Csl families appear to be missing from some species. For example, no examples of CslG ESTs have been found in rice or maize. Microarray data and reporter constructs are being used to determine the expression pattern of the CesA and Csl genes in Arabidopsis. Mutations and insertion events have been identified in a majority of the genes in the Arabidopsis CesA superfamily and are being characterized by phenotypic and biochemical analysis. While we cannot yet link the function of any of the Csl genes to their respective products, the expression and localization of these genes is consistent with the expected expression pattern of polysaccharide synthases that contribute to the primary cell wall.     

Embryo induction and regeneration from root explants of Medicago truncatula after osmotic pre-treatment

Embryo induction and regeneration from suspension culture of two Medicago truncatula cvs. (cv. R 108 1 and cv. Jemalong) have been studied. The influence of osmotic pre-treatment (1 M solution of sucrose for 48 h and 72 h) of roots as an initial explant, on embryogenic efficiency of the suspension culture was assessed. In comparison to the control, the level of abscisic acid (ABA) increased significantly after osmotic stress. The increased ABA level did not correlate with the induction of embryogenesis neither with the improved embryogenic potential of cv. R 108 1. The shortest regeneration period and the highest percent of conversion to plants were found in cv. R 108 1 after 72-h pre-treatment of roots. The efficiency of somatic embryo conversion was less after 48-h pre-treatment and much less for the untreated control. Osmotic stress did not positively affect the process of embryogenesis from root explants of cv. Jemalong, confirming its cultivar dependence. A single cell suspension fraction was produced in both Medicago trunacatula cvs. during the somatic embryo maturation stage. A higher embryogenic potential than the initial suspension culture was established only for the cell suspension originating from 72-h pre-treated roots of cv. R 108 1. The data confirms that the process of somatic embryo induction and embryo conversion from root explants of cv. R 108 1 could be promoted by osmotic stress pre-treatment.     

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

蒺藜苜蓿DGAT1基因的克隆和功能鉴定

该研究采用RT-PCR与电子克隆的方法,从蒺藜苜蓿cDNA中克隆得到2个编码二脂酰甘油酰基转移酶(diacylglycerol acyltransferase,DGAT)的基因MtDGAT1-1和MtDGAT1-2。MtDGAT1-1长1 620bp,编码539个氨基酸;MtDGAT1-2长1 524bp,编码507个氨基酸。多序列比对显示,MtDGAT1-1和MtDGAT1-2编码蛋白具有典型的植物DGAT1结构域。表达分析显示,MtDGAT1-1和MtDGAT1-2在根、茎、叶、花、种子中都有表达,在种子发育中高表达,且MtDGAT1-1于种子发育的中前期高表达,而MtDGAT1-2于种子发育的中后期高表达。酵母互补实验证实,MtDGAT1-2编码蛋白具有DGAT酶活性,能够恢复H1246的TAG合成和油体形成;而MtDGAT1-1编码蛋白不能恢复H1246的TAG合成和油体形成。     

Defensin gene family in <Emphasis Type="Italic">Medicago truncatula:</Emphasis> structure,expression and induction by signal molecules

A large gene family encoding the putative cysteine-rich defensins was discovered in Medicago truncatula. Sixteen members of the family were identified by screening a cloned seed defensin from M. sativa (Gao et al. 2000) against the Institute for Genomic Research’s (TIGR) M. truncatula gene index (MtGI version 7). Based on the comparison of their amino acid sequences, M. truncatula defensins fell arbitrarily into three classes displaying extensive sequence divergence outside of the eight canonical cysteine residues. The presence of Class II defensins is reported for the first time in a legume plant. In silico as well as Northern blot and RT-PCR analyses indicated these genes were expressed in a variety of tissues including leaves, flowers, developing pods, mature seed and roots. The expression of these genes was differentially induced in response to a variety of biotic and abiotic stimuli. For the first time, a defensin gene (TC77480) was shown to be induced in roots in response to infection by the mycorrhizal fungus, Glomus versiforme. Northern blot analysis indicated that the tissue-specific expression patterns of the cloned Def1 and Def2 genes differed substantially between M. truncatula and M. sativa. Furthermore, the induction profiles of the Def1 and Def2 genes in response to the signaling molecules methyl jasmonate, ethylene and salicylic acid differed markedly between these two legumes.     

Expressed sequence tags from stem tissue of the American chestnut, Castanea dentata

Subtracted and size-selected unsubtracted cDNA libraries were created to examine gene expression in the woody tissues of Castanea dentata. A total of 50 clones were sequenced and comparisons were made to the GenBank database. Expression analysis of 20 selected clones revealed that 13 were expressed predominantly in the stem and leaf tissues, while the other seven were present in all tissues examined.     

Lectin genes from the legume Medicago truncatula

We report the cloning and characterization of two lectin genes from Medicago truncatula, designated Mtlec1 and Mtlec2. The two genes show a high degree of homology and apparently belong to a small multigene family. Mtlec1 appears to encode a functional lectin with 277 amino acids, whereas Mtlec2 is probably non-functional, since a frameshift mutation (insertion of two nucleotides) leads to premature translation termination after only 98 amino acids. The deduced amino acid sequence of the polypeptide MtLEC1 suggests that this lectin is a metalloprotein with Glc/Man specificity.     

Phylogenetics of New World Astragalus: Screening of novel nuclear loci for the reconstruction of phylogenies at low taxonomic levels

This study explores methods to use information gathered from genomics technology to understand evolutionary relationships in the hyperdiverse legume group Neo-Astragalus. These species inhabit deserts and mountains of North and South America, and even though the monophyly of the group is well established, relationships within it are still poorly understood. Plastid genes, commonly used to infer phylogenies in plants, are usually not useful for closely related taxa because of low levels of genetic variation. TheMedicago truncatula genome project provided a suite of candidate nuclear loci with high levels of variation that might prove suitable for low-level phylogenetics. This paper reports the development of methods for screening a large number of these nuclear loci, and detailed analysis of four of them. Four different patterns of phylogenetic diversification occur in the loci sampled from these genomes ofAstragalus species. One locus (CNGC4) was single copy and could be directly used in phylogenetic analyses. Two loci (ARG10 and FENR) showed patterns strongly suggestive of duplication events in some taxa, and one locus (tRALS) has apparently undergone a cryptic duplication, making it very difficult to diagnose. Potential methods for using the information provided by these loci are discussed.     

Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants

From a pool of Medicago truncatula mutants—obtained by gamma-irradiation or ethyl methanesulfonate mutagenesis—impaired in symbiosis with the N-fixing bacterium Sinorhizobium meliloti, new mutants are described and genetically analysed, and for already reported mutants, complementary data are given on their phenotypic and genetic analysis. Phenotypic data relate to nodulation and mycorrhizal phenotypes. Among the five new mutants, three were classified as [Nod⁺ Fix^– Myc⁺] and the mutations were ascribed to two loci, Mtsym20 (TRV43, TRV54) and Mtsym21 (TRV49). For the two other new mutants, one was classified as [Nod^–/+ Myc⁺] with a mutation ascribed to gene Mtsym15 (TRV48), and the other as [Nod^– Myc^-/+] with a mutation ascribed to gene Mtsym16 (TRV58). Genetic analysis of three previously described mutants has shown that [Nod^–/+ Myc⁺] TR74 mutant can be ascribed to gene Mtsym14, and that [Nod^–/+ Myc^–/+] TR89 and TRV9 mutants are ascribed to gene Mtsym2 (dmi2). Using a detailed analysis of mycorrhizal phenotype, we have observed a delayed typical arbuscular mycorrhizal formation on some mutants that present thick lens-shaped appressoria. This phenotype was called [Myc^–/+] and mutants TR25, TR26, TR89, TRV9, P1 and Y6 were reclassified as [Myc^–/+]. Mutant P1 was reclassified as [Nod^–/+] because of a late nodulation observed on roots of this mutant.     

蒺藜苜蓿叶片光合作用对盐胁迫的响应

为阐明蒺藜苜蓿(Medicago truncatula)叶片光合效率对盐胁迫的响应规律,明确其土壤盐分阈值,该研究以盆栽蒺藜苜蓿幼苗为研究对象,采用加盐的方式人工模拟盐胁迫环境,设置不同浓度NaCl处理(0、50、100、150、200、250、300、400mmol·L-1),利用Li-6400便携式光合测定仪分析了蒺藜苜蓿幼苗光合效率参数对土壤盐分浓度的响应特征。结果表明:(1)蒺藜苜蓿叶片净光合速率(Pn)和光合作用特征参数等具有明显的土壤盐分临界效应。在NaCl浓度为100~200mmol·L-1时,蒺藜苜蓿可维持较高光合生产力,此盐分范围内适宜的光合有效辐射(PAR)为600~1 300μmol·m-2·s-1,出现Pn最大值(20.7μmol·m-2·s-1)的NaCl浓度为150mmol·L-1,对应PAR为1 200μmol·m-2·s-1左右。(2)在NaCl浓度150mmol·L-1时,随着NaCl浓度的增加,表观量子效率(AQY)、光补偿点(LCP)、暗呼吸速率(Rd)和最大光合速率(Pnmax)逐渐增大;在NaCl浓度为150mmol·L-1时,AQY、Rd和Pnmax分别达到最大值0.030、0.605 7μmol·m-2·s-1、19.4μmol·m-2·s-1,而LCP达到最小值19.8μmol·m-2·s-1。(3)NaCl浓度为150mmol·L-1可作为导致蒺藜苜蓿净光合速率下降的气孔限制和非气孔限制因素的转折点,并且随着NaCl浓度升高,其光合速率由气孔限制转为非气孔限制的PAR降低。以上结果表明,蒺藜苜蓿对盐胁迫具有较强的适应性,在较高盐分浓度下可获得较高的光合生产力。     

Molecular Evolution and Positive Selection of the Symbiotic Gene NORK in Medicago truncatula

Understanding the selective constraints of partner specificity in mutually beneficial symbiosis is a significant, yet largely unexplored, prospect of evolutionary biology. These selective constraints can be explored through the study of nucleotide polymorphism at loci controlling specificity. The membrane-anchored receptor NORK (nodulation receptor kinase) of the legume Medicago truncatula controls early steps of root infection by two symbiotic microorganisms: nitrogen-fixing bacteria (rhizobia) and endomycorrhizal fungi (Glomales). We analyzed the diversity of the gene NORK by sequencing 4 kilobases in 28 inbred lines sampled from natural populations. We detected 33 polymorphic sites with only one nonsynonymous change. Analysis based on Tajima’s D and Fay and Wu’s H summary statistics revealed no departure from the neutral model. We analyzed divergence using sequences from the closely related species M. coerulea. The McDonald-Kreitman test indicated a significant excess of nonsynonymous changes contributing to this divergence. Furthermore, maximum-likelihood analysis of a molecular phylogeny of a few legume species indicated that a number of amino acid sites, likely located in the receptor domain of the protein, evolved under the regime of positive selection. Further research should focus on the rate and direction of molecular coevolution between microorganisms’ signaling molecules and legumes’ receptors. [Reviewing Editor: Dr. Deborah Charlesworth] Sequence data were deposited in the GenBank database under accession nos. AY676428 to AY676457 and AJ884582.     

Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism

Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.