The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive

BACKGROUND The Medicago truncatula 2HA seed line is highly embryogenic while the parental line Jemalong rarely produces embryos. The 2HA line was developed from one of the rare Jemalong regenerates and this method for obtaining a highly regenerable genotype in M. truncatula is readily reproducible suggesting an epigenetic mechanism. Microarray transcriptomic analysis showed down regulation of an ETHYLENE INSENSITIVE 3-like gene in 2HA callus which provided an approach to investigating epigenetic regulation of genes related to ethylene signalling and the 2HA phenotype. Ethylene is involved in many developmental processes including somatic embryogenesis (SE) and is associated with stress responses. RESULTS Microarray transcriptomic analysis showed a significant number of up-regulated transcripts in 2HA tissue culture, including nodule and embryo specific genes and transposon-like genes, while only a few genes were down-regulated, including an EIN3-like gene we called MtEIL1. This reduced expression was associated with ethylene insensitivity of 2HA plants that was further investigated. The weak ethylene insensitivity affected root and nodule development. Sequencing of MtEIL1 found no difference between 2HA and wild-type plants. DNA methylation analysis of MtEIL1 revealed significant difference between 2HA and wild-type plants. Tiling arrays demonstrated an elevated level of miRNA in 2HA plants that hybridised to the antisense strand of the MtEIL1 gene. AFLP-like methylation profiling revealed more differences in DNA methylation between 2HA and wild-type. Segregation analysis demonstrated the recessive nature of the eil1 phenotype and the dominant nature of the SE trait. CONCLUSIONS We have demonstrated that EIL1 of Medicago truncatula (MtEIL1) is epigenetically silenced in the 2HA seed line. The possible cause is an elevated level of miRNA that targets its 3'UTR and is also associated with DNA methylation of MtEIL1. Down regulation of MtEIL1 makes it possible to form nodules in the presence of ethylene and affects root growth under normal conditions. Segregation analysis showed no association between MtEIL1 expression and SE in culture but the role and mechanism of ethylene signalling in the process of plant regeneration through SE requires further investigation. The work also suggests that epigenetic changes to a particular gene induced in culture can be fixed in regenerated plants.This work was funded by the Australian Research Council (CEO348212) through the ARC Centre of Excellence for Integrative Legume Research (CILR)

Characterisation of the legume SERK-NIK gene superfamily including splice variants: Implications for development and defence

<p>Abstract</p> <p>Background</p> <p><it>SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE </it>(<it>SERK</it>) genes are part of the regulation of diverse signalling events in plants. Current evidence shows SERK proteins function both in developmental and defence signalling pathways, which occur in response to both peptide and steroid ligands. <it>SERKs </it>are generally present as small gene families in plants, with five <it>SERK </it>genes in Arabidopsis. Knowledge gained primarily through work on Arabidopsis SERKs indicates that these proteins probably interact with a wide range of other receptor kinases and form a fundamental part of many essential signalling pathways. The <it>SERK1 </it>gene of the model legume, <it>Medicago truncatula </it>functions in somatic and zygotic embryogenesis, and during many phases of plant development, including nodule and lateral root formation. However, other <it>SERK </it>genes in <it>M. truncatula </it>and other legumes are largely unidentified and their functions unknown.</p> <p>Results</p> <p>To aid the understanding of signalling pathways in <it>M. truncatula</it>, we have identified and annotated the <it>SERK </it>genes in this species. Using degenerate PCR and database mining, eight more <it>SERK</it>-like genes have been identified and these have been shown to be expressed. The amplification and sequencing of several different PCR products from one of these genes is consistent with the presence of splice variants. Four of the eight additional genes identified are upregulated in cultured leaf tissue grown on embryogenic medium. The sequence information obtained from <it>M. truncatula </it>was used to identify <it>SERK </it>family genes in the recently sequenced soybean (<it>Glycine max</it>) genome.</p> <p>Conclusions</p> <p>A total of nine <it>SERK </it>or <it>SERK-like </it>genes have been identified in <it>M. truncatula </it>and potentially 17 in soybean. Five <it>M. truncatula SERK </it>genes arose from duplication events not evident in soybean and Lotus. The presence of splice variants has not been previously reported in a <it>SERK </it>gene. Upregulation of four newly identified <it>SERK </it>genes (in addition to the previously described <it>MtSERK1</it>) in embryogenic tissue cultures suggests these genes also play a role in the process of somatic embryogenesis. The phylogenetic relationship of members of the <it>SERK </it>gene family to closely related genes, and to development and defence function is discussed.</p

Evolution of the magnetic phase transition in MnO confined to channel type matrices. Neutron diffraction study

Neutron diffraction studies of antiferromagnetic MnO confined to MCM-41 type matrices with channel diameters 24-87 A demonstrate a continuous magnetic phase transition in contrast to a discontinuous first order transition in the bulk. The character of the magnetic transition transforms with decreasing channel diameter, showing the decreasing critical exponent and transition temperature, however the latter turns out to be above the N\'eel temperature for the bulk. This enhancement is explained within the framework of Landau theory taking into consideration the ternary interaction of the magnetic and associated structural order parameters.Comment: 6 pages pdf file, including 4 figures, uses revtex4.cl

ESR of MnO embedded in silica nanoporous matrices with different topologies

Electron spin resonance (ESR) experiments were performed with antiferromagnetic MnO confined within a porous vycor-type glass and within MCM-type channel matrices. A signal from confined MnO shows two components from crystallized and amorphous MnO and depends on the pore topology. Crystallized MnO within a porous glass shows a behavior having many similarities to the bulk. In contrast with the bulk the strong ESR signal due to disordered "surface" spins is observed below the magnetic transition. With the decrease of channel diameter the fraction of amorphous MnO increases while the amount of crystallized MnO decreases. The mutual influence of amorphous and crystalline MnO is observed in the matrices with a larger channel diameter. In the matrices with a smaller channel diameter the ESR signal mainly originates from amorphous MnO and its behavior is typical for the highly disordered magnetic system.Comment: 7 pages pdf file, 5 figure

Structure of MnO nanoparticles embedded into channel-type matrices

X-ray diffraction experiments were performed on MnO confined in mesoporous silica SBA-15 and MCM-41 matrices with different channel diameters. The measured patterns were analyzed by profile analysis and compared to numerical simulations of the diffraction from confined nanoparticles. From the lineshape and the specific shift of the diffraction reflections it was shown that the embedded objects form ribbon-like structures in the SBA-15 matrices with channels diameters of 47-87 {\AA}, and nanowire-like structures in the MCM-41 matrices with channels diameters of 24-35 {\AA}. In the latter case the confined nanoparticles appear to be narrower than the channel diameters. The physical reasons for the two different shapes of the confined nanoparticles are discussed.Comment: 8 pages, including 9 postscript figures, uses revtex4.cl

Use of Physicochemical Method for Evaluation of Mucilage Producing Ability of the Linum Usitatissimum L. Seeds

Abstract In the modern medicine of many European countries flax is used as a medicament with a wide range of use. Wholesome effect of flax seeds is determined by the large amount of enveloping substances. This property is connected with content of mucilage up to 10% and glycoside linamarin. Flaxseed polysaccharides also possess antiinflammatory effect. Furthermore, mucilage production can be a chemosystematic characteric of intraspecific taxons. In literature intervarietal variability data is limited. Therefore, comparative evaluation of mucilage producing ability of flax seeds with different morphotypes is of interest. The research of micromorphological characteristics of seed coat and mucilage production dynamics was carried out and it was established that mucilage-producing cells are localized predominantly in the external layer of seed coat. It was established that Bahmalskiy, Nebesnyj, Kustanayskiy yantar varieties possess the highest level of mucilage production. Morphotype and varietal specificity of mucilage production are determined, consequently it can be used as a marker feature of L. usitatissimum new forms. The proposed technique is based on the determination of seed physicochemical characteristics and can be used for express analysis of the vegetal samples and their differentiation by the directions of use: as a fatty oil or mucilage-containing raw material. Keywords: Linum usitatissimum L. varieties, seeds, mucilage production, hydration dynamics, physicochemical method