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)

Somatic Embryogenesis in the Medicago truncatula Model: Cellular and Molecular Mechanisms

Medicago truncatula is now widely regarded as a legume model where there is an increasing range of genomic resources. Highly regenerable lines have been developed from the wild-type Jemalong cultivar, most likely due to epigenetic changes. These lines with high rates of somatic embryogenesis (SE) can be compared with wild-type where SE is rare. Much of the research has been with the high SE genotype Jemalong 2HA (2HA). SE can be induced from leaf tissue explants or isolated mesophyll protoplasts. In 2HA, the exogenous phytohormones 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) are central to SE. However, there are interactions with ethylene, abscisic acid (ABA), and gibberellic acid (GA) which produce maximum SE. In the main, somatic embryos are derived from dedifferentiated cells, undergo organellar changes, and produce stem-like cells. There is evidence that the SE is induced as a result of a stress and hormone interaction and this is discussed. In M. truncatula, there are connections between stress and specific up-regulated genes and specific hormones and up-regulated genes during the SE induction phase. Some of the transcription factors have been knocked down using RNAi to show they are critical for SE induction (MtWUSCHEL, MtSERF1). SE research in M. truncatula has utilized high throughput transcriptomic and proteomic studies and the more detailed investigation of some individual genes. In this review, these studies are integrated to suggest a framework and timeline for some of the key events of SE induction in M. truncatula

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

Partial anomalous pulmonary venous return mimics arterial positioning after central line placement for plasmapheresis

Case: A 61-year-old male who was status post right single lung transplant complicated by grade three primary graft dysfunction was admitted and mechanically ventilated for hypoxic respiratory failure. Donor specific antibody testing to human leukocyte antigens was positive. The protocol for antibody-mediated transplant rejection was initiated. One aspect of the protocol was a series of plasmapheresis procedures. In advance of the first plasmapheresis, central line placement was attempted via the left internal jugular vein. A chest x-ray showed a line that terminated to the left of the midline and raised concern for arterial placement (Figure 1). Blood gases from the line were: pH 7.72, pCO2 21 mmHg, pO2 369 mmHg, bicarbonate 28.4 mmol/L.Alex J. Griffith (Department of Pathology and Laboratory Medicine, University of Wisconsin Hospital), Victoria Ray (MD, Department of Anesthesiology, University of Wisconsin Hospital), William N. Rose (MD, Department of Emergency Medicine, University of Wisconsin Hospital)Includes bibliographical reference

Heat Stress in Legume Seed Setting: Effects, Causes, and Future Prospects

Grain legumes provide a rich resource of plant nutrition to human diets and are vital for food security and sustainable cropping. Heat stress during flowering has a detrimental effect on legume seed yield, mainly due to irreversible loss of seed number. To start with, we provide an overview of the developmental and physiological basis of controlling seed setting in response to heat stress. It is shown that every single process of seed setting including male and female gametophyte development, fertilization, and early seed/fruit development is sensitive to heat stress, in particular male reproductive development in legume crops is especially susceptible. A series of physiochemical processes including heat shock proteins, antioxidants, metabolites, and hormones centered with sugar starvation are proposed to play a key role in regulating legume seed setting in response to heat stress. The exploration of the molecular mechanisms underlying reproductive heat tolerance is in its infancy. Medicago truncatula, with a small diploid genome, and well-established transformation system and molecular platforms, has become a valuable model for testing gene function that can be applied to advance the physiological and molecular understanding of legume reproductive heat tolerance.The work is supported by National Key R & D Program of China (No. 2017YFD0301307 and 2017YFD0300204-3) and Anhui Agricultural University startup funds for YS, and National Natural Science Foundation of China (No. 31760579) and Hainan University startup funds (No. kyqd1663) for YL

Time evolution of the Partridge-Barton Model

The time evolution of the Partridge-Barton model in the presence of the pleiotropic constraint and deleterious somatic mutations is exactly solved for arbitrary fecundity in the context of a matricial formalism. Analytical expressions for the time dependence of the mean survival probabilities are derived. Using the fact that the asymptotic behavior for large time $t$ is controlled by the largest matrix eigenvalue, we obtain the steady state values for the mean survival probabilities and the Malthusian growth exponent. The mean age of the population exhibits a $t^{-1}$ power law decayment. Some Monte Carlo simulations were also performed and they corroborated our theoretical results.Comment: 10 pages, Latex, 1 postscript figure, published in Phys. Rev. E 61, 5664 (2000

Exact Solution of an Evolutionary Model without Ageing

We introduce an age-structured asexual population model containing all the relevant features of evolutionary ageing theories. Beneficial as well as deleterious mutations, heredity and arbitrary fecundity are present and managed by natural selection. An exact solution without ageing is found. We show that fertility is associated with generalized forms of the Fibonacci sequence, while mutations and natural selection are merged into an integral equation which is solved by Fourier series. Average survival probabilities and Malthusian growth exponents are calculated indicating that the system may exhibit mutational meltdown. The relevance of the model in the context of fissile reproduction groups as many protozoa and coelenterates is discussed.Comment: LaTeX file, 15 pages, 2 ps figures, to appear in Phys. Rev.