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

Lactate production is a prioritized feature of adipocyte metabolism

Adipose tissue is essential for whole-body glucose homeostasis, with a primary role in lipid storage. It has been previously observed that lactate production is also an important metabolic feature of adipocytes, but its relationship to adipose and whole-body glucose disposal remains unclear. Therefore, using a combination of metabolic labeling techniques, here we closely examined lactate production of cultured and primary mammalian adipocytes. Insulin treatment increased glucose uptake and conversion to lactate, with the latter responding more to insulin than did other metabolic fates of glucose. However, lactate production did not just serve as a mechanism to dispose of excess glucose, because we also observed that lactate production in adipocytes did not solely depend on glucose availability and even occurred independently of glucose metabolism. This suggests that lactate production is prioritized in adipocytes. Furthermore, knocking down lactate dehydrogenase specifically in the fat body of Drosophila flies lowered circulating lactate and improved whole-body glucose disposal. These results emphasize that lactate production is an additional metabolic role of adipose tissue beyond lipid storage and release

Dissection of the Complex Phenotype in Cuticular Mutants of Arabidopsis Reveals a Role of SERRATE as a Mediator

Mutations in LACERATA (LCR), FIDDLEHEAD (FDH), and BODYGUARD (BDG) cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis), of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE), which encodes a protein of RNA–processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway

Pain-causing stinging nettle toxins target TMEM233 to modulate NaV1.7 function

Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons

SHINE Transcription Factors Act Redundantly to Pattern the Archetypal Surface of Arabidopsis Flower Organs

Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ surface patterning and the broader control of flower development and biological functions

Conditions for the effective functioning of the system of internalization of economic damage from emissions in the territory

The results of the study are derived from a framework for estimating and recovering damage from emissions, the assimilation potential and sustainable development goals taken into account as well. The identified conditions for the establishment of an effective system for internalizing economic damage from emissions in the territory are limited to ensuring the fairness and consistency of the decisions taken, formation of incentives for continuous improvement of the situation and achievement of the goals of sustainable development in the territories. The main conditions are to reduce the limitations of traditional methods and to overcome uncertainty in the analysis of data. The development of assimilation potential should generate income for its owner (the source of income is the polluter). If payments for cap-and-trade payments are insufficient, the state must subsidize the activity. Since the estimation of the cost of cap-and-overs is done in some part in isolation, it is possible to identify the economic feasibility of its operation within the framework of the model. Due to importance and complexity, one of the main elements and conditions of an effective system of internalization of externalities in the territory is the solution of the problems of road transport

Contradictions and barriers to sustainable development of territories

The aim of the study is to identify contradictions and obstacles to the transition to sustainable development in the territories. The methodological framework is analysis and synthesis, the concept of sustainable development, institutional analysis approaches, limited rationality and opportunism in behaviour, ideas about stakeholders, and systematic and scenario-based approaches. The Sustainable Development Goals reflect interests of the world’s population. However, the search for consensus on a strategy to achieve them is hampered by existing and emerging contradictions. The paper describes the practice of formulating a compromise on the environmental situation in the territory. The analysis of factors is presented in the context of theoretical constraints, interests of various territories and social groups. Gaps and various contradictions have been identified in the implementation of sustainable development concept in the territories. The contradictions between components of sustainable development; the essence of sustainable development tools; the competitiveness of the territory’s products and an effective system of internalization of externalities; various stakeholders are considered. The territories’ transition to sustainable development requires the removal of considered and potential contradictions. Possible directions for this are presented

DNA Methylation Analysis: Choosing the Right Method

In the burgeoning field of epigenetics, there are several methods available to determine the methylation status of DNA samples. However, choosing the method that is best suited to answering a particular biological question still proves to be a difficult task. This review aims to provide biologists, particularly those new to the field of epigenetics, with a simple algorithm to help guide them in the selection of the most appropriate assay to meet their research needs. First of all, we have separated all methods into two categories: those that are used for: (1) the discovery of unknown epigenetic changes; and (2) the assessment of DNA methylation within particular regulatory regions/genes of interest. The techniques are then scrutinized and ranked according to their robustness, high throughput capabilities and cost. This review includes the majority of methods available to date, but with a particular focus on commercially available kits or other simple and straightforward solutions that have proven to be useful