Molecular cloning and characterization of OsCDase, a ceramidase enzyme from rice

SUMMARY: Sphingolipids are a structurally diverse group of molecules based on long-chain sphingoid bases that are found in animal, fungal and plant cells. In contrast to the situation in animals and yeast, much less is known about the spectrum of sphingolipid species in plants and the roles they play in mediating cellular processes. Here, we report the cloning and characterization of a plant ceramidase from rice (Oryza sativa spp. Japonica cv. Nipponbare). Sequence analysis suggests that the rice ceramidase (OsCDase) is similar to mammalian neutral ceramidases. We demonstrate that OsCDase is a bona fide ceramidase by heterologous expression in the yeast double knockout mutant Deltaypc1Deltaydc1 that lacks the yeast ceramidases YPC1p and YDC1p. Biochemical characterization of OsCDase showed that it exhibited classical Michaelis-Menten kinetics, with optimum activity between pH 5.7 and 6.0. OsCDase activity was enhanced in the presence of Ca(2+), Mg(2+), Mn(2+) and Zn(2+), but inhibited in the presence of Fe(2+). OsCDase appears to use ceramide instead of phytoceramide as a substrate. Subcellular localization showed that OsCDase is localized to the endoplasmic reticulum and Golgi, suggesting that these organelles are sites of ceramide metabolism in plants.     

Two receptor-like genes, Vfa1 and Vfa2, confer resistance to the fungal pathogen Venturia inaequalis inciting apple scab disease

The Vf locus, originating from the crabapple species Malus floribunda 821, confers resistance to five races of the fungal pathogen Venturia inaequalis, the causal agent of apple scab disease. Previously, a cluster of four receptor-like genes, Vfa1, Vfa2, Vfa3, and Vfa4, was identified within the Vf locus. Because the amino-acid sequence of Vfa3 is truncated, it was deemed nonfunctional. In this study, each of the three full-length Vfa genes was introduced into a plant cloning vector, pCAMBIA2301, and used for Agrobacterium-mediated transformation of two apple cultivars, Galaxy and McIntosh, to assess functionality of these genes and to characterize their roles in resistance to V. inaequalis. Transformed apple lines carrying each of Vfa1, Vfa2, or Vfa4 were developed, analyzed for the presence of the transgene using polymerase chain reaction and Southern blotting, and assayed for resistance to apple scab following inoculation with V. inaequalis. Transformed lines expressing Vfa4 were found to be susceptible to apple scab, whereas those expressing either Vfa1 or Vfa2 exhibited partial resistance to apple scab. Based on Western blot analysis as well as microscopic analysis of plant resistance reactions, the roles of Vfa1 and Vfa2 in apple scab disease resistance response are discussed.     

An on-farm investigation of beef suckler herds using an animal welfare index (AWI)

Background  

Beef suckler farms (194 farms throughout 13 counties) were assessed once with housed cattle and once with cattle at grass using an animal welfare index (AWI). Twenty-three of the 194 farms were revisited a year later and re-evaluated using the AWI and the Tier-Gerechtheits-Index 35L/2000 (TGI35L/2000). Thirty-three indicators were collected in five categories: locomotion (5 indicators); social interactions (between animals) (7), flooring (5), environment (7) and Stockpersonship (9). Three indicators relating to the size of the farm were also collected.     

An integrative approach to identify hexaploid wheat miRNAome associated with development and tolerance to abiotic stress

Background

Wheat is a major staple crop with broad adaptability to a wide range of environmental conditions. This adaptability involves several stress and developmentally responsive genes, in which microRNAs (miRNAs) have emerged as important regulatory factors. However, the currently used approaches to identify miRNAs in this polyploid complex system focus on conserved and highly expressed miRNAs avoiding regularly those that are often lineage-specific, condition-specific, or appeared recently in evolution. In addition, many environmental and biological factors affecting miRNA expression were not yet considered, resulting still in an incomplete repertoire of wheat miRNAs.

Results

We developed a conservation-independent technique based on an integrative approach that combines machine learning, bioinformatic tools, biological insights of known miRNA expression profiles and universal criteria of plant miRNAs to identify miRNAs with more confidence. The developed pipeline can potentially identify novel wheat miRNAs that share features common to several species or that are species specific or clade specific. It allowed the discovery of 199 miRNA candidates associated with different abiotic stresses and development stages. We also highlight from the raw data 267 miRNAs conserved with 43 miRBase families. The predicted miRNAs are highly associated with abiotic stress responses, tolerance and development. GO enrichment analysis showed that they may play biological and physiological roles associated with cold, salt and aluminum (Al) through auxin signaling pathways, regulation of gene expression, ubiquitination, transport, carbohydrates, gibberellins, lipid, glutathione and secondary metabolism, photosynthesis, as well as floral transition and flowering.

Conclusion

This approach provides a broad repertoire of hexaploid wheat miRNAs associated with abiotic stress responses, tolerance and development. These valuable resources of expressed wheat miRNAs will help in elucidating the regulatory mechanisms involved in freezing and Al responses and tolerance mechanisms as well as for development and flowering. In the long term, it may help in breeding stress tolerant plants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1490-8) contains supplementary material, which is available to authorized users.     

An inter-laboratory comparison demonstrates that [1H]-NMR metabolite fingerprinting is a robust technique for collaborative plant metabolomic data collection

In any metabolomics experiment, robustness and reproducibility of data collection is of vital importance. These become more important in collaborative studies where data is to be collected on multiple instruments. With minimisation of variance in sample preparation and instrument performance it is possible to elucidate even subtle differences in metabolite fingerprints due to genotype or biological treatment. In this paper we report on an inter laboratory comparison of plant derived samples by [¹H]-NMR spectroscopy across five different sites and within those sites utilising instruments with different probes and magnetic field strengths of 9.4 T (400 MHz), 11.7 T (500 MHz) and 14.1 T (600 MHz). Whilst the focus of the study is on consistent data collection across laboratories, aspects of sample stability and the requirement for sample rotation within the NMR magnet are also discussed. Comparability of the datasets from participating laboratories was exceptionally good and the data were amenable to comparative analysis by multivariate statistics. Field strength differences can be adjusted for in the data pre-processing and multivariate analysis demonstrating that [¹H]-NMR fingerprinting is the ideal technique for large scale plant metabolomics data collection requiring the participation of multiple laboratories.     

Endurance of the endolithic desert cyanobacterium Chroococcidiopsis under UVC radiation

Desert cyanobacteria of the genus Chroococcidiopsis are extremely resistant to desiccation and ionizing radiation. When an endolithic strain was exposed to UVC radiation cell lysis, genome damage, photosynthetic pigment bleaching and reduced photochemical performance occurred. Nevertheless, survivors were scored after UVC doses as high as 13?kJ/m² and their endurance ascribed to multicellular aggregates enveloped in thick envelopes, so that attenuated UVC radiation reached the inner cells. In addition, the accumulation of carotenoids contributed to UVC resistance by providing protection against oxidative stress. Finally, in survivors repair mechanisms were responsible for the recovery of the induced damage to genome and photosynthetic apparatus.     

Genetic Features of Resident Biofilms Determine Attachment of Listeria monocytogenes

Planktonic Listeria monocytogenes cells in food-processing environments tend most frequently to adhere to solid surfaces. Under these conditions, they are likely to encounter resident biofilms rather than a raw solid surface. Although metabolic interactions between L. monocytogenes and resident microflora have been widely studied, little is known about the biofilm properties that influence the initial fixation of L. monocytogenes to the biofilm interface. To study these properties, we created a set of model resident Lactococcus lactis biofilms with various architectures, types of matrices, and individual cell surface properties. This was achieved using cell wall mutants that affect bacterial chain formation, exopolysaccharide (EPS) synthesis and surface hydrophobicity. The dynamics of the formation of these biofilm structures were analyzed in flow cell chambers using in situ time course confocal laser scanning microscopy imaging. All the L. lactis biofilms tested reduced the initial immobilization of L. monocytogenes compared to the glass substratum of the flow cell. Significant differences were seen in L. monocytogenes settlement as a function of the genetic background of resident lactococcal biofilm cells. In particular, biofilms of the L. lactis chain-forming mutant resulted in a marked increase in L. monocytogenes settlement, while biofilms of the EPS-secreting mutant efficiently prevented pathogen fixation. These results offer new insights into the role of resident biofilms in governing the settlement of pathogens on food chain surfaces and could be of relevance in the field of food safety controls.Listeria monocytogenes is a food pathogen that has been implicated in numerous food-borne disease outbreaks (5, 58). This organism is found not only in food products but also on surfaces in food-processing plants (18). It is well documented that L. monocytogenes is able to adhere and form persistent biofilms on a variety of solid materials, such as stainless steel, glass, or polymers (18, 48, 51, 52). However, in food-manufacturing plants (and particularly in fermented-food-processing environments), it is most likely that the first contact between a pathogen and a surface will concern a resident microbial biofilm covering the solid surface (10, 35, 46). In this context, such a resident biofilm may be regarded as a “conditioning film” that modifies the topographic and physicochemical characteristics of the surface and hence the adhesion capability of planktonic microorganisms coming into contact with this substratum (6).Once the pathogens are immobilized on the surface, interactions between the pathogens and their environment (physiological interactions with resident flora, nutrient availability, pH, water activity, temperature, and cleaning and disinfection procedures) govern the long-term settlement and persistence of the pathogens on the surface. Various studies have demonstrated the inhibition of L. monocytogenes development by natural “protective” biofilms (10, 66). Competition for nutrients has been demonstrated as a major mechanism underlying the inhibition of pathogen development (25, 27). The production of antimicrobial agents (bacteriocins, acids, and hydrogen peroxide) has also been reported as being of importance to such interactions (13, 20, 36). For example, Lactococcus lactis has been described as being exceptionally efficient in controlling the development of L. monocytogenes on food-processing surfaces by means of competitive exclusion (66) or bacteriocin production (35). It has been reported that treating a surface with a bacterial polysaccharide prevented the adhesion of different nosocomial pathogens (60). Furthermore, alginate-overexpressing Pseudomonas aeruginosa biofilms reduced the retention of Cryptosporidium parvum oocysts (54). Other recent studies have shown that the composition and quantity of specific exopolysaccharides (EPS) in Pseudomonas biofilms can inhibit the fixation of Escherichia coli or Erwinia chrysanthemi planktonic cells in porous media (37, 38).The present study investigated those properties of resident biofilms that could affect the settlement of L. monocytogenes. L. lactis was used as a model resident biofilm strain, as this is widely used in dairy fermentations and its cell wall properties have been the subject of considerable study (22, 23). Cell wall mutants of L. lactis MG1363 were used to create a set of model biofilms that differed in terms of their architecture, EPS synthesis, and cell surface hydrophobicity. These biofilms were used to evaluate the attachment of fluorescent inert polystyrene microbeads and of two reference strains of L. monocytogenes (LO28 and EGDe) using in situ confocal fluorescence imaging.     

Inhibition of autophagy by TAB2 and TAB3

Autophagic responses are coupled to the activation of the inhibitor of NF-κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFβ-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via their coiled-coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C-terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy-stimulatory 'switch' whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1.