Does Methyl Jasmonate Effectively Protect Plants under Heavy Metal Contamination? Fatty Acid Content in Wheat Leaves Exposed to Cadmium with or without Exogenous Methyl Jasmonate Application.

The effect of methyl jasmonate (MJ) (1 µM) on wheat (Triticum aestivum L. cv. Moskovskaya 39), seedlings and the fatty acid (FA) content of leaves under optimal and cadmium (Cd) (100 µM) stress conditions wasinvestigated. Height and biomass accumulation was studied traditionally; the netphotosynthesis rate (Pn) was studied using a photosynthesis system, FAs'profile-GS-MS. No effect on the height and Pn rate of the MJ pre-treatment wheat at optimum growth conditions was found. MJ pre-treatment led to a decrease in the total amount of saturated (about 11%) and unsaturated (about 17%) identified FAs, except α-linoleic FA (ALA), which is probably associated with its involvement in energy-dependent processes. Under Cd impact, the MJ-treated plants had a higher biomass accumulation and Pn rate compared to untreated seedlings. Both MJ and Cd caused stress-induced elevation of palmitic acid (PA) versus an absence of myristic acid (MA), which is used for elongation. It is suggested that PA participates in alternative adaptation mechanisms (not only as a constituent of the lipid bilayer of biomembrane) of plants under stress. Overall, the dynamics of FAs showed an increase in the saturated FA that is important in the packing of the biomembrane. It is supposed that the positive effect of MJ is associated with lower Cd content in plants and a higher ALA content in leaves.

The Effects of Low-Level Helium-Neon (He-Ne) Laser Irradiation on Lipids and Fatty Acids, and the Activity of Energetic Metabolism Enzymes and Proteome in the Blastula Stage and Underyearlings of the Atlantic Salmon Salmo salar: A Novel Approach in Salmonid Restoration Procedures in the North.

The effect of He-Ne laser irradiation on fishery parameters as well as on biochemical state, including the lipids and fatty acids, the activity of energy metabolism enzymes and the proteome in the blastula stage and in underyearlings of wild Atlantic salmon after irradiation at the cleavage stage/early blastula (considered as the stages when the cell has a high potential for differentiation) was studied. Low mortality rates of eggs were determined during embryogenesis, as well as increased weight gain and lower morality rates of underyearlings in the experimental group. This is confirmed by changes in a number of interrelated indicators of lipid metabolism: a decrease in total lipids content, including diacylglycerols, triacylglycerols, cholesterol esters, and the phospholipids content remained unchanged. The embryos in the blastula stage (experimental group) had higher aerobic capacity and an increase in pentose phosphate pathway activity. The proteome profiles of eggs in the blastula stage were 131 proteins, of which 48 were significantly identified. The major protein was found to be phosvitin. The proteomes of underyearlings were represented by 2018 proteins, of which 49 were unique for the control and 39 for the experimental group. He-Ne laser irradiation had a strong effect on the contents of histone proteins.

Amniotic fluid stem cells and the cell source repertoire for non-invasive prenatal testing.

Cell-free fetal DNA (cffDNA)-based non-invasive prenatal testing (NIPT) is considered to be a very promising screening tool for pregnant women with an increased risk of fetal aneuploidy. Already millions of women worldwide underwent NIPT. However, due to the observed false-positive and false-negative results, this screening approach does not fulfil the criteria of a diagnostic test. Accordingly, positive results still require risk-carrying invasive prenatal testing, such as amniocentesis or chorionic villus sampling (CVS), for confirmation. Such hurdles need to be overcome before NIPT could become a diagnostic approach widely used in the general population. Here we discuss new evidence that besides the placenta amniotic fluid stem cells (AFSCs) could also represent an origin of cffDNA in the mother's blood. A comprehensive picture of the involved cell source repertoire could pave the way to more reliable interpretations of NIPT results and ameliorate counselling of advice-seeking patients.

Into the Deep: New Data on the Lipid and Fatty Acid Profile of Redfish Sebastes mentella Inhabiting Different Depths in the Irminger Sea.

New data on lipid and fatty acid profiles are presented, and the dynamics of the studied components in muscles in the males and females of the beaked redfish, Sebastes mentella, in the depth gradient of the Irminger Sea (North Atlantic) is discussed. The contents of the total lipids (TLs), total phospholipids (PLs), monoacylglycerols (MAGs), diacylglycerols (DAGs), triacylglycerols (TAGs), cholesterol (Chol), Chol esters, non-esterified fatty acids (NEFAs), and wax esters were determined by HPTLC; the phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine (PC), and lysophosphatidylcholine (LPC) were determined by HPLC; and fatty acids of total lipids were determined using GC. The Chol esters prevailed in muscles over the storage TAGs, and the wax ester content was high, which is a characteristic trait of vertically migrating species. Specific dynamics in certain PL in redfish were found to be depended on depth, suggesting that PLs are involved in the re-arrangement of the membrane physicochemical state and the maintenance of motor activity under high hydrostatic pressure. The high contents of DHA and EPA were observed in beaked redfish muscles is the species' characteristic trait. The MUFAs in muscles include dietary markers of zooplankton (copepods)-20:1(n-9) and 22:1(n-11), whose content was found to be lower in fish sampled from greater depths.

The Effect of the Photoperiod on the Fatty Acid Profile and Weight in Hatchery-Reared Underyearlings and Yearlings of Atlantic Salmon Salmo salar L.

The influence of two light regimes, 16:8 h light/dark (LD 16:8) and 24:0 h light/dark (LD 24:0), in comparison to a usual hatchery light regime (HL), on the fatty acids content and weight gain in hatchery-reared underyearlings (at 0+ age) and yearlings (at 1+ age) of Atlantic salmon in the summer-autumn period was studied. The total lipids were analyzed by Folch method, the lipid classes using HPTLC, and the fatty acids of total lipids using GC. The increase in EPA and DHA observed in October in underyearlings and yearlings salmon (especially under LD 24:0) suggests they were physiologically preparing for overwintering. The changes in fatty acids and their ratios in juvenile Atlantic salmon can be used as biochemical indicators of the degree to which hatchery-reared fish are ready to smoltify. These associated with an increase in marine-type specific DHA and EPA, an increase in the 16:0/18:1(n-9) ratio, in correlation with a reduction in MUFAs (mainly 18:1(n-9)). These biochemical modifications, accompanied by fish weight gain, were more pronounced in October in yearlings exposed to continuous light (LD 24:0). The mortality rate was lower in experimental groups of underyearliings with additional lighting. Exposure to prolonged and continuous light did not affect yearlings mortality rate.

JAK-STAT inhibition impairs K-RAS-driven lung adenocarcinoma progression.

Oncogenic K-RAS has been difficult to target and currently there is no K-RAS-based targeted therapy available for patients suffering from K-RAS-driven lung adenocarcinoma (AC). Alternatively, targeting K-RAS-downstream effectors, K-RAS-cooperating signaling pathways or cancer hallmarks, such as tumor-promoting inflammation, has been shown to be a promising therapeutic strategy. Since the JAK-STAT pathway is considered to be a central player in inflammation-mediated tumorigenesis, we investigated here the implication of JAK-STAT signaling and the therapeutic potential of JAK1/2 inhibition in K-RAS-driven lung AC. Our data showed that JAK1 and JAK2 are activated in human lung AC and that increased activation of JAK-STAT signaling correlated with disease progression and K-RAS activity in human lung AC. Accordingly, administration of the JAK1/2 selective tyrosine kinase inhibitor ruxolitinib reduced proliferation of tumor cells and effectively reduced tumor progression in immunodeficient and immunocompetent mouse models of K-RAS-driven lung AC. Notably, JAK1/2 inhibition led to the establishment of an antitumorigenic tumor microenvironment, characterized by decreased levels of tumor-promoting chemokines and cytokines and reduced numbers of infiltrating myeloid derived suppressor cells, thereby impairing tumor growth. Taken together, we identified JAK1/2 inhibition as promising therapy for K-RAS-driven lung AC.

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Genome Sequencing Reveals the Origin of the Allotetraploid Arabidopsis suecica.

Polyploidy is an example of instantaneous speciation when it involves the formation of a new cytotype that is incompatible with the parental species. Because new polyploid individuals are likely to be rare, establishment of a new species is unlikely unless polyploids are able to reproduce through self-fertilization (selfing), or asexually. Conversely, selfing (or asexuality) makes it possible for polyploid species to originate from a single individual-a bona fide speciation event. The extent to which this happens is not known. Here, we consider the origin of Arabidopsis suecica, a selfing allopolyploid between Arabidopsis thaliana and Arabidopsis arenosa, which has hitherto been considered to be an example of a unique origin. Based on whole-genome re-sequencing of 15 natural A. suecica accessions, we identify ubiquitous shared polymorphism with the parental species, and hence conclusively reject a unique origin in favor of multiple founding individuals. We further estimate that the species originated after the last glacial maximum in Eastern Europe or central Eurasia (rather than Sweden, as the name might suggest). Finally, annotation of the self-incompatibility loci in A. suecica revealed that both loci carry non-functional alleles. The locus inherited from the selfing A. thaliana is fixed for an ancestral non-functional allele, whereas the locus inherited from the outcrossing A. arenosa is fixed for a novel loss-of-function allele. Furthermore, the allele inherited from A. thaliana is predicted to transcriptionally silence the allele inherited from A. arenosa, suggesting that loss of self-incompatibility may have been instantaneous.

DNA methylation in Arabidopsis has a genetic basis and shows evidence of local adaptation.

Epigenome modulation potentially provides a mechanism for organisms to adapt, within and between generations. However, neither the extent to which this occurs, nor the mechanisms involved are known. Here we investigate DNA methylation variation in Swedish Arabidopsis thaliana accessions grown at two different temperatures. Environmental effects were limited to transposons, where CHH methylation was found to increase with temperature. Genome-wide association studies (GWAS) revealed that the extensive CHH methylation variation was strongly associated with genetic variants in both cis and trans, including a major trans-association close to the DNA methyltransferase CMT2. Unlike CHH methylation, CpG gene body methylation (GBM) was not affected by growth temperature, but was instead correlated with the latitude of origin. Accessions from colder regions had higher levels of GBM for a significant fraction of the genome, and this was associated with increased transcription for the genes affected. GWAS revealed that this effect was largely due to trans-acting loci, many of which showed evidence of local adaptation.

A tobacco homolog of DCN1 is involved in pollen development and embryogenesis.

KEY MESSAGE: We show that DCN1 binds ubiquitin and RUB/NEDD8, associates with cullin, and is functionally conserved. DCN1 activity is required for pollen development transitions and embryogenesis, and for pollen tube growth. Plant proteomes show remarkable plasticity in reaction to environmental challenges and during developmental transitions. Some of this adaptability comes from ubiquitin-mediated protein degradation regulated by cullin-RING E3 ubiquitin ligases (CRLs). CRLs are activated through modification of the cullin subunit with the ubiquitin-like protein RUB/NEDD8 by an E3 ligase called defective in cullin neddylation 1 (DCN1). Here we show that tobacco DCN1 binds ubiquitin and RUB/NEDD8 and associates with cullin. When knocked down by RNAi, tobacco pollen formation was affected and zygotic embryogenesis was blocked around the globular stage. Additionally, we found that RNAi of DCN1 inhibited the stress-triggered reprogramming of cultured microspores from their intrinsic gametophytic mode of development to an embryogenic state. This stress-induced developmental switch is a known feature in many important crops and leads ultimately to the formation of haploid embryos and plants. Compensating the RNAi effect by re-transformation with a promoter-silencing construct restored pollen development and zygotic embryogenesis, as well as the ability for stress-induced formation of embryogenic microspores. Overexpression of DCN1 accelerated pollen tube growth and increased the potential for microspore reprogramming. These results demonstrate that the biochemical function of DCN1 is conserved in plants and that its activity is involved in transitions during pollen development and embryogenesis, and for pollen tube growth.

Massive genomic variation and strong selection in Arabidopsis thaliana lines from Sweden.

Despite advances in sequencing, the goal of obtaining a comprehensive view of genetic variation in populations is still far from reached. We sequenced 180 lines of A. thaliana from Sweden to obtain as complete a picture as possible of variation in a single region. Whereas simple polymorphisms in the unique portion of the genome are readily identified, other polymorphisms are not. The massive variation in genome size identified by flow cytometry seems largely to be due to 45S rDNA copy number variation, with lines from northern Sweden having particularly large numbers of copies. Strong selection is evident in the form of long-range linkage disequilibrium (LD), as well as in LD between nearby compensatory mutations. Many footprints of selective sweeps were found in lines from northern Sweden, and a massive global sweep was shown to have involved a 700-kb transposition.

A novel strategy for obtaining kanamycin resistance in Arabidopsis thaliana by silencing an endogenous gene encoding a putative chloroplast transporter.

The use of bacterial antibiotic resistance markers in transgenic plants raises concerns about horizontal gene transfer to soil bacteria. We report here that kanamycin resistance in Arabidopsis thaliana can be achieved by silencing an endogenous gene encoding a putative chloroplast transporter, which presumably imports kanamycin into chloroplasts to interfere with ribosomal RNA. Homologs of the transporter exist in other plant species, suggesting this strategy may be generally useful for selecting transformed plant cells.

Evolutionary conservation and regulation of particular alternative splicing events in plant SR proteins.

Alternative splicing is an important mechanism for fine tuning of gene expression at the post-transcriptional level. SR proteins govern splice site selection and spliceosome assembly. The Arabidopsis genome encodes 19 SR proteins, several of which have no orthologues in metazoan. Three of the plant specific subfamilies are characterized by the presence of a relatively long alternatively spliced intron located in their first RNA recognition motif, which potentially results in an extremely truncated protein. In atRSZ33, a member of the RS2Z subfamily, this alternative splicing event was shown to be autoregulated. Here we show that atRSp31, a member of the RS subfamily, does not autoregulate alternative splicing of its similarly positioned intron. Interestingly, this alternative splicing event is regulated by atRSZ33. We demonstrate that the positions of these long introns and their capability for alternative splicing are conserved from green algae to flowering plants. Moreover, in particular alternative splicing events the splicing signals are embedded into highly conserved sequences. In different taxa, these conserved sequences occur in at least one gene within a subfamily. The evolutionary preservation of alternative splice forms together with highly conserved intron features argues for additional functions hidden in the genes of these plant-specific SR proteins.

Methotrexate is a new selectable marker for tobacco immature pollen transformation.

We report here a new selectable marker for tobacco immature pollen transformation based on the expression of dihydrofolate reductase (dhfr) gene which confers resistance to methotrexate (Mtx). Two immature pollen transformation approaches, i.e., male germ line transformation and particle bombardment of embryogenic mid-bicellular pollen have been used for the production of stable transgenic tobacco plants. In the first method, two methotrexate-resistant plants were selected from a total of 7161 seeds recovered after transformation experiments. In the second method, four methotrexate-resistant plants were obtained from 29 bombardments using 3.7 x 10(5) pollen grains per bombardment. Southern analysis confirmed the transgenic nature of T0 and T1 candidate transgenic plants, and a genetic analysis showed that the transgenes are transmitted to subsequent generations.

Identification of a villin-related tobacco protein as a novel cross-reactive plant allergen.

In a paradigmatic approach we identified cross-reactive plant allergens for allergy diagnosis and treatment by screening of a tobacco leaf complementary DNA (cDNA) library with serum IgE from a polysensitized allergic patient. Two IgE-reactive cDNA clones were isolated which code for proteins with significant sequence similarity to the actin-binding protein, villin. Northern- and Western-blotting demonstrate expression of the villin-related allergens in pollen and somatic plant tissues. In addition, villin-related proteins were detected in several plant allergen sources (tree-, grass-, weed pollen, fruits, vegetables, nuts). A recombinant C-terminal fragment of the villin-related protein was expressed in Escherichia coli, purified and shown to react specifically with allergic patients IgE. After profilin, villin-related proteins represent another family of cytoskeletal proteins, which has been identified as cross-reactive plant allergens. They may be used for the diagnosis and treatment of patients suffering from multivalent plant allergies.

The MAP kinase kinase NtMEK2 is involved in tobacco pollen germination.

The tobacco ntf4 mitogen-activated protein (MAP) kinase gene (and its encoded protein p45(Ntf4)) is expressed at later stages of pollen maturation. We have found that the highly related MAP kinase SIPK is also expressed in pollen and, like p45(Ntf4), is activated upon pollen hydration. The MAP kinase kinase NtMEK2 activates SIPK, and here we show that it can also activate p45(Ntf4). In an attempt to inhibit the function of both MAP kinases simultaneously we constructed a loss-of-function mutant version of NtMEK2, which, in transient transformation assays, led to an inhibition of germination in the transformed pollen grains. These data indicate that NtMEK2, and by inference its substrates p45(Ntf4) and/or SIPK, are involved in pollen germination.