The relaxation dynamics of the photoexcited hydrated electron have been subject to conflicting interpretations. Here, we report time-resolved photoelectron spectra of hydrated electrons in a liquid microjet with the aim of clarifying ambiguities from previous experiments. A sequence of three ultrashort laser pulses (~100 femtosecond duration) successively created hydrated electrons by charge-transfer-to-solvent excitation of dissolved anions, electronically excited these electrons via the s→p transition, and then ejected them into vacuum. Two distinct transient signals were observed. One was assigned to the initially excited p-state with a lifetime of ~75 femtoseconds, and the other, with a lifetime of ~400 femtoseconds, was attributed to s-state electrons just after internal conversion in a nonequilibrated solvent environment. These assignments support the nonadiabatic relaxation model.

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Fluorocarbon film deposition onto Si and its influence on the measured Si etch rate in CF4/H2 reactive ion etching in a symmetric two electrode reactor has been studied as a function of CF4/H2 feed gas composition, total gas flow, and applied rf power. For reactive ion etching, the fluorocarbon film thickness on Si increases as the percentage x of H2 in CF4/x% H2 is increased. The fluorocarbon film thickness depends on the total gas flow and is greater for greater gas flows. The observed Si etch rate is controlled by the fluorocarbon film. The Si etch rate is directly proportional to the inverse of the F,C-film thickness for fluorocarbon films thicker than ∼10 Å, which may indicate a diffusion-limited mechanism. Both in-diffusion of fluorine and out-diffusion of SiF4 etch product through the fluorocarbon film are consistent with the decrease of the Si etch rate. The relative importance of the lowering of the atomic F concentration in the gas phase on the Si etch rate, e.g., by the H scavenging mechanism, has also been studied. This reaction can be important for conditions where either the inner walls of the etching apparatus can be maintained free of C,F film throughout the etching experiment, and/or a low hydrogen concentration (≤20%) in the CF4/H2 feed gas is used. For a fixed gas composition of CF4/40% H2, the rf-power dependence of the C,F-film thickness and of the Si etch rate was studied. In reactive ion etching, i.e., if rf power is supplied to the bottom (substrate) electrode, at first a monotonic rise in deposited fluorocarbon film thickness with increasing rf power is observed; at high rf-power levels a dramatic decrease in the C,F-layer thickness occurs, which is concomitant with a greater intensity of near surface lattice disorder (from ion channeling studies) and Si etching. Silicon etching is not observed for lower rf powers. In cases where rf power was supplied to the top electrode only, C,F-film deposition has been observed and no Si etching. These data are consistent with a recombinant model of etch anisotropy.

Limber pine (Pinus flexilis) is a keystone species of high-elevation forest ecosystems of western North America, but some parts of the geographic range have high infection and mortality from the non-native white pine blister rust caused by Cronartium ribicola. Genetic maps can provide essential knowledge for understanding genetic disease resistance as well as local adaptation to changing climates. Exome-seq was performed to construct high-density genetic maps in two seed families. Composite maps positioned 9612 unigenes across 12 linkage groups (LGs). Syntenic analysis of genome structure revealed that the majority of orthologs were positional orthologous genes (POGs) with localization on homologous LGs among conifer species. Gene ontology (GO) enrichment analysis showed relatively fewer constraints for POGs with putative roles in adaptation to environments and relatively more conservation for POGs with roles in basic cell function and maintenance. The mapped genes included 639 nucleotide-binding site leucine-rich repeat genes (NBS-LRRs), 290 receptor-like protein kinase genes (RLKs), and 1014 genes with potential roles in the defense response and induced systemic resistance to attack by pathogens. Orthologous loci for resistance to rust pathogens were identified and were co-positioned with multiple members of the R gene family, revealing the evolutionary pressure acting upon them. This high-density genetic map provides a genomic resource and practical tool for breeding and genetic conservation programs, with applications in genomewide association studies (GWASs), the characterization of functional genes underlying complex traits, and the sequencing and assembly of the full-length genomes of limber pine and related Pinus species.

BackgroundThe fungus Cronartium ribicola (Cri) is an economically and ecologically important forest pathogen that causes white pine blister rust (WPBR) disease on five-needle pines. To cause stem cankers and kill white pine trees the fungus elaborates a life cycle with five stages of spore development on five-needle pines and the alternate host Ribes plants. To increase our understanding of molecular WP-BR interactions, here we report genome-wide transcriptional profile analysis of C. ribicola using RNA-seq.ResultscDNA libraries were constructed from aeciospore, urediniospore, and western white pine (Pinus monticola) tissues post Cri infection. Over 200 million RNA-seq 100-bp paired-end (PE) reads from rust fungal spores were de novo assembled and a reference transcriptome was generated with 17,880 transcripts that were expressed from 13,629 unigenes. A total of 734 unique proteins were predicted as a part of the Cri secretome from complete open reading frames (ORFs), and 41 % of them were Cronartium-specific. This study further identified a repertoire of candidate effectors and other pathogenicity determinants. Differentially expressed genes (DEGs) were identified to gain an understanding of molecular events important during the WPBR fungus life cycle by comparing Cri transcriptomes at different infection stages. Large-scale changes of in planta gene expression profiles were observed, revealing that multiple fungal biosynthetic pathways were enhanced during mycelium growth inside infected pine stem tissues. Conversely, many fungal genes that were up-regulated at the urediniospore stage appeared to be signalling components and transporters. The secreted fungal protein genes that were up-regulated in pine needle tissues during early infection were primarily associated with cell wall modifications, possibly to mask the rust pathogen from plant defenses.ConclusionThis comprehensive transcriptome profiling substantially improves our current understanding of molecular WP-BR interactions. The repertoire of candidate effectors and other putative pathogenicity determinants identified here are valuable for future functional analysis of Cri virulence and pathogenicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1861-1) contains supplementary material, which is available to authorized users.

ATP is copackaged and coreleased with adrenergic, serotonergic, and cholinergic neurotransmitters, suggesting a possible interaction between the signaling pathways for ATP and these coreleased neurotransmitters. Muscarinic m2 and m4, a2-adrenergic, and D2-dopaminergic neurotransmitter receptors, which have in common their ability to inhibit adenylate cyclase through the inhibitory guanine nucleotide binding protein Gi, were transfected and expressed in Chinese hamster ovary (CHO) cells that contain endogenous ATP receptors coupled to the release of arachidonic acid. Normal functional coupling of m2, m4, a2, and D2 receptors was demonstrated by their ability to inhibit forskolin-stimulated cAMP accumulation with dose-response activities consistent with previous reports for these GI-coupled receptors. Stimulation of m2, m4, a2, and D2 receptors resulted in an augmentation of ATP-stimulated arachidonic acid release. With the exception of the m4 receptor, none of the receptors tested was able to stimulate arachidonic acid release in the absence of ATP. Potentiation of ATP-stimulated arachidonic acid release was independent of changes in cAMP. The augmentation of ATP-stimulated arachidonic acid release and the inhibition of cAMP accumulation were both blocked by pertussis toxin, an inhibitor of G,, but with different dose-response characteristics. Inhibition of protein kinase C with staurosporine or long-term pretreatment of the cells with the phorbol ester phorbol 12-myristate 13-acetate blocked the augmentation response. This demonstrates that Gr-coupled inhibitory receptors can amplify ATP-receptor-stimulated arachidonic acid release through a pertussis-toxin-sensitive G protein, independent of their ability to inhibit adenylate cyclase activity.After neural stimulation, neurotransmitters are released from storage vesicles and bind to cell surface receptors to initiate transmembrane signaling. ATP has been shown to be packaged with cholinergic, serotonergic, and adrenergic neurotransmitters in storage vesicles and coreleased during the process of nerve stimulation (1). ATP, in addition to its role in intermediary metabolism and cellular energy, can itself act as a neurotransmitter by binding with high affinity to cell surface purinergic receptors on both excitable and nonexcitable cells. The corelease of ATP with neurotransmitters suggests a possible interaction between their signaling pathways.The cascade of guanine nucleotide binding protein (G protein)-coupled receptor signaling involves the sequential stimulation of receptors, G proteins, and effector enzymes to generate intracellular second messengers, such as cAMP, arachidonic acid, and inositol phosphates. ATP, acting through P2-purinergic receptors, stimulates phospholipase A2 and phospholipase C, which results in the liberation of arachidonic acid and inositol phosphates, respectively (25). Arachidonic acid and many ofits eicosanoid metabolites have been shown to be important second messengers in both neural and nonneural cells (2, 3) and may play...

BackgroundLinkage of DNA markers with phenotypic traits provides essential information to dissect clustered genes with potential phenotypic contributions in a target genome region. Pinus flexilis E. James (limber pine) is a keystone five-needle pine species in mountain-top ecosystems of North America. White pine blister rust (WPBR), caused by a non-native fungal pathogen Cronartium ribicola (J.C. Fisch.), has resulted in mortality in this conifer species and is still spreading through the distribution. The objective of this research was to develop P. flexilis transcriptome-wide single nucleotide polymorphism (SNP) markers using RNA-seq analysis for genetic mapping of the major gene (Cr4) that confers complete resistance to C. ribicola.ResultsNeedle tissues of one resistant and two susceptible seedling families were subjected to RNA-seq analysis. In silico SNP markers were uncovered by mapping the RNA-seq reads back to the de novo assembled transcriptomes. A total of 110,573 in silico SNPs and 2,870 indels were identified with an average of 3.7 SNPs per Kb. These SNPs were distributed in 17,041 unigenes. Of these polymorphic P. flexilis unigenes, 6,584 were highly conserved as compared to the genome sequence of P. taeda L (loblolly pine). High-throughput genotyping arrays were designed and were used to search for Cr4-linked genic SNPs in megagametophyte populations of four maternal trees by haploid-segregation analysis. A total of 32 SNP markers in 25 genes were localized on the Cr4 linkage group (LG). Syntenic relationships of this Cr4-LG map with the model conifer species P. taeda anchored Cr4 on Pinus consensus LG8, indicating that R genes against C. ribicola have evolved independently in different five-needle pines. Functional genes close to Cr4 were annotated and their potential roles in Cr4-mediated resistance were further discussed.ConclusionsWe demonstrated a very effective, low-cost strategy for developing a SNP genetic map of a phenotypic trait of interest. SNP discovery through transcriptome comparison was integrated with high-throughput genotyping of a small set of in silico SNPs. This strategy may be applied to mapping any trait in non-model plant species that have complex genomes. Whole transcriptome sequencing provides a powerful tool for SNP discovery in conifers and other species with complex genomes, for which sequencing and annotation of complex genomes is still challenging. The genic SNP map for the consensus Cr4-LG may help future molecular breeding efforts by enabling both Cr4 positional characterization and selection of this gene against WPBR.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3079-2) contains supplementary material, which is available to authorized users.