Detoxification-related gene expression accompanies anhydrobiosis in the foliar nematode (Aphelenchoides fragariae).

The foliar nematode (Aphelenchoides fragariae) is a quarantined pest that infects a broad range of herbaceous and woody plants. Previous work has demonstrated its remarkable ability to survive rapid and extreme desiccation, although the specific molecular mechanisms underlying its anhydrobiotic response have not been characterized. The authors used RNA sequencing and de novo transcriptome assembly to compare patterns of gene expression between hydrated and 24-hr desiccated nematodes. In total, 2,083 and 953 genes were significantly up- and downregulated, respectively, in desiccated nematodes. Of the 100 annotated genes with the largest positive fold-changes, more than one third encoded putative detoxification-related proteins. Genes encoding enzymes of Phase I and Phase II detoxification systems were among the most strongly upregulated in the transcriptome, including 35 cytochrome p450s, 23 short chain dehydrogenase/reductases, 5 glutathione-S-transferases, and 22 UDP-glucuronosyltransferases. Genes encoding heat shock proteins, unfolded protein response enzymes, and intrinsically disordered proteins were also upregulated. Anhydrobiosis in A. fragariae appears to involve both strategies to minimize protein misfolding and aggregation, and wholesale induction of the cellular detoxification machinery. These processes may be controlled in part through the activity of forkhead transcription factors similar to Caenorhabditis elegans' daf-16, a number of which were differentially expressed under desiccation.The foliar nematode (Aphelenchoides fragariae) is a quarantined pest that infects a broad range of herbaceous and woody plants. Previous work has demonstrated its remarkable ability to survive rapid and extreme desiccation, although the specific molecular mechanisms underlying its anhydrobiotic response have not been characterized. The authors used RNA sequencing and de novo transcriptome assembly to compare patterns of gene expression between hydrated and 24-hr desiccated nematodes. In total, 2,083 and 953 genes were significantly up- and downregulated, respectively, in desiccated nematodes. Of the 100 annotated genes with the largest positive fold-changes, more than one third encoded putative detoxification-related proteins. Genes encoding enzymes of Phase I and Phase II detoxification systems were among the most strongly upregulated in the transcriptome, including 35 cytochrome p450s, 23 short chain dehydrogenase/reductases, 5 glutathione-S-transferases, and 22 UDP-glucuronosyltransferases. Genes encoding heat shock proteins, unfolded protein response enzymes, and intrinsically disordered proteins were also upregulated. Anhydrobiosis in A. fragariae appears to involve both strategies to minimize protein misfolding and aggregation, and wholesale induction of the cellular detoxification machinery. These processes may be controlled in part through the activity of forkhead transcription factors similar to Caenorhabditis elegans' daf-16, a number of which were differentially expressed under desiccation.

Thermal cues drive plasticity of desiccation resistance in montane salamanders with implications for climate change.

Organisms rely upon external cues to avoid detrimental conditions during environmental change. Rapid water loss, or desiccation, is a universal threat for terrestrial plants and animals, especially under climate change, but the cues that facilitate plastic responses to avoid desiccation are unclear. We integrate acclimation experiments with gene expression analyses to identify the cues that regulate resistance to water loss at the physiological and regulatory level in a montane salamander (Plethodon metcalfi). Here we show that temperature is an important cue for developing a desiccation-resistant phenotype and might act as a reliable cue for organisms across the globe. Gene expression analyses consistently identify regulation of stem cell differentiation and embryonic development of vasculature. The temperature-sensitive blood vessel development suggests that salamanders regulate water loss through the regression and regeneration of capillary beds in the skin, indicating that tissue regeneration may be used for physiological purposes beyond replacing lost limbs.

Multiple Nodulation Genes Are Up-Regulated During Establishment of Reniform Nematode Feeding Sites in Soybean.

The semi-endoparastic reniform nematode (Rotylenchulus reniformis) infects over 300 plant species. Females penetrate host roots and induce formation of complex, multinucleate feeding sites called syncytia. While anatomical changes associated with reniform nematode infection are well documented, little is known about their molecular basis. We grew soybean (Glycine max) in a split-root growth system, inoculated half of each root system with R. reniformis, and quantified gene expression in infected and control root tissue at four dates after inoculation. Over 6,000 genes were differentially expressed between inoculated and control roots on at least one date (false discovery rate [FDR] = 0.01, |log2FC| ≥ 1), and 507 gene sets were significantly enriched or depleted in inoculated roots (FDR = 0.05). Numerous genes up-regulated during syncytium formation had previously been associated with rhizobia nodulation. These included the nodule-initiating transcription factors CYCLOPS, NSP1, NSP2, and NIN, as well as multiple nodulins associated with the plant-derived peribacteroid membrane. Nodulation-related NIP aquaporins and SWEET sugar transporters were induced, as were plant CLAVATA3/ESR-related (CLE) signaling proteins and cell cycle regulators such as CCS52A and E2F. Nodulins and nodule-associated genes may have ancestral functions in normal root development and mycorrhization that have been co-opted by both parasitic nematodes and rhizobial bacteria to promote feeding site and nodule formation.

Transcriptional Responses in Root and Leaf of Prunus persica under Drought Stress Using RNA Sequencing.

Prunus persica L. Batsch, or peach, is one of the most important crops and it is widely established in irrigated arid and semi-arid regions. However, due to variations in the climate and the increased aridity, drought has become a major constraint, causing crop losses worldwide. The use of drought-tolerant rootstocks in modern fruit production appears to be a useful method of alleviating water deficit problems. However, the transcriptomic variation and the major molecular mechanisms that underlie the adaptation of drought-tolerant rootstocks to water shortage remain unclear. Hence, in this study, high-throughput sequencing (RNA-seq) was performed to assess the transcriptomic changes and the key genes involved in the response to drought in root tissues (GF677 rootstock) and leaf tissues (graft, var. Catherina) subjected to 16 days of drought stress. In total, 12 RNA libraries were constructed and sequenced. This generated a total of 315 M raw reads from both tissues, which allowed the assembly of 22,079 and 17,854 genes associated with the root and leaf tissues, respectively. Subsets of 500 differentially expressed genes (DEGs) in roots and 236 in leaves were identified and functionally annotated with 56 gene ontology (GO) terms and 99 metabolic pathways, which were mostly associated with aminobenzoate degradation and phenylpropanoid biosynthesis. The GO analysis highlighted the biological functions that were exclusive to the root tissue, such as "locomotion," "hormone metabolic process," and "detection of stimulus," indicating the stress-buffering role of the GF677 rootstock. Furthermore, the complex regulatory network involved in the drought response was revealed, involving proteins that are associated with signaling transduction, transcription and hormone regulation, redox homeostasis, and frontline barriers. We identified two poorly characterized genes in P. persica: growth-regulating factor 5 (GRF5), which may be involved in cellular expansion, and AtHB12, which may be involved in root elongation. The reliability of the RNA-seq experiment was validated by analyzing the expression patterns of 34 DEGs potentially involved in drought tolerance using quantitative reverse transcription polymerase chain reaction. The transcriptomic resources generated in this study provide a broad characterization of the acclimation of P. persica to drought, shedding light on the major molecular responses to the most important environmental stressor.

A genome-wide analysis of MADS-box genes in peach [Prunus persica (L.) Batsch].

BACKGROUND: MADS-box genes encode a family of eukaryotic transcription factors distinguished by the presence of a highly-conserved ~58 amino acid DNA-binding and dimerization domain (the MADS-box). The central role played by MADS-box genes in peach endodormancy regulation led us to examine this large gene family in more detail. We identified the locations and sequences of 79 MADS-box genes in peach, separated them into established subfamilies, and broadly surveyed their tissue-specific and dormancy-induced expression patterns using next-generation sequencing. We then focused on the dormancy-related SVP/AGL24 and FLC subfamilies, comparing their numbers and phylogenetic relationships with those of other sequenced woody perennial genomes. RESULTS: We identified 79 MADS-box genes distributed across all eight peach chromosomes and frequently located in clusters of two or more genes. They encode proteins with a mean length of 248 ± 72 amino acids and include representatives from most of the thirteen Type II (MIKC) subfamilies, as well as members of the Type I Mα, Mß, and Mγ subfamilies. Most Type I genes were present in species-specific monophyletic lineages, and their expression in the peach sporophyte was low or absent. Most Type II genes had Arabidopsis orthologs and were expressed at much higher levels throughout vegetative and fruit tissues. During short-day-induced growth cessation, seven Type II genes from the SVP/AGL24, AGL17, and SEP subfamilies showed significant changes in expression. Phylogenetic analyses indicated that multiple, independent expansions have taken place within the SVP/AGL24 and FLC lineages in woody perennial species. CONCLUSIONS: Most Type I genes appear to have arisen through tandem duplications after the divergence of the Arabidopsis and peach lineages, whereas Type II genes appear to have increased following whole genome duplication events. An exception to the latter rule occurs in the FLC and SVP/AGL24 Type II subfamilies, in which species-specific tandem duplicates have been retained in a number of perennial species. These subfamilies comprise part of a genetic toolkit that regulates endodormancy transitions, but phylogenetic and expression data suggest that individual orthologs may not function identically across all species.

HDAC3 is essential for DNA replication in hematopoietic progenitor cells.

Histone deacetylase 3 (HDAC3) contributes to the regulation of gene expression, chromatin structure, and genomic stability. Because HDAC3 associates with oncoproteins that drive leukemia and lymphoma, we engineered a conditional deletion allele in mice to explore the physiological roles of Hdac3 in hematopoiesis. We used the Vav-Cre transgenic allele to trigger recombination, which yielded a dramatic loss of lymphoid cells, hypocellular bone marrow, and mild anemia. Phenotypic and functional analysis suggested that Hdac3 was required for the formation of the earliest lymphoid progenitor cells in the marrow, but that the marrow contained 3-5 times more multipotent progenitor cells. Hdac3(-/-) stem cells were severely compromised in competitive bone marrow transplantation. In vitro, Hdac3(-/-) stem and progenitor cells failed to proliferate, and most cells remained undifferentiated. Moreover, one-third of the Hdac3(-/-) stem and progenitor cells were in S phase 2 hours after BrdU labeling in vivo, suggesting that these cells were impaired in transit through the S phase. DNA fiber-labeling experiments indicated that Hdac3 was required for efficient DNA replication in hematopoietic stem and progenitor cells. Thus, Hdac3 is required for the passage of hematopoietic stem/progenitor cells through the S phase, for stem cell functions, and for lymphopoiesis.

Inhibition of histone deacetylase 3 causes replication stress in cutaneous T cell lymphoma.

Given the fundamental roles of histone deacetylases (HDACs) in the regulation of DNA repair, replication, transcription and chromatin structure, it is fitting that therapies targeting HDAC activities are now being explored as anti-cancer agents. In fact, two histone deacetylase inhibitors (HDIs), SAHA and Depsipeptide, are FDA approved for single-agent treatment of refractory cutaneous T cell lymphoma (CTCL). An important target of these HDIs, histone deacetylase 3 (HDAC3), regulates processes such as DNA repair, metabolism, and tumorigenesis through the regulation of chromatin structure and gene expression. Here we show that HDAC3 inhibition using a first in class selective inhibitor, RGFP966, resulted in decreased cell growth in CTCL cell lines due to increased apoptosis that was associated with DNA damage and impaired S phase progression. Through isolation of proteins on nascent DNA (iPOND), we found that HDAC3 was associated with chromatin and is present at and around DNA replication forks. DNA fiber labeling analysis showed that inhibition of HDAC3 resulted in a significant reduction in DNA replication fork velocity within the first hour of drug treatment. These results suggest that selective inhibition of HDAC3 could be useful in treatment of CTCL by disrupting DNA replication of the rapidly cycling tumor cells, ultimately leading to cell death.

Differential expression of a ß-1,4-endoglucanase induced by diet change in the foliar nematode Aphelenchoides fragariae.

We identified and characterized a ß-1,4-endoglucanase, Afr-ENG-1, in the foliar nematode Aphelenchoides fragariae that is differentially expressed when the nematode feeds on fungi or plants. When individuals from hosta plants were transferred to a fungus culture, expression of the enzyme decreased 1,812-fold after five generations on the fungus diet. Afr-eng-1 was readily detected in the genome of 75% of nematodes from the plant population but only in 38% of the diet-changed population. The gene cannot be detected in nematodes maintained on fungus for over 100 generations. Diet was also associated with changes in nematode body size and in the severity of symptoms caused on hosta leaves. Plant-diet nematodes caused larger lesions and were longer and thinner than fungus-diet nematodes. Nematodes moved from a plant diet to a fungus diet for five generations had the same body size as the nematodes that had fed on the fungus for 100 generations. Full-length sequences of Afr-eng-1 were obtained and found to encode a glycosyl hydrolase family 5 protein. This is the first ß-1,4-endoglucanase and plant-parasitism-related gene described in the genus Aphelenchoides.

Induction of Glutaredoxin Expression in Response to Desiccation Stress in the Foliar Nematode Aphelenchoides fragariae.

Desiccation tolerance plays an important role in the overwinter survival of the foliar nematode Aphelenchoides fragariae. Survival rates of A. fragariae were compared with those of the anhydrobiotic soil-dwelling nematode Aphelenchus avenae after desiccation (90% RH), cold (4°C) and osmotic (500 mM sucrose) stress treatments. A. fragariae formed aggregates during desiccation and showed higher survival rates than A. avenae under desiccation and osmotic stress. Analysis of transcripts with Illumina RNA-seq indicated that glutaredoxin and other antioxidant-related genes were up-regulated under desiccation stress. Quantitative RT-PCR demonstrated 2.8 fold and 1.3 fold up-regulation of a glutaredoxin gene under desiccated and osmotic stress, respectively, suggesting the participation of antioxidant mechanisms in desiccation tolerance of A. fragariae.

Hdac3 is essential for the maintenance of chromatin structure and genome stability.

Hdac3 is essential for efficient DNA replication and DNA damage control. Deletion of Hdac3 impaired DNA repair and greatly reduced chromatin compaction and heterochromatin content. These defects corresponded to increases in histone H3K9,K14ac; H4K5ac; and H4K12ac in late S phase of the cell cycle, and histone deposition marks were retained in quiescent Hdac3-null cells. Liver-specific deletion of Hdac3 culminated in hepatocellular carcinoma. Whereas HDAC3 expression was downregulated in only a small number of human liver cancers, the mRNA levels of the HDAC3 cofactor NCOR1 were reduced in one-third of these cases. siRNA targeting of NCOR1 and SMRT (NCOR2) increased H4K5ac and caused DNA damage, indicating that the HDAC3/NCOR/SMRT axis is critical for maintaining chromatin structure and genomic stability.

Binding energies for alkane molecules on a carbon surface from gas-solid chromatography and molecular mechanics.

Gas-solid chromatography was used to determine B(2s) (gas-solid virial coefficient) values for 12 alkanes (10 branched and 2 cyclic) interacting with a carbon powder (Carbopack B, Supelco). B(2s) values were determined by multiple size variant injections within the temperature range of 393 to 623 K with each alkane measured at 5 or 6 different temperatures. The temperature variations of the gas-solid virial coefficients were used to find the experimental adsorption energy or binding energy values (E( *)) for each alkane. A molecular mechanics based, rough-surface model was used to calculate the molecule-surface binding energy (E(cal)( *)) using augmented MM2 parameters. The surface model consisted of three parallel graphene layers with each layer containing 127 interconnected benzene rings and two separated nanostructures each containing 17 benzene rings arranged in a linear strip. As the parallel nanostructures are moved closer together, the surface roughness increases and molecule-surface interactions are enhanced. A comparison of the experimental and calculated binding energies showed excellent agreement with an average difference of 3.8%. Linear regressions of E( *) versus E(cal)( *) for the current data set and a combined current and prior alkane data set both gave excellent correlations. For the combined data set with 18 linear, branched and cyclic alkanes; a linear regression of E( *)=0.9848E(cal)( *) and r(2)=0.976 was obtained. The results indicate that alkane-surface binding energies may be calculated from MM2 parameters for some gas-solid systems.

Reducing airborne ectomycorrhizal fungi and growing non-mycorrhizal loblolly pine (Pinus taeda L.) seedlings in a greenhouse.

Atmospheric spores of ectomycorrhizal (ECM) fungi are a potential source of contamination when mycorrhizal studies are performed in the greenhouse, and techniques for minimizing such contamination have rarely been tested. We grew loblolly pine (Pinus taeda L.) from seed in a greenhouse and inside a high-efficiency particulate air-filtered chamber (HFC) constructed within the same greenhouse. Seedlings were germinated in seven different sand- or soil-based and artificially based growth media. Seedlings grown in the HFC had fewer mycorrhizal short roots than those grown in the open greenhouse atmosphere. Furthermore, the proportion of seedlings from the HFC that were completely non-mycorrhizal was higher than that of seedlings from the greenhouse atmosphere. Seedlings grown in sterilized, artificially based growth media (>50% peat moss, vermiculite, and/or perlite by volume) had fewer mycorrhizal short roots than those grown in sand- or soil-based media. The HFC described here can minimize undesirable ECM colonization of host seedlings in greenhouse bioassays. In addition, the number of non-mycorrhizal seedlings can be maximized when the HFC is used in combination with artificially based growth media.

Automatic discrimination of fine roots in minirhizotron images.

Minirhizotrons provide detailed information on the production, life history and mortality of fine roots. However, manual processing of minirhizotron images is time-consuming, limiting the number and size of experiments that can reasonably be analysed. Previously, an algorithm was developed to automatically detect and measure individual roots in minirhizotron images. Here, species-specific root classifiers were developed to discriminate detected roots from bright background artifacts. Classifiers were developed from training images of peach (Prunus persica), freeman maple (Acer x freemanii) and sweetbay magnolia (Magnolia virginiana) using the Adaboost algorithm. True- and false-positive rates for classifiers were estimated using receiver operating characteristic curves. Classifiers gave true positive rates of 89-94% and false positive rates of 3-7% when applied to nontraining images of the species for which they were developed. The application of a classifier trained on one species to images from another species resulted in little or no reduction in accuracy. These results suggest that a single root classifier can be used to distinguish roots from background objects across multiple minirhizotron experiments. By incorporating root detection and discrimination algorithms into an open-source minirhizotron image analysis application, many analysis tasks that are currently performed by hand can be automated.

Evaluation of molecular mechanics calculated binding energies for isolated and monolayer organic molecules on graphite.

The calculated molecule-surface binding energy, E(cal)( *), for physical adsorption was determined using molecular mechanics MM2 parameters for a model graphite surface and various organic molecules. The results for E(cal)( *) were compared to published experimental binding energy values, E( *), from gas chromatography (GC) or thermal desorption (TD). The binding energies from GC were for isolated molecules in the Henry's law region of adsorption, and the binding energies from TD were for molecules in monolayer coverage on a highly oriented pyrolytic graphite (HOPG). A simple desorption model was used to allow the calculation of monolayer coverage to include both molecule-surface and molecule-molecule interactions and then the results were compared to experimental values. For the 14 TD organic adsorbates (polyaromatic hydrocarbons, alcohols, benzene, substituted benzenes, methane, chloroalkanes, N,N-dimethylformamide, and C(60) Buckyball), the experimental versus calculated binding energies were E( *)=1.1193E(cal)( *) and r(2)=0.967. The GC E( *) values were also well correlated by calculated E(cal)( *) values for a set of 11 benzene and methyl substituted benzenes and for another set of 10 alkanes and haloalkanes. The TD E(cal)( *) mechanics computation provides a useful comparison to the one for GC data since adsorbate-adsorbate interactions as well as adsorbate-surface must be considered.

Changes in the risk of fine-root mortality with age: a case study in peach, Prunus persica (Rosaceae).

Previous studies suggest that younger roots are more vulnerable to mortality than older roots. We analyzed minirhizotron data using a mixed-age, proportional hazards regression approach to determine whether the risk of mortality (or "hazard") was higher for younger roots than for older roots in a West Virginia peach orchard. While root age apparently had a strong effect on the hazard when considered alone, this effect was largely due to different rates of mortality among roots of different orders, diameters, and depths. Roots with dependent laterals (higher order roots) had a lower hazard than first-order roots in 1996 and 1997. Greater root diameter was also associated with a decreased hazard in both 1996 and 1997. In both years, there was a significant decrease in the hazard with depth. When considered alone, age appeared to be a strong predictor of risk: a 1-d increase in initial root age was associated with a 1.26-2.62% decrease in the hazard. However, when diameter, order, and depth were incorporated into the model, the effect of root age disappeared or was greatly reduced. Baseline hazard function plots revealed that the timing of high-risk periods was generally related to seasonal factors rather than individual root age.

MASS-MORTALITY LAYERS OF FOSSIL STICKLEBACK FISH: CATASTROPHIC KILLS OF POLYMORPHIC SCHOOLS.

Stickleback fish (Gasterosteus doryssus) from late middle-Miocene lacustrine deposits of the Truckee Formation in west-central Nevada are abundant and well preserved. They occasionally occur in unusually high densities on single annual laminations (varves) in "mass-mortality layers." We demonstrate that stickleback mass-mortality layers consist of members of schools and may be used as population samples. Comparison of stickleback mass-mortality samples to time-averaged samples, which accumulated over hundreds or thousands of years in the same deposit, indicates that, for some purposes, the time-averaged samples are acceptable surrogates for instantaneous population samples from the G. doryssus lineage. Mass-mortality and time-averaged samples are similar for variation of pelvic structure and dorsal-spine number, but associations between states of different characters may be weaker in mass-mortality samples than in time-averaged samples. Thus, character variances in time-averaged samples of G. doryssus are comparable to those of living populations, but character correlations are suspect. Character variances and correlations in time-averaged fossil samples must be evaluated on a case-by-case basis and interpreted with caution.