Small GTP-Binding Protein Gene Is Associated with QTL for Submergence Tolerance in Rice

Small GTP-binding proteins play critical roles in signal transduction in mammalian and plant systems. In this study, sequence variation of a small GTP-binding protein identified in the subgenomic region was analyzed. The major quantitative trait locus (QTL) controlling submergence tolerance on the 6.5-cM region of chromosome 9 was previously mapped, sequenced, and annotated. One of the most interesting candidate genes located in this QTL was a 5.2-kb sequence, which included a coding sequence consisting of two exons and a promoter. The deduced amino acid sequence corresponded to a 24.8 kD protein consisting of 226 amino acids, with 98% identity to RGP1, a small GTP-binding protein involved in a signal pathway responding to hormones, such as cytokinin and ethylene. According to the amino acid sequence, a putative small G-protein was classified as a small Ras-related GTP-binding protein. DNA gel blot analysis showed that the putative gene encoding the Ras-related GTP-binding protein was present as a single copy in the rice genome. Comparison of genomic sequences from several rice cultivars tolerant to submergence identified single nucleotide polymorphisms located in the TATA box of the Ras promoter region. Linkage analysis showed that the putative gene for GTP-binding protein was tightly linked to the peak of the QTL previously mapped on the long arm of chromosome 9. The single strand conformation polymorphism of the putative GTP-binding protein gene can be used for allele discrimination and marker assisted selection for tolerance to flash flooding.     

Two Horizontally Transferred Xenobiotic Resistance Gene Clusters Associated with Detoxification of Benzoxazolinones by Fusarium Species

Microbes encounter a broad spectrum of antimicrobial compounds in their environments and often possess metabolic strategies to detoxify such xenobiotics. We have previously shown that Fusarium verticillioides, a fungal pathogen of maize known for its production of fumonisin mycotoxins, possesses two unlinked loci, FDB1 and FDB2, necessary for detoxification of antimicrobial compounds produced by maize, including the γ-lactam 2-benzoxazolinone (BOA). In support of these earlier studies, microarray analysis of F. verticillioides exposed to BOA identified the induction of multiple genes at FDB1 and FDB2, indicating the loci consist of gene clusters. One of the FDB1 cluster genes encoded a protein having domain homology to the metallo-β-lactamase (MBL) superfamily. Deletion of this gene (MBL1) rendered F. verticillioides incapable of metabolizing BOA and thus unable to grow on BOA-amended media. Deletion of other FDB1 cluster genes, in particular AMD1 and DLH1, did not affect BOA degradation. Phylogenetic analyses and topology testing of the FDB1 and FDB2 cluster genes suggested two horizontal transfer events among fungi, one being transfer of FDB1 from Fusarium to Colletotrichum, and the second being transfer of the FDB2 cluster from Fusarium to Aspergillus. Together, the results suggest that plant-derived xenobiotics have exerted evolutionary pressure on these fungi, leading to horizontal transfer of genes that enhance fitness or virulence.     

Whole Genome Transcript Profiling of Drug Induced Steatosis in Rats Reveals a Gene Signature Predictive of Outcome

Drug induced steatosis (DIS) is characterised by excess triglyceride accumulation in the form of lipid droplets (LD) in liver cells. To explore mechanisms underlying DIS we interrogated the publically available microarray data from the Japanese Toxicogenomics Project (TGP) to study comprehensively whole genome gene expression changes in the liver of treated rats. For this purpose a total of 17 and 12 drugs which are diverse in molecular structure and mode of action were considered based on their ability to cause either steatosis or phospholipidosis, respectively, while 7 drugs served as negative controls. In our efforts we focused on 200 genes which are considered to be mechanistically relevant in the process of lipid droplet biogenesis in hepatocytes as recently published (Sahini and Borlak, 2014). Based on mechanistic considerations we identified 19 genes which displayed dose dependent responses while 10 genes showed time dependency. Importantly, the present study defined 9 genes (ANGPTL4, FABP7, FADS1, FGF21, GOT1, LDLR, GK, STAT3, and PKLR) as signature genes to predict DIS. Moreover, cross tabulation revealed 9 genes to be regulated ≥10 times amongst the various conditions and included genes linked to glucose metabolism, lipid transport and lipogenesis as well as signalling events. Additionally, a comparison between drugs causing phospholipidosis and/or steatosis revealed 26 genes to be regulated in common including 4 signature genes to predict DIS (PKLR, GK, FABP7 and FADS1). Furthermore, a comparison between in vivo single dose (3, 6, 9 and 24 h) and findings from rat hepatocyte studies (2 h, 8 h, 24 h) identified 10 genes which are regulated in common and contained 2 DIS signature genes (FABP7, FGF21). Altogether, our studies provide comprehensive information on mechanistically linked gene expression changes of a range of drugs causing steatosis and phospholipidosis and encourage the screening of DIS signature genes at the preclinical stage.     

Metagenomic Analysis Reveals Diverse Polyketide Synthase Gene Clusters in Microorganisms Associated with the Marine Sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up <1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.     

Comparative Transcriptome Analysis Reveals Potential Gene Modules Associated with Cold Tolerance in Sugarcane (Saccharum officinarum L.)

Journal of Plant Growth Regulation - Sugarcane is an important crop worldwide, and most sugar is derived directly from sugarcane. Due to its thermophilic nature, the yield of sugarcane is largely...     

Dynamic Precision Phenotyping Reveals Mechanism of Crop Tolerance to Root Herbivory

The western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) is a major pest of maize (Zea mays) that is well adapted to most crop management strategies. Breeding for tolerance is a promising alternative to combat WCR but is currently constrained by a lack of physiological understanding and phenotyping tools. We developed dynamic precision phenotyping approaches using ¹¹C with positron emission tomography, root autoradiography, and radiometabolite flux analysis to understand maize tolerance to WCR. Our results reveal that WCR attack induces specific patterns of lateral root growth that are associated with a shift in auxin biosynthesis from indole-3-pyruvic acid to indole-3-acetonitrile. WCR attack also increases transport of newly synthesized amino acids to the roots, including the accumulation of Gln. Finally, the regrowth zones of WCR-attacked roots show an increase in Gln turnover, which strongly correlates with the induction of indole-3-acetonitrile-dependent auxin biosynthesis. In summary, our findings identify local changes in the auxin biosynthesis flux network as a promising marker for induced WCR tolerance.The western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte; Supplemental Fig. S1) is a voracious pest of maize (Zea mays). Larvae hatch in the soil during late spring and immediately begin feeding on the crop’s root system. Over time, active feeding can result in substantial root damage with significant loss of water and/or nutrient uptake, thus weakening plants (Flint-Garcia et al., 2009). Plants also become highly susceptible to lodging when major damage is inflicted upon the anchoring root system. Taken together, these effects can result in significant corn yield losses and management costs totaling between $650 million to $1 billion in the U.S. annually (Flint-Garcia et al., 2009; Gray et al., 2009).History reveals the enormous resilience and adaptability of this pest and just how quickly it can evolve to overcome management strategies. For example, resistance to application of chemical pesticides, including cyclodienes (benzene hexachloride, aldrin) and organophosphates (methyl parathion), was seen over just a 10-year period of their use in Nebraska’s cornfields during the 1950s and 1990s, respectively (Ball and Weekman, 1963; Meinke et al., 1998). Alternate management practices, including rotation of corn with other crops on a seasonal basis, was generally considered the best choice for management since 1909 (Levine et al., 2002). In east/central Illinois, 95% to 98% of cropland had adopted a management strategy using only soybean as the rotation crop. Unfortunately, the enthusiastic adoption of this strategy over a broad area combined with the efficacy of the technique created a strong selection that favored a less common D. v. virgifera phenotype with reduced egg laying fidelity to cornfields. Over time, natural selection afforded a strong reproductive advantage to females laying their eggs in soybean fields. Since the late 1990s, a strain of the western corn rootworm with resistance to crop rotation can be found in parts of Illinois, Indiana, and parts of bordering states (Gray et al., 2009; Levine et al., 2002).More recently, D. v. virgifera resistance to deployed genetically modified organisms has been reported. First introduced into the market to target this pest back in 2003, genetically altered Bt-maize expressing one or more proteins from the soil bacteria Bacillus thuringiensis provided enhanced plant defenses to larval feeding. When a vulnerable insect ate the Bt-containing plant, the protein became activated in its gut, forming a toxin that paralyzed the digestive system and caused it to stop feeding. Unfortunately, resistance began to show within three generations of selection (Meihls et al., 2008).An alternative strategy to reduce the negative impact of D. v. virgifera attack without triggering counter adaptations in the pest is plant tolerance, which relies on a plant’s capacity to maintain growth and yield even in the presence of substantial damage. While D. v. virgifera-tolerant maize germplasms exhibiting slight to moderate tolerances to D. v. virgifera have been reported (Flint-Garcia et al., 2009), more effective lines are needed. Unfortunately, we know very little about the underlying mechanisms for crop tolerance. Over the years, one resounding message has been that the physiological processes affected by herbivory should be better characterized before breeding tools can be leveraged in a rational way to generate improved varieties that maintain high yields under herbivore pressure (Riedell, 1990). Rational decision making in the breeding selection process requires rigorous phenotyping; however, present phenotyping tools tell us little about the plasticity of root systems, especially when it comes to understanding mechanisms for crop tolerance to attack belowground. It was recently suggested that the timing for allocation of newly fixed carbon resources as soluble sugars between leaves, stalks, and root systems, and their coordination with mobilization of other resources including amino acids, may play significant roles in determining the ability of maize plants to survive an attack by D. v. virgifera (Orians et al., 2011; Robert et al., 2014).In this work, our systematic evaluation of the physiological, metabolic, and genetic basis for root regrowth as a tolerance trait sheds new light on the regulation of the growth hormone auxin (indole-3-acetic acid [IAA]) and its role in this process. Radioactive decay of ¹¹C (β⁺ emitter, t_1/2 = 20.4 min), dynamic whole-plant positron emission tomography, root autoradiography, and radiometabolite flux analyses allowed us to map the transport, allocation and metabolism of carbon and nitrogen resources against genetic and radiolabeled biochemical markers including [¹¹C]IAA, [¹¹C]indole, [¹¹C]indole-3-acetonitrile ([¹¹C]IAN), [¹¹C]indole-3-acetamide ([¹¹C]IAM), and l-[5-¹¹C]Gln (Supplemental Fig. S2). Taken together, these tools enabled us for the first time, to our knowledge, to rigorously map out the auxin biosynthesis flux network at regional tissue levels and in turn provide new insights on auxin regulation and its coordination with the availability of a key amino acid, l-Gln. The developed phenotyping tools can now be employed for the rapid identification and selection of D. v. virgifera-tolerant maize germplasm.     

Metabolomic Profiling Reveals Biochemical Pathways and Biomarkers Associated with Pathogenesis in Cystic Fibrosis Cells

Cystic fibrosis (CF) is a life-shortening disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To gain an understanding of the epithelial dysfunction associated with CF mutations and discover biomarkers for therapeutics development, untargeted metabolomic analysis was performed on primary human airway epithelial cell cultures from three separate cohorts of CF patients and non-CF subjects. Statistical analysis revealed a set of reproducible and significant metabolic differences between the CF and non-CF cells. Aside from changes that were consistent with known CF effects, such as diminished cellular regulation against oxidative stress and osmotic stress, new observations on the cellular metabolism in the disease were generated. In the CF cells, the levels of various purine nucleotides, which may function to regulate cellular responses via purinergic signaling, were significantly decreased. Furthermore, CF cells exhibited reduced glucose metabolism in glycolysis, pentose phosphate pathway, and sorbitol pathway, which may further exacerbate oxidative stress and limit the epithelial cell response to environmental pressure. Taken together, these findings reveal novel metabolic abnormalities associated with the CF pathological process and identify a panel of potential biomarkers for therapeutic development using this model system.     

Peroxidase Profiling Reveals Genetic Linkage between Peroxidase Gene Clusters and Basal Host and Non-Host Resistance to Rusts and Mildew in Barley

Background

Higher plants possess a large multigene family encoding secreted class III peroxidase (Prx) proteins. Peroxidases appear to be associated with plant disease resistance based on observations of induction during disease challenge and the presence or absence of isozymes in resistant vs susceptible varieties. Despite these associations, there is no evidence that allelic variation of peroxidases directly determines levels of disease resistance.

Methodology/Principal Findings

The current study introduces a new strategy called Prx-Profiling. We showed that with this strategy a large number of peroxidase genes can be mapped on the barley genome. In order to obtain an estimate of the total number of Prx clusters we followed a re-sampling procedure, which indicated that the barley genome contains about 40 peroxidase gene clusters. We examined the association between the Prxs mapped and the QTLs for resistance of barley to homologous and heterologous rusts, and to the barley powdery mildew fungus. We report that 61% of the QTLs for partial resistance to P. hordei, 61% of the QTLs for resistance to B. graminis and 47% of the QTLs for non-host resistance to other Puccinia species co-localize with Prx based markers.

Conclusions/Significance

We conclude that Prx-Profiling was effective in finding the genetic location of Prx genes on the barley genome. The finding that QTLs for basal resistance to rusts and powdery mildew fungi tend to co-locate with Prx clusters provides a base for exploring the functional role of Prx-related genes in determining natural differences in levels of basal resistance.     

Metabolite Profiling Reveals Abiotic Stress Tolerance in Tn5 Mutant of Pseudomonas putida

Pseudomonas is an efficient plant growth–promoting rhizobacteria (PGPR); however, intolerance to drought and high temperature limit its application in agriculture as a bioinoculant. Transposon 5 (Tn5) mutagenesis was used to generate a stress tolerant mutant from a PGPR Pseudomonas putida NBRI1108 isolated from chickpea rhizosphere. A mutant NBRI1108T, selected after screening of nearly 10,000 transconjugants, exhibited significant tolerance towards high temperature and drought. Southern hybridization analysis of EcoRI and XhoI restricted genomic DNA of NBRI1108T confirmed that it had a single Tn5 insertion. The metabolic changes in the polar and non-polar extracts of NBRI1108 and NBRI1108T were examined using ¹H, ³¹P nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Thirty six chemically diverse metabolites consisting of amino acids, fatty acids and phospholipids were identified and quantified. Insertion of Tn5 influenced amino acid and phospholipid metabolism and resulted in significantly higher concentration of aspartic acid, glutamic acid, glycinebetaine, glycerophosphatidylcholine (GPC) and putrescine in NBRI1108T as compared to that in NBRI1108. The concentration of glutamic acid, glycinebetaine and GPC increased by 34%, 95% and 100%, respectively in the NBRI1108T as compared to that in NBRI1108. High concentration of glycerophosphatidylethanolamine (GPE) and undetected GPC in NBRI1108 indicates that biosynthesis of GPE may have taken place via the methylation pathway of phospholipid biosynthesis. However, high GPC and low GPE concentration in NBRI1108T suggest that methylation pathway and phosphatidylcholine synthase (PCS) pathway of phospholipid biosynthesis are being followed in the NBRI1108T. Application of multivariate principal component analysis (PCA) on the quantified metabolites revealed clear variations in NBRI1108 and NBRI1108T in polar and non-polar metabolites. Identification of abiotic stress tolerant metabolites from the NBRI1108T suggest that Tn5 mutagenesis enhanced tolerance towards high temperature and drought. Tolerance to drought was further confirmed in greenhouse experiments with maize as host plant, where NBRI1108T showed relatively high biomass under drought conditions.     

Genome-Wide Association Study Reveals Multiple Novel QTL Associated with Low Oxygen Tolerance in Hybrid Catfish

Hypoxic condition is common in aquaculture, leading to major economic losses. Genetic analysis of hypoxia tolerance, therefore, is not only scientifically significant, but also economically important. Catfish is generally regarded as being highly tolerant to low dissolved oxygen, but variations exist among various populations, strains, and species. In this study, we conducted a genome-wide association study (GWAS) using the catfish 250 K SNP array to identify quantitative trait locus (QTL) associated with tolerance to low dissolved oxygen in the channel catfish × blue catfish interspecific system. Four linkage groups (LG2, LG4, LG23, and LG29) were found to be associated with low oxygen tolerance in hybrid catfish. Multiple significant SNPs were found to be physically linked in genomic regions containing significant QTL for low oxygen tolerance on LG2 and LG23, and in those regions containing suggestively significant QTL on LG2, LG4, LG23, and LG29, suggesting that the physically linked SNPs were genuinely segregating and related with low oxygen tolerance. Analysis of genes within the associated genomic regions suggested that many of these genes were involved in VEGF, MAPK, mTOR, PI3K-Akt, P53-mediated apoptosis, and DNA damage checkpoint pathways. Comparative analysis indicated that most of the QTL at the species level, as analyzed by using the interspecific system, did not overlap with those identified from six strains of channel catfish, confirming the complexity of the genetic architecture of hypoxia tolerance in catfish.     

Profiling of Volatile Compounds and Associated Gene Expression and Enzyme Activity during Fruit Development in Two Cucumber Cultivars

Changes in volatile content, as well as associated gene expression and enzyme activity in developing cucumber fruits were investigated in two Cucumis sativus L. lines (No. 26 and No. 14) that differ significantly in fruit flavor. Total volatile, six-carbon (C6) aldehyde, linolenic and linoleic acid content were higher during the early stages, whereas the nine-carbon (C9) aldehyde content was higher during the latter stages in both lines. Expression of C. sativus hydroperoxide lyase (CsHPL) mirrored 13-hydroperoxide lyase (13-HPL) enzyme activity in variety No. 26, whereas CsHPL expression was correlated with 9-hydroperoxide lyase (9-HPL) enzyme activity in cultivar No. 14. 13-HPL activity decreased significantly, while LOX (lipoxygenase) and 9-HPL activity increased along with fruit ripening in both lines, which accounted for the higher C6 and C9 aldehyde content at 0-6 day post anthesis (dpa) and 9-12 dpa, respectively. Volatile compounds from fruits at five developmental stages were analyzed by principal component analysis (PCA), and heatmaps of volatile content, gene expression and enzyme activity were constructed.     

Genetic Diversity of Nostoc Symbionts Endophytically Associated with Two Bryophyte Species

The diversity of the endophytic Nostoc symbionts of two thalloid bryophytes, the hornwort Anthoceros fusiformis and the liverwort Blasia pusilla, was examined using the tRNA^Leu (UAA) intron sequence as a marker. The results confirmed that many different Nostoc strains are involved in both associations under natural conditions in the field. The level of Nostoc diversity within individual bryophyte thalli varied, but single DNA fragments were consistently amplified from individual symbiotic colonies. Some Nostoc strains were widespread and were detected from thalli collected from different field sites and different years. These findings indicate a moderate level of spatial and temporal continuity in bryophyte-Nostoc symbioses.     

寄生垂叶榕的两种非传粉榕小蜂的产卵行为

通过对两种果外产卵非传粉榕小蜂Philotrypesissp.和Sycoscaptersp.产卵行为的详细观察,发现两种小蜂产卵期都集中在榕果发育的间花期,并且只在进过传粉榕小蜂的榕果内产卵。这两种小蜂的产卵行为基本都可以分为寻找产卵位点、刺壁、产卵和回收产卵器等步骤。落在果面上的两种小蜂的繁殖雌蜂都通过触角敲击果面寻找产卵位点。产卵结束后,Philotrypesissp.大多数情况在原位收回产卵针,而Sycoscaptersp.必须向前爬行才能将产卵针从果内收回。为了争夺产卵位点,在同一榕果产卵的Philotrypesissp.繁殖雌蜂之间会进行打斗。而Sycoscaptersp.繁殖雌蜂之间未观察到打斗行为。两种小蜂产卵器长度虽显著长于各自产卵时榕果果壁厚度,但却显著短于其产卵时榕果果壁和子房层的总厚度,说明这两种小蜂采用产卵针直接刺穿小花子房的产卵模式。