High density and limiting nitrogen response in rice
ABSTRACT: Physiological changes underlying high density stress were examined in Oryza sativa plants over the course of a life cycle by assessing differences in gene expression. Moreover, the nitrogen limitation was examined in parallel with high density stress to gain a better understanding of physiological responses specific to high density stress. RNA was extracted from 21 and 31 days old plants. The plants were grown under four conditions: sufficient nitrogen (10mM N) and low density (six plants per bin), limiting nitrogen (3mM N) and low density, sufficient nitrogen and high density (40 plants per bin), limiting nitrogen and high density. Three biological replicates were sampled from each growth condition.
Project description:A custom high density tiling path was designed to a 1.5Mb region of the canFam2.0 reference genome with probes tiled at 6bp or 26bp offsets. 60mer oligonucleotides were synthesized in situ to one 8 x 60K Agilent microarray and hybridized with canine genomic DNA. A single-channel hybridization protocol was employed using eight canine samples.
Project description:Understanding the environmental factors that shape microbial communities is crucial, especially in extreme environments, like Antarctica. Two main forces were reported to influence Antarctic soil microbes: birds and plants. Both birds and plants are currently undergoing unprecedented changes in their distribution and abundance due to global warming. However, we need to clearly understand the relationship between plants, birds and soil microorganisms. We therefore collected rhizosphere and bulk soils from six different sampling sites subjected to different levels of bird influence and colonized by Colobanthus quitensis and Deschampsia antarctica in the Admiralty Bay, King George Island, Maritime Antarctic. Microarray and qPCR assays targeting 16S rRNA genes of specific taxa were used to assess microbial community structure, composition and abundance and analyzed with a range of soil physico-chemical parameters. The results indicated significant rhizosphere effects in four out of the six sites, including areas with different levels of bird influence. Acidobacteria were significantly more abundant in soils with little bird influence (low nitrogen) and in bulk soil. In contrast, Actinobacteria were significantly more abundant in the rhizosphere of both plant species. At two of the sampling sites under strong bird influence (penguin colonies), Firmicutes were significantly more abundant in D. antarctica rhizosphere but not in C. quitensis rhizosphere. The Firmicutes were also positively and significantly correlated to the nitrogen concentrations in the soil. We conclude that the microbial communities in Antarctic soils are driven both by bird and plants, and that the effect is taxa-specific. The study was carried out at the Brazilian Antarctic Station Comandante Ferraz (EACF, 62°04’S, 58°21’W), located in Martel Inlet, Admiralty Bay, King George Island, Antarctic Peninsula, which is part of the South Shetlands Archipelago in Maritime Antarctica. It is a medium sized research station with a population of 10 to 15 people during the winter months (March to November) and about 60 people during the austral summer months (November to March). During the austral summers of 2006 – 2007 and 2008 – 2009, the vascular plants D. antarctica or C. quitensis were sampled, where both plants were found, in triplicate at six different sites: A – Arctowski (2006 – 2007), Q – Quimica (2006 – 2007), I – Ipanema (2006 – 2007), M – North Mountain (2008 – 2009), D – Demay Point (2008 – 2009), C – Copacabana (2008 – 2009) (Figure 1). Points A, C and D were located inside an environmental protected area. Point A is close to the Arctowski Polish Station and next to a colony of Adelie penguins (Pygoscelis adeliae), point C is next to the USA summer station Copacabana in a Gentoo penguin (P. papua) colony, and point D is near to a Polish refuge next to a colony of Chinstrap penguins (P. antarcticus). At point I, there were no penguin colonies present, but this section was used as a nesting site by local species of flying birds. Point Q was located in the vicinity of the EACF; thus there has been (and continues to be) an intense anthropogenic influence on this spot, which is not the case at the other sampling sites. Point M was located at the top of North Mountain, around 200 m altitude. This site has no influence from penguin colonies and only a few nests of skua (Catharacta sp.) were observed. At each sampling site, triplicate soil samples were taken for chemical and biological analyses, with the exception of the Arctowski site (A) where we only took two replicates. Each vascular plant sample was frozen (-20°C) at the EACF.
Project description:Arabidopsis thaliana plants (ecotype Landsberg erecta) were grown at 20˚C constant temperature, 8 hr/16 hr Light/Dark photoperiod at 50-60% ambient humidity, for 8 to 9 weeks. Short day conditions prevented the onset of flowering and the plants were thus maintained in growth stage 1 (leaf production) with 13 to 15 rosette leaves larger than 1mm (stage 1.13 to 1.14). Diamondback moth (DBM, Plutella xylostella) larvae were maintained on cabbage (Brassica oleracea) plants in a climate-controlled room at 25ºC, 12 hr photoperiod with 50%-60% relative humidity. Two days before exposing A. thaliana plants to herbivore treatment, plants were transferred to a climate-controlled room (22ºC, 50-60% humidity, 12 hr photoperiod). For insect treatment, seven Diamondback moth (DBM, Plutella xylostella) larvae (third to fifth instars) were placed on a group of four or five plants until time of harvest, for each time point separately. All rosette leaves were harvested at 1h, 4h, 12h, and 24h after onset of continuous herbivory and flash frozen in liquid nitrogen. Control plants were maintained under the same conditions and harvested in parallel. Keywords: time course, stress response, herbivory response For each time point (1h, 4h, 12h, and 24h) two biological replicates (DBM treatment) were performed. Control plants were harvested in parallel. Total RNA from each sample was used for two labelling reactions using dye-flips (Cy5 or Cy3) and labelled cDNA from treatment and control plants were co-hybridized. Thus, for each time-point four replicates were analyzed each comparing treatment with the corresponding control (rep1 and rep2 are dye-flips of biological replicate 1, and rep3 and rep4 are dye-flips of biological replicate 2). For background correction, we defined the mean of the lowest 10% of spot intensities from a particular subgrid as the background for that subgrid. This mean was subtracted from each spot in the subgrid. Signal intensities that did not exceed the background plus 3 standard deviations thereof were defined as not detectable and were excluded from further analyses. We normalized using loess curves thus generating log2 transformed expression ratios comparing DBM treatment with control. These ratios are given in the VALUE columns of each array.
Project description:Salt responsive genes were identified in chinese willow (Salix matsudana) after the plants were treated with 100 mM NaCl. for 48 hours We used microarrays to identify genes responsible for combating salt stress. Those up-regulated during the NaCl treatment may protect the plants from damages caused by salt stress. 2 month-old S. matsudana plants which were treated with 100 mM NaCl and control plants were used for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain salt responsive genes that protect the plants from stress injury.Those differentially expressed genes identified by the microarray would help to understand the mechanism of S. matsudana reacting to salt stress.
Project description:In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on the spatiotemporal expression patterns and gene ontology (GO) categories of anther-expressed genes, some noteworthy expression patterns are discussed in connection with various important biological events of anther development. The separated transcriptomes of rice microspore/pollen and tapetum were measured at the premeiosis, meiosis, tetrad, uninuclear, bicellular, and tricelluar stages by using laser microdissection (LM)-mediated microarray.
Project description:Objective - The TRIB1 locus has been linked to hepatic triglyceride metabolism in mice and to plasma triglycerides and coronary artery disease (CAD) in humans. The lipid associated SNPs identified by genome-wide association studies (GWAS) are located ~ 30 kb downstream from TRIB1 suggesting complex regulatory effects on genes or pathways relevant to hepatic triglyceride metabolism. The goal of this study was to investigate the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits. Methods & Results - Characterization of the risk locus reveals that it encompasses a gene, TRIB1 associated locus (TRIBAL) comprised of a well conserved promoter region and an alternatively spliced transcript. Bioinformatic analysis and re-sequencing identified a single nucleotide polymorphism (SNP), rs2001844, within the promoter region that associates with increased plasma triglycerides, reduced HDL-C and CAD risk. Furthermore, we show that rs2001844 is an expression trait locus (eQTL) for TRIB1 expression in blood and alters TRIBAL promoter activity in a reporter assay model. The TRIBAL transcript has features typical of long noncoding RNAs (lncRNA), including poor sequence conservation. Modulation of TRIBAL expression had limited impact on either TRIB1 or lipid regulatory genes mRNA levels in human hepatocyte models. In contrast, TRIB1 knockdown markedly increased TRIBAL expression in HepG2 cells and primary human hepatocytes. Conclusions - These studies demonstrate an interplay between a novel locus,TRIBAL, and TRIB1. TRIBAL is located in the GWAS identified risk locus, responds to altered expression of TRIB1, harbors a risk SNP that is an eQTL for TRIB1 expression and associates with plasma triglyceride concentrations. HepG2 hepatoma cells were stably infected with TRIBAL1 or no insert carrying lentiviruses
Project description:Non-symbiotic hemoglobins are ubiquitously expressed proteins known to interact with nitric oxide, an inhibitor of mitochondrial respiration and an important signalling component. We evaluated the underlying molecular mechanisms of AtHb1 (also referred as AtGLB1 or AHb1) function, its effects on stress response and the interplay with nitric oxide. For this purpose, AtHb1 was overexpressed in Arabidopsis thaliana under control of the seed-specific promoter LeB4. We performed comparative transcriptome analysis of developing siliques from wild type (WT, Col-0) and transgenic plants subjected to control and moderate hypoxic conditions. The experimental design was used to analyze the underlying molecular mechanisms of AtHb1 function and to assess differences in the hypoxic response between WT and AtHb1-overexpressing seeds/siliques. Hypoxic and control (normoxic) treatment was carried out with complete transgenic (T3) and WT plants 45 days after germination (DPG). Plants were aerated with a gas mixture containing 10.5% oxygen or ambient gas containing 21% oxygen for control samples in darkness. After one hour, plants were decapitated, immediately frozen in liquid nitrogen, and siliques were dissected in liquid nitrogen for RNA extraction and hybridization to Affymetrix microarrays. Hypoxic and control treatment runs with WT and AtHb1-overexpressing plants were repeated twice to provide 3 biological replicates (total number of samples = 12).
Project description:Following 20wk of either chow or high fat feeding, tissues were collected from C57BL/6J mice. Gene expression in four tissues that have been associated with obesity-related metabolic complications: white adipose tissue (WAT), skeletal muscle, the liver, and the heart were examined. Data suggest that some tissues may be attempting to reduce the metabolic perturbations that occur in the early stages of obesity. Also, many identified genes were associated with a variety of disorders, thereby serving as potential links between obesity and related health risks. After 3 wk of weaning, male C57BL/6J littermates were randomly separated into control (n=6) and treatment (n=6) groups for each tissue. After an acclimatization period of 1wk, animals were given free access to a chow diet (23% protein, 21% fat, and 55% carbohydrate) or a high fat (HF) diet (15% protein, 59% fat, and 26% carbohydrate) for 20wk. Following the feeding period, animals were fasted for 6h, anesthetized with pentobarbital, and weighed. Blood glucose was assessed in anesthetized mice. Non-esterified fatty acids and immunoreactive insulin was assayed with a double antibody method. Samples of WAT, the liver, gastrocnemius muscle and heart were obtained, frozen with liquid nitrogen, and stored at -80oC until subsequent use. Tissue samples were homogenized at short intervals of time and total RNA was isolated using cold TRIzol (Invitrogen, Ontario, Canada) to prevent RNA degradation. Cdna was prepared to perform the microarrays. For every treatment sample and its corresponding control, two microarrays were performed in a dye-swap normalization experiment. Mouse M7K microarray chip from the Southern Alberta Microarray Facility (University of Calgary, Calgary, AB) were used (GEO Accession: Platform GPL3965).
Project description:Background: The change from juvenile to mature phase in woody plants is often accompanied by a gradual loss of rooting ability, as well as by reduced microRNA (miR) 156 and increased miR172 expression. Results: We characterized the population of miRNAs of Eucalyptus grandis and compared by Northern blot the gradual reduction in miR156 and increase in miR172 expression during development to the loss of rooting ability. Forty known and eight novel miRNAs were discovered and their predicted targets are listed. The expression pattern of nine miRNAs was determined during adventitious root formation in juvenile and mature cuttings. While the expression levels of miR156 and miR172 were inverse in juvenile and mature tissues, no mutual relationship was found between high miR156 expression and rooting ability, or high miR172 expression and loss of rooting ability. This is shown both in E. grandis and also in E. brachyphylla, in which explants that underwent rejuvenation in tissue culture conditions were also examined. Conclusions: It is suggested that in these Eucalyptus species, there is no correlation between the switch of miR156 with miR172 expression in the stems and the loss of rooting ability. Examination of microRNA in seedlings of Eucalyptus grandis