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Transcriptional and metabolomic analysis of
Plant genomics
We have previously shown that lipophilic components (LPC) of the brown seaweed
Gene expression studies revealed that the accumulation of proline was mediated by an increase in the expression of the proline synthesis genes
Taken together, the results of the experiments presented in this paper provide evidence to support LPC mediated freezing tolerance enhancement through a combination of the priming of plants for the increased accumulation of osmoprotectants and alteration of cellular fatty acid composition.
Environmental factors such as low temperature, drought, and salinity are the major abiotic stress factors that adversely affect the growth and development of plants limiting crop productivity
A number of chemicals have been tested for inducing freezing tolerance. Whereas exogenous application of cryoprotectants such as sorbitol and polyethylene glycol were found to be either phyto-toxic or only marginally effective in imparting frost tolerance
Extracts of
The role of proline in low temperature tolerance in plants has been reported
In an earlier study, we demonstrated that
Several expression profile studies in Arabidopsis have established that large scale changes occur in gene expression during cold acclimation and subsequent freezing tolerance
Microarrays provide an advantage of allowing parallel quantification of the gene expression at the whole genome level. Here, we report changes in the metabolome and global transcript profile in plants, after treatment with the LPC and subsequent exposure to freezing temperatures and post-freezing recovery.
Proline estimation
Plants treated with ANE or LPC accumulated higher amounts of proline during freezing stress, as compared to water controls (Figure
Figure 1
Proline accumulation and expression of proline metabolism genes in wild-type (WT)
Proline accumulation and expression of proline metabolism genes in wild-type (WT)
Transcriptional analysis of proline biosynthesis and degradation genes
Analysis of transcript abundance for genes involved in proline biosynthesis (
Proline mutant studies
The role of proline in ANE-mediated freezing tolerance in Arabidopsis, was confirmed by using
Figure 2
Soluble sugar estimation and sugar mutant studies
Wild-type plants treated with ANE or LPC accumulated higher amounts of total soluble sugars in response to freezing stress, as compared to untreated controls (Figure
Figure 3
Accumulation of soluble sugars in wild-type Arabidopsis plants treated with ANE or LPC under different freezing and thawing regimes
Accumulation of soluble sugars in wild-type Arabidopsis plants treated with ANE or LPC under different freezing and thawing regimes. Data are the means ± SE, Bar with * is significantly different (P ≤ 0.05) from that of the control treatment.
Confirmation of the role of soluble sugars in ANE-mediated freezing tolerance using Arabidopsis mutant
The extent of tissue damage in
Figure 4
Sugar accumulation is required for ANE-mediated freezing tolerance in
Sugar accumulation is required for ANE-mediated freezing tolerance in
1H NMR metabolite profiles of ANE extracts
Application of LPC elicited significant changes in the metabolome of Arabidopsis in response to freezing stress. At ambient temperature, the application of ANE or LPC had minimal effect on the metabolic profile of Arabidopsis. However, when exposed to −2°C for 24 h, significant increases in peaks with 1H NMR chemical shifts (δ) at 0.8 to 1.6, 2.1 to 2.8, 3.2 to 3.9, and 5.2 to 5.5 ppm were observed in the plants treated with LPC, as compared to control (Figure
Figure 5
1H-NMR metabolite profile spectrum of wild-type Arabidopsis plants treated with LPC during freezing temperature of −2°C for 24 h
1 H-NMR metabolite profile spectrum of wild-type Arabidopsis plants treated with LPC during freezing temperature of −2°C for 24 h.
Figure 6
2D NMR spectra of metabolites extracted from LPC-treated plants. upon exposure to freezing stress of −2.5°C for 24 h
2D NMR spectra of metabolites extracted from LPC-treated plants, upon exposure to freezing stress of −2.5°C for 24 h: (A) 2D NMR TOCSY spectrum showing characteristic proton correlations in unsaturated fatty acids and (B) 2D NMR HSQC spectrum showing characteristic proton and carbon signals of unsaturated fatty acids and sugars or sugar alcohols.
Global transcriptional changes elicited by ANE
The transcript level changes during the post freezing recovery period were analyzed in ANE-treated Arabidopsis plants thawed at 4°C for 24h after a freezing treatment of −2°C for 24h (Additional file
Additional file 1
Figure S1. Global gene expression of Arabidopsis plants treated with lipophilic fraction of ANE (LPC) during freezing stress and post freezing recovery period. Volcano plot of differential expression pattern during (a) freezing stress and (b) post freezing recovery with ≥1.5 foldchange and P≤0.05 cut off. Scatter plot of log2 signal intensities of three replicate samples from LPC-treated plants during (c) freezing stress and (d) post freezing recovery. Figure S2. Functional categorization of differentially expressed genes. Normalized frequency (Provart and Zhu, 2003) categories of (a) up, or (c) down-regulated genes in LPC-treated plants during freezing. (b&d) functional categorization (b) up or (d) down-regulated genes during freezing according to the actual number of genes. Figure S3. Functional categorization of differentially expressed genes. Normalized frequency (Provart and Zhu, 2003) categories of (a) up, or (c) down-regulated genes in LPC-treated plants during post-freezing recovery period. (b& d) functional categorization (b) up or (d) down-regulated genes according to the actual number of genes during post-freezing recovery period. Figure S4. Heat map of selected differentially expressed genes in LPC treated plants during (FR) freezing stress and (TH) post freezing recovery period. Expression-level change is shown in a color relative to the expression level, as indicated in the color scale bar.
Click here for file
Functional classification
The genes that were differentially expressed during freezing or post-freezing recovery were classified into different functional categories, using the web-based tool Classification Super-Viewer, BLAST searches, and literature reports. The gene lists were classified according to the absolute gene number and normalized frequency. Genes associated with abiotic or biotic stimuli, transcription factor activity, response to stress, etc. ranked highest among the up-regulated genes (Additional file
When the genes were classified according to the actual number of genes which were differentially expressed, 'unknown category' (other molecular functions, other biological processes, other metabolic processes, other cellular processes, etc.) ranked at the top (Additional file
Pathway analysis
LPC-mediated differential gene expression in Arabdopsis
Standardized AraCyc-defined metabolic pathways were used to study the metabolic changes in LPC-treated Arabidopsis plants during freezing and post-freezing recovery. To examine the similarities and differences in the concerned metabolic pathways during the freezing stress and post-freezing recovery periods, entire biosynthetic pathways of Arabidopsis were analyzed. During freezing, out of the total 1113 genes which were differentially expressed (either up-regulated or down-regulated), 334 genes were identified by the Pathway Tools Omics Viewer, and of these 179 genes were assigned specific pathway. On the other hand, 138 genes out of total 398 genes were identified and 66 genes were assigned pathway during the post freezing recovery period. We found that specific pathways were affected significantly by LPC treatment, with little overlap during freezing and post-freezing recovery (Figure
Figure 7
Diagram of representative metabolic pathways regulated by the LPC treatment during freezing stress
Diagram of representative metabolic pathways regulated by the LPC treatment during freezing stress. Each pathway is shown as a glyph consisting of nodes and lines, which represent the metabolites and reactions, respectively. Expression-level change of each reaction is shown in a color relative to the expression level, as indicated in the color scale bar. Triangle, amino acids; square, carbohydrates; diamond, proteins; open circle, others; closed circle, phosphorylated.
Figure 8
Venn diagram showing the number of significantly (a) up or (b) down-regulated and the shared genes during freezing stress and post-freezing recovery period
Venn diagram showing the number of significantly (a) up or (b) down-regulated and the shared genes during freezing stress and post-freezing recovery period.
Major changes observed were the multi-step promotion of the starch degradation pathway (SEX1, SEX4, AT1G03310, AT4G09020, AT4G15210, AT3G46970), inactivation of sucrose degradation (AT1G12240.2.12), induction of galactose degradation pathways I & II (MUR4, AT2G34850), stimulation of raffinose/stachyose biosynthesis (UGE2, UGE5, GolS2, GolS3), and down-regulation of trehalose synthesis (TPS8, TPS9), and gluconeogenesis (AT1G09780) (Figure
A major pathway involved in galactolipid biosynthesis and lipid trafficking in thylakoid membranes (DGD1) was specifically affected (Figure
During freezing, the hormonal balance pathway responsible for conjugation of cytokinins involving
A polyamine biosynthetic gene (AT5G15950) involved in spermine/spermidine biosynthesis was up-regulated in LPC treated plants. Pathway analysis of proline dehydrogenase (
Specificity of LPC treatment at the transcriptional level
A rather limited gene overlap was found between the ANE treatment and the control, as shown in the Venn diagram (Figure
Additional file 2
Table S1. Primer sets and the PCR conditions used for gene expression studies. This material is available as part of the online article from:
Click here for file
Heat map
To get a broad overview of the expression pattern of individual genes during freezing and post-freezing recovery, a heat map was generated using the web-based tool, Heat Mapper Plus. A representative sample of the heat map of the global changes in the transcript levels of genes during freezing and the post-freezing recovery period is given in Additional file
Verification of microarray data by quantitative RT–PCR
Several transcripts of selected up-regulated genes based on microarray analysis were quantified independently by RT–PCR (Additional file
The results presented in this paper show that lipophilic components of
Proline is one of several compounds with roles as compatible solutes that accumulate in response to freezing and drought stress,
In higher plants, proline is synthesized via the glutamate pathway or the ornithine pathway
The results of our experiments with
Several studies have suggested that cold-induced sugar accumulation enhances the degree of plant freezing tolerance. In Arabidopsis, a large increase in the degree of freezing tolerance is positively correlated with soluble sugar content
Recently, Uemura et al.
A positive correlation between sugar accumulation and freezing tolerance has been widely documented in many plant species, including Arabidopsis
1H NMR analysis of the Arabidopsis metabolome, during freezing, revealed that the application of LPC altered biochemical pathways resulting in the accumulation of specific metabolites, leading to enhanced freezing tolerance. The 1H NMR spectra of plants treated with LPC was dominated by peaks with chemical shifts (δ) around 0.8 to 4.0 ppm. These chemical shifts primarily represented 2 major groups of compounds; 0.8 – 2.8 ppm represents lipophilic components like fatty acids and sterols, while resonance at 3.0 to 4.0 ppm represented carbohydrates, sugars, sugar alcohols and organic acids
1H NMR peaks with chemical shifts (δ) around 0.8 to 2.8 ppm, represented the lipophilic components such as fatty acids and sterols, were another major group of metabolites which showed specific changes during freezing stress in LPC-treated plants. Fatty acids are major components of cellular membranes, suberin, and cutin waxes that provide structural barriers to the environment. At low temperature, plant membranes undergo a transition from a liquid crystalline state to a gel-like phase with reduced fluidity contributing to ion leakage and deactivation of membrane proteins
Metabolite profiling revealed an increase in unsaturated fatty acids in plants treated with LPC when exposed to freezing. The spectral peaks (or peak groups) at δ = 5.4-5.2 ppm (protons on double bond carbons), 2.8, 2.3, 2.1, 1.6, 1.4-1.2 ppm (protons on alkyl chain), and 1.0-0.8 ppm (terminal methyl group of alkyl chain) are indicative of unsaturation of fatty acid. We have previously reported that application of ANE or LPC resulted in less tissue damage and electrolyte leakage, as compared to controls, thereby improving the LT50 values
Chemical components in the extracts of
Further, we used a whole-genome approach to determine the ANE-mediated freezing tolerance in Arabidopsis plants. Application of LPC significantly altered gene expression for 1% of the Arabidopsis genes. In this study, we focused on gene expression related to three aspects: (1) response to stress and/or stress stimulus; (2) compatible osmolyte accumulation; (3) changes in membrane lipid profile in response to stress.
Around 40 annotated genes were found to be up-regulated in our focus category with four genes (At1g10760, At1g55920, At1g09780, At3g22840) found to be directly involved in low temperature stress tolerance in Arabidopsis
Genes activated in response to biotic stress and/or stimuli were also activated owing to the fact that both biotic and abiotic stress stimuli share a common signaling cascade to bring about physiological responses
Similarly, several studies have suggested that cold-induced sugar accumulation enhances the degree of plant freezing tolerance. In Arabidopsis, a large increase in the degree of freezing tolerance is positively correlated with soluble sugar content
Starch is the main carbohydrate store in plants. Regulation of starch metabolism, in particular in response to environmental cues, is of primary importance for carbon and energy flow in plants
Raffinose family oligosaccharides (RFOs) such as raffinose and stachyose are accumulated during the process of cold acclimation, when plants acquire increased frost tolerance
LPC treatment specifically induced a major pathway involved in galactolipid biosynthesis and lipid trafficking in thylakoid membranes controlled by the gene DGD1. Other changes include down-regulation of sterol/cholesterol biosynthesis, regulation of Acyl-CoA thioesterases and down regulation of long chain fatty acid synthesis (Figure
Membranes are the major injury sites during freezing stress
An early response to low temperature stimulus, by tolerant plant species like winter barley oats, is decreased in membrane fluidity
We found that lipophilic components of ANE (LPC) modulated specific metabolic pathways in
Plant material and
Seeds of
Preparation of
A commercial formulation of powdered, alkaline extract of
The lipophilic fraction (hereafter referred to as LPC) was prepared from a methanol extract of ANE by sequential fractionation with hexane, chloroform and ethyl acetate as described previously by Rayirath et al.
Phenotypic studies and sample collection
Petri dish freezing tolerance assay
Ten days after germination, Petri dishes were transferred to a temperature-controlled incubator, set to −2 ± 0.1°C. To achieve uniform freezing, the Petri dishes were equilibrated at −2°C for 24 h before further lowering the temperature. After equilibration, the temperature of the chamber was progressively lowered at the rate of 1°C per day until the desired sub-zero temperatures were attained. The temperature was monitored by wireless temperature sensor modules (Traceable® Remote sensor module, Fisher Scientific, Ottawa, ON, Canada) placed inside the Petri plates. At each temperature, five Petri plates per treatment were withdrawn from the chamber, thawed at 4°C for 12 h, in the dark, and returned to the original growth conditions (16:8 photoperiod, 22/18°C day/night temperatures). Survival of plants (by degree of chlorosis and shoot damage) was recorded visually, 48 h after returning to the original growth conditions.
In vivo freezing assay
The Arabidopsis ecotype Col-0 plants were grown in peat pellets (Jiffy-7®, Jiffy Products Ltd, NB, Canada) in a greenhouse set at 22/18°C day/night temperatures and 16:8 photoperiod with an irradiance of 100 μmol photons m-1 s-1. Three-week-old plants were used in the experiments. Extract treatments were administered 48 h prior to freezing treatment by irrigating with 1.0 g L-1 of ANE or LPC (1.0 g L-1 equivalent made up with sterile distilled water) at the rate of 20 ml per plant. The control plants were grown under identical conditions as the plants used in the extract treatment, except they received an equal volume of methanol (100 μl/g equivalent), made up with distilled water, but without the LPC. Forty eight hours after treatment, plants were transferred to a low temperature incubator set to 0°C. Freezing was initiated by spraying ice cold water, and the temperature of the chamber was progressively lowered at the rate of 1°C every 24 h until the desired sub-zero temperature was attained. Temperature was monitored through wireless temperature sensor modules placed at different points inside the freezing cabinet. At each treatment temperature, fifteen randomly selected plants from each treatment were withdrawn from the chamber, thawed at 4°C for 12 h in the dark, and then returned to the original growth conditions of the green house. Two days later, survival of plants was recorded visually according to the degree of chlorosis and leaf damage. Leaf samples, for biochemical assays, collected from plants undergoing the peat pellet freezing tolerance, were flash frozen in liquid nitrogen, ground to fine powder and stored at −80°C until further analysis.
Proline estimation
To study the role of proline in ANE-mediated freezing tolerance, the proline content was measured in acidic extracts and quantified spectrophotometrically using an acid-ninhydrin reagent with proline as the standard
Proline mutant studies
Petri dish freezing tolerance assays, and peat pellet freezing tolerance assays, were carried out following the methods previously described by Xin and Browse
Transcriptional analysis of proline biosynthesis and degradation genes
The expression of proline biosynthesis genes,
Total soluble sugars estimation and sugar mutant studies
Total soluble sugars were estimated by the colorimetric assay developed by Farrar
Plant growth conditions, extract treatments and freezing temperatures were as described in the peat pellet freezing assay, as described in Xin and Browse
Metabolite profiling
Global profiling
Global metabolite profiling of freeze-stressed Arabidopsis plants was carried out using a method described by Ward et al.
NMR spectra were acquired on a Bruker Avance III 600 MHz NMR spectrometer (Bruker Corporation, East Milton, ON), operating at 600.28 MHz 1H observation frequency, and a temperature of 25±0.2°C. Spectra were obtained with a gradient broadband inverse 5 mm probe (optimized for 1H), auto-tuned, matched and shimmed on each sample, at a flip angle of 30° pulse, followed by a 2.66 s acquisition time and 2 s relaxation delay. Each spectrum consisted of 32 scans of 64 k data points, with a spectral width of 12335.52 Hz. 1H NMR chemical shifts in the spectra were referenced to CD2H peak of methanol at δ 3.31 ppm, and that peak intensity was also used as a reference for comparison of the level of metabolites level between samples. The signals were acquired, processed and analyzed using TopSpin® NMR data acquisition and processing software (Bruker Biospin Ltd, East Milton, ON) integrated with the spectrometer. The experiment was conducted with three biological replicates and the experiment was repeated twice.
Targeted profiling
For targeted metabolite profiling, representative samples used in the NMR experiments above were dried and re-suspended with 20 mM phosphase buffer (pH 7.0), 1 mM NaN3 and 0.49 mM DSS (3-(trimethylsilyl)-1-propanesulfonic acid) in D2O. NMR spectra were acquired on the same Bruker Avance III 600 NMR spectrometer, as described above, using a 5 mm inverse gradient probe set to 25.0±0.1°C. 1D NOESY spectra with pre-saturation on water were acquired with 256 scans, an acquisition time of 2.73s, a sweep width of 20 ppm, a mixing time of 100 ms, and relaxation delay of 5s. All spectra were referenced using the internal DSS standard set to 0 ppm. NMR spectra were processed and binned for the region of d 0–10 ppm (water peak region d 4.7-4.83 ppm excluded) within 0.04 ppm chemical shift window by using the NMR software package NMR Suite 5.0 (Chenomx, Edmonton, Canada). The same software package was used to do targeted profiling. Multivariate data analysis (PCA and PLS-DA) was performed by SIMCA-P+ 12.01.0 software package (Umetrics, Umea, Sweden). The experiment had three biological replicates.
Microarray analysis
RNA extraction and cRNA preparation
Total RNA was isolated using an RNA
Hybridization and signal acquisition
Equal quantities of purified cRNA were hybridized to GeneChip® Arabidopsis ATH1 Genome Arrays (Affymetrix Inc. Santa Clara, CA). Subsequent steps included washing, blocking, and streptavadin-Cy3:Cy5 staining. Fluorescence emission was quantitatively detected usingthe iScan® System (Illumina Inc. Hayward, CA) with Illumina Beadstudio® software (Illumina Inc. Hayward, CA). Each treatment had three biological replicates.
Microarray data analysis
Statistical analysis
The raw data were analyzed using Flex Array statistical software (Genome Quebec, Montreal, QC) to obtain a measure of gene expression. Background correction, normalization, and summarization of probe-level gene chip data was performed using the RMA (
Functional classification of differentially expressed genes
The differentially expressed gene lists were distributed into functional categories using the web based tool, Classification Super-Viewer (
Furthermore, the input set was bootstrapped one hundred times by sampling the input set (with repeats) and then reclassifying each set generated. The standard deviation for the scores generated from the bootstrap sets was displayed along with the normalized class score. In this way classes represented by small numbers of genes on the chip were easily identified
Pathway analysis
The Pathway Tools Omics Viewer (formerly the Pathway Tools Expression Viewer) (
Heat map and Venn diagram
Heat mapping was perfromed using the web based tool, Heat-mapper
Validation of microarray experiment using RT-PCR
A 'Two-step RT-PCR' was used for quantifying transcript yield and comparing differential gene expression. Ten micrograms of total RNA were treated with DNase using the TURBO DNA-
Competing interests
The authors declare no competing interests.
Authors' contributions
BP, PR conceived the experiment. BP, PR, JZ, CK, MDH, BB planned the study. PR,JZ, XJ, CK conducted experiments. PR, JZ, CK, MDH, BB, ATC, DH, SK, BP analyzed, interpreted data and wrote the manuscript. All authors read and approved the final manuscript.
BP's lab is supported by the grants from Natural Sciences and Engineering Research Council of Canada (NSERC), Nova Scotia Department of Agriculture & Marketing (NSDAF) and Acadian Seaplants Limited. Authors are grateful to Dr. Laszlo Szabados (Institute of Plant Biology, Biological Research Center, Szeged, Hungary) for providing P5CS1 mutants and Dr. Matsuo Uemura & Dr. Glenn Thorlby (School of Biological Sciences, University of London, UK) for providing FR67 (
Responses to abiotic stresses
Protection of plants from frost using hydrophobic particle film and acrylic polymer
The cytological responses of tomato seedling cotyledons to chilling and the influence of membrane modifications upon these responses
Inhibition of chilling-induced photooxidative damage to leaves of Cucumis sativus L. by treatment with amino alcohols
Effects of Kelp (Macrocystis integrifolia and Eklonia maxima) foliar applications on bean crop growth
Agronomic uses of seaweed and microalgae
The effect of seaweed concentrate on the growth of tomatoes in nematode infested soil
Alleviation of salinity stress in Kentucky bluegrass by plant growth regulators and Iron
Frost Management in Cool Climate Vineyards
Effect of cold hardening, wilting and exogenously applied proline on leaf proline content and frost tolerance of several genotypes of Solanum
Evaluation of polyamine and proline levels during low temperature acclimation of citrus
Heritable improvement of frost tolerance in winter wheat by in vitro-selection of hydroxyproline-resistant proline overproducing mutants
Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant
Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana
Regulation of levels of proline as an osmolyte in plants under water stress
Effects of three stabilizing agents – proline, betaine, and trehalose on membrane phospholipids
Products of proline catabolism can induce osmotically regulated genes in rice
Proline accumulates in plants exposed to UV radiation and protects them against UV induced peroxidation
Metabolic implications of stress induced proline accumulation in plants
Glutamine synthetase in the phloem plays a major role in controlling proline production
Lipophilic components of the brown seaweed, Ascophyllum nodosum, enhance freezing tolerance in Arabidopsis thaliana
Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway
Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high salinity stresses using a full-length cDNA microarray
Down-Regulating α-Galactosidase. Enhances freezing tolerance in transgenic petunia
Molecular cloning and relationship to freezing tolerance of cold-acclimation-specific genes of alfalfa
Cold acclimation and freezing stress tolerance: role of protein metabolism
The cold shock response: a hot topic
Chilling sensitivity in plants and Cyanobacteria: the crucial contribution of membrane lipid
Plant cold acclimation: freezing tolerance genes and regulatory mechanisms
Proline biosynthesis and osmoregulation in plants
Reciprocal regulation of delta 1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants
Proline synthesis and degradation: a model system for elucidating stress-related signal transduction
Cold induced freezing tolerance in Arabidopsis
The leaf-order-dependent enhancement of freezing tolerance in cold-acclimated Arabidopsisrosettes is not correlated with the transcript levels of the cold-inducible transcription factors of CBF/DREB1
Altering flux through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana modifies photosynthetic acclimation at low temperatures and the development of freezing tolerance
Cold responses of Arabidopsis mutants impaired in freezing tolerance
Freezing sensitivity in the sfr4 mutant of Arabidopsis is due to low sugar content and is manifested by loss of osmotic responsiveness
Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves
Impact of soluble sugar concentrations on the acquisition of freezing tolerance in accessions of Arabidopsis thaliana with contrasting cold adaptation: Evidence for a role of raffinose in cold acclimation
Changes in leaf ultrastructure and carbohydrates in Arabidopsis thaliana L. (Heynh) cv. Columbia during rapid cold acclimation
Assessment of 1H NMR spectroscopy and multivariate analysis as a technique for metabolite fingerprinting of Arabidopsis thaliana
Acclimative response to temperature stress in higher plants: approaches of gene engineering for temperature tolerance
Stressful "memories" of plants: Evidence and possible mechanisms
Enhancing arabidopsis salt and drought stress tolerance by chemical priming for its abscisic acid responses
Starch-related α-glucan/water dikinase is involved in the cold-induced development of freezing tolerance in Arabidopsis
The cold-induced early activation of phospholipase C and D pathways determines the response of two distinct clusters of genes in Arabidopsis cell suspensions
Proteome analysis of cold stress response in Arabidopsis thaliana using DIGE-technology
A cyclophilin links redox and light signals to cysteine biosynthesis and stress responses in chloroplasts
The sigma factor RpoS is required for stress tolerance and environmental fitness of Pseudomonas fluorescens Pf-5
Role of the plasma membrane in freezing injury and cold acclimation
Molecular responses of plants to cold shock and cold acclimation
Induction of abiotic stress tolerance by salicylic acid signalling
Role of salicylic acid in plants
Nitric oxide and salicylic acid signaling in plant defense
The role of salicylic acid and jasmonic acid in pathogen defence
Effects of salicylic acid and cold on freezing tolerance in winter wheat leaves
Effects of salicylic acid and cold treatments on protein levels and on the activities of antioxidant enzymes in the apoplast of winter wheat leaves
Jasmonate and salicylate induce the expression of pathogenesis-related-protein genes and increase resistance to chilling injury in tomato fruit
Salicylate accumulation inhibits growth at chilling temperature in Arabidopsis
A plastid-localized glycogen synthase kinase 3 modulates stress tolerance and carbohydrate metabolism
β-Amylase induction and the protective role of maltose during temperature shock
A redox-regulated chloroplast protein phosphatase binds to starch diurnally and functions in its accumulation
Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation
Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana
The role of raffinose in the cold acclimation response of Arabidopsis thaliana
Differential effects of galactose containing saccharides on mechanical freeze-thaw damage to isolated thylakoid membranes
Galactinol synthase activity and soluble sugars in developing seeds of four soybean genotypes
Cold-acclimation of Arabidopsis thaliana: effect of plasma membrane composition and freeze induced lesions
Biochemical and biophysical properties of thylakoid acyl lipids
Cold acclimation of the Arabidopsis dgd1 mutant results in recovery from photosystem I-limited photosynthesis
Profiling membrane lipids in plant stress responses: role of phospholipase D{alpha} in freezing-induced lipid changes in Arabidopsis
Differential degradation of extraplastidic and plastidic lipids during freezing and post-freezing recovery in Arabidopsis thaliana
The role of lipids in plastid protein transport
Galactolipids rule in seed plants
Arabidopsis galactolipid biosynthesis and lipid trafficking mediated by DGD1
Physical properties of membranes of cold hardened and non hardened winter rape plants
A revised medium for rapid growth and bio-assays with tobacco tissue cultures
Extracts of brown seaweed, Ascophyllum nodosum, induce gibberellic acid (GA3) independent amylase activity in barley
Ammonia assimilation, photorespiration and amino acid biosynthesis
Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis
Carbon Partitioning
Isolation of mutants affecting the development of freezing tolerance in Arabidopsis thaliana (L.) Heynh
The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth
A Browser-based Functional Classification SuperViewer for Arabidopsis Genomics
A random variance model for detection of differential gene expression in small microarray experiments
AraCyc: a biochemical pathway database for Arabidopsis

subset: commoncrawl


text: Bundesliga Record-breaking Pulisic on song again for USMNT vor 2 Stunden Two goals in 45 minutes saw Christian Pulisic set two more USMNT records, against Saint Vincent and the Grenadines. Summary Pulisic scored twice for the US in World Cup qualifying.Youngest US international to score in qualifier, and to score a brace.17-year-old already the holder of many records for club and country. Borussia Dortmund's attacking midfielder Christian Pulisic is racking up the records for the USMNT as he helped them to a comfortable 6-0 win over Saint Vincent and the Grenadines in a 2018 FIFA World Cup qualifier. Click here for an overview of all international matches involving Bundesliga players.The 17-year-old, who already made history as the youngest USMNT goalscorer when he struck in a friendly against Bolivia last year, became not only the youngest player to score for his country in an official World Cup qualifier, he then added a second for good measure to be the youngest scorer of a brace.Watch: Pulisic's indelible mark on the Bundesliga: He entered the game at half-time with his team already 3-0 ahead, replacing Borussia Mönchengladbach's versatile star Fabian Johnson. His goal made it 4-0 before he set up the fifth and, in stoppage time, put the cherry on top of another show-stopping performance.Got Pulisic in your Official Fantasy Bundesliga team? Click here to pick him now!Pulisic became the youngest player in the Bundesliga to score two goals in a season last term and he is looking forward to skittling even more records for club and country in years to come. "I'm delighted that, for example, I can train with the likes of Mario Götze and Andre Schürrle - I learn a great deal from them since they've already experienced a lot in their careers," Pulisic told the German newspaper,Ruhrnachrichten.Watch: Pulisic's keepy-uppy challenge during BVB's media days: "I'm convinced we're going to have a really, really strong team and I'm looking forward to fighting for my place on it. The most important thing for me is that we're successful and if I can play as many minutes as possible at the same time, that would be perfect."And the more minutes Pulisic gets to play, the more records he can break for club and country. More news Bundesliga Moment of Matchday 8 Bundesliga Moment of Matchday 8 Bundesliga Plentiful praise for Pulisic Free! Get the Bundesliga newsletter delivered straight to your inbox. Sign up today! Subscribe


text:
Join Anne-Marie Concepción for an in-depth discussion in this video 4. Creating and applying masters intelligently, part of InDesign CS4: 10 Habits of Highly Effective Pros.
Highly effective InDesign pros are essentially lazy. It's true! They are savvy…users who are always on the lookout for opportunities to make InDesign do the…work instead of them. A prime example of this is master pages.…Now why should I tediously add page numbers to every single page in this…document when I could just go to the master page and add a page number there?…So I am just going to create a text frame, go to Type > Insert Special…Character > Marker > Current Page Number. I will select it and make it really…large with Ctrl+Shift or Command+Shift+> and put that centered and then I am…just going to Alt+Drag or Option+Drag it to the right. And then when I go to…my document pages, every single page is automatically numbered. That is a typical…use for a master page and that's just a beginning of master pages.…
Now, I am always surprise to encounter people who actually never touch…the master page and they just create everything from scratch on the document pages.…But to become a true superuser of InDesign, you have got to use master pages. …
- Exploring styles to help with design
- Building custom defaults
- Working with auto bullets and syncing to save time
- Using keystrokes and Quick Apply
- Creating and applying Masters intelligently
- Mastering autoflow in InDesign
- Managing links effectively


text:
Susan Lacovara
Green The Grasses Seem - Poem by Susan Lacovara
If you like the lace and lateness,
Then prepare your bed in lust
If you crave a winding wooded trail,
Then shade your eyes from dust
If you light the match, to brighten,
Then brave the growing flame
If you dare to ride the current
Then tell me... who's to blame....
If her lips reveal some flavor
Which newly tempts your taste
And the secrets of the season
Are secured, and safely placed
Return not, to this fortress
That hides and heals your scars
For no one gains free entrance,
If not guided by the stars.
Poet's Notes about The Poem
Comments about Green The Grasses Seem by Susan Lacovara
Loke Kok Yee (9/24/2015 4:21:00 AM)
Now my appetite is whetted,
I'll read more of your poems-10 (Report) Reply
Anthony Burkett (2/3/2014 9:42:00 PM)
These lines have a graceful gliding pace that is easy to walk beside... delighting the senses like a stroll down a country lane... as surprising as a stolen kiss... delightful! (Report) Reply
Ramesh Rai (11/10/2013 3:30:00 AM)
every question followed by a suitable answer. if you write such beautiful poem be prepared to read the comment. thnx for sharing.10/10 (Report) Reply
Marcus Mckinley (9/5/2013 10:54:00 AM)
it seems that love has ventured for greener pastures only too dream of home. but, as fate would have it any trails leading back have already been overgrown. nicely written and full of imagery. (Report) Reply
Khairul Ahsan (9/5/2013 9:15:00 AM)
A wonderful poem! Straight talk.
For no one gains free entrance,
If not guided by the stars.
Stars? ? ? (Report) Reply
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Susan Lacovara's Other Poems
Famous Poems
Phenomenal Woman
Maya Angelou
Still I Rise
Maya Angelou
The Road Not Taken
Robert Frost
If You Forget Me
Pablo Neruda
Langston Hughes
Annabel Lee
Edgar Allan Poe
Caged Bird
Maya Angelou
Rudyard Kipling
Stopping By Woods On A Snowy Evening
Robert Frost
A Dream Within A Dream
Edgar Allan Poe


text: Show Hide image Politics 28 April 2010 Italian charity may sue Sunday Times for libel Medical charity Emergency likely to sue The Sunday Times for libel over a report claiming three of i Sign up for our weekly email * The report, published on 19 April, claimed the charity workers - Matteo Dell'Aira, Marco Garatti and Matteo Pagani - had confessed their involvement in a plot to kill Helmand governor Gulab Mangal. But the organisation's vice president Alessandro Bertani told Adnkronos International (AKI) that the report was baseless. Italian newspapers II Giornale and Libero had followed the British report. Bertani said Emergency will sue the Italian newspapers for continuing with the slandering report despite the organisation denying The Sunday Times story. He also did not rule out suing the Times. However, he indicated that a word of apology from the UK paper would stop them from taking legal action. Subscribe from just £1 per issue More


text:
Coach Mike Shanahan said Mobley would be re-examined in a month or so, but doctors were not sure anything would change.
Mobley has been out since bruising his spinal cord Oct. 26, when he collided with teammate Kelly Herndon and Baltimore running back Jamal Lewis.
When Mobley was examined by Dr. Robert Watkins of Los Angeles last week, an X-ray showed a bruise remains. Watkins recommended that Mobley sit out.
"Right now there is a question mark if he will play a year from now or what direction he will go," Shanahan said Wednesday. "But no decision has to be made immediately, so we will just have to wait and see."
Mobley will remain on the Broncos roster, Shanahan said, but two others are leaving.
Shanahan said defensive lineman Marco Coleman, 34, plans to retire, and the Broncos have cut guard Calvin Collins, who signed with the team Dec. 31 after being out of football since 2001.


text:
How Do I Know If My Injury Constitutes Medical Malpractice?
Medical professionals are not expected to be infallible and there may be poor results from treatment that do not amount to malpractice. Medical professionals are expected to exercise the basic knowledge, skills, and care ordinarily possessed and exercised by other members of the profession acting under similar conditions and circumstances. This basic knowledge and skill is called a standard of practice, or standard of care. When a medical professional treats a patient and fails to use this basic knowledge, skill, and care whether the failure consists of doing something incorrectly, or failing to do something that should be done, that failure is a deviation from the standard of practice or care.
The injured party has the burden of proving that the health care provider negligently failed to meet the applicable professional standard of care for that health care provider. That means the health care provider failed to act as a reasonably prudent health care provider in New Mexico would under similar circumstances. Health care providers are judged in comparison with similarly trained and qualified providers of the same class in the same community and in the same field of medical specialization. Likewise, a hospital owes their patients a duty of ordinary care to provide equipment and facilities reasonably suited to the intended uses and such as are in general use in similar hospitals under similar circumstances. The injured patient must then prove that the health care provider`s breach of the standard of care was a proximate cause of the injuries to the patient. In other words, there must be a close connection between the action of the health care provider and the harm to the patient. Finally, you have to show what damages resulted form the alleged mistake. This may include medical bills, lost wages, pain and suffering, etc.
No medical malpractice action may be filed against a qualifying health care provider (one who participate in the state sponsored excess insurance plan) before application is made to the medical review commission and a decision is rendered. The panel will decide whether there is substantial evidence that the acts complained of occurred and that they constitute malpractice and whether there is a reasonable medical probability that the patient was injured thereby. The panel`s findings are neither binding nor admissible in any subsequent court proceedings.
Expert testimony is generally required in a medical malpractice action unless the alleged negligence is so apparent as to be within a layman`s comprehension. Medical malpractice claims are very difficult to prevail in and are also extremely expensive partly due to the need for qualified expert review and testimony. Whether or not you have a good case depends on the professional judgment of attorneys and medical experts. If you are told that you do not have a case with merit, you should seek another opinion from one or more other attorneys.
Speak to an Experienced Medical Malpractice Attorney Today
This article is intended to be helpful and informative. But even common legal matters can become complex and stressful. A qualified medical malpractice lawyer can address your particular legal needs, explain the law, and represent you in court. Take the first step now and contact a local medical malpractice attorney to discuss your specific legal situation.
Additional Medical Malpractice Articles
- What Happens If I Am Injured In The Course Of Medical Treatment?
- Is There A Time Limit In Which I Need To File A Lawsuit For Medical Malpractice?
- Who Can Be Held Accountable For The Medical Malpractice?
- Does Signing A Consent Form Waive My Rights To File A Lawsuit For Medical Malpractice?
- How Much Can I Expect An Attorney To Charge To Handle A Medical Malpractice Case?
- How Can I Determine How Much My Claim Is Worth?


text:
We both knew that we wanted to be together forever. We wanted to get married as soon as possible! I wanted the ring on my finger as soon as possible too! However, we still needed to be curteous to my parents, (that's what he said!) Even though we had already picked out the ring, he said that he wouldn't buy it till later, and that he wanted to travel to Arkansas to meet my parents and ask for my hand in marriage, the right way.
Boy, was I sad!
One day, in between two of my classes at Brigham Young University Idaho,
he said that he wanted to go somewhere on Campus and talk to me, like
we always do. So we walk around, looking for a place to go. We find a
small rock inside the Campus Ricks Gardens with a sweet smelling rose
bush nearby. We sit down together on the rock and start talking about
the future. Then his questions got really specific. Questions such as "
So is there a beautiful place in Arkansas where I can propose to you." I
peered off into the distance, broken-hearted.
"Yes," I replied.
"Have you thought about what you are going to say when I ask you?" He inquired.
" Of course, I have thought of it." I pondered for a moment. I had to ask the question:
"Have you thought about what you are going to say, how you are
going to ask?" I stared at him, waiting for an answer. My hope
was fleeting and time was drawing close to walk back across the campus
to another class. He replied comfortably:
"No." That was it for me! I couldn't stand it any longer. And I think
he noticed that that was enough teasing about proposal for the day
because I said:
"I have to get back to class, will you walk with me?"
"Sure, but I have one more question." .... My heart stopped. He made
sure I was still sitting down on the rock. He stood up, then knelt down
in front of me. He pulled out a small item from his pocket. At this
point it didn't register that that was the ring I had picked out just a
week before. He then said, in a firm, confident voice... holding the
item infront of me with one hand:
"Madeline, will you marry me?" I didn't even answer before I blurted out:
" You're joking! HOW?! WHAT?! I can't believe....! YOU! GOOBER!?!"
I jumped on him as he was trying to stand back up. These mix of words
were the only crops of words that could be uttered from my mouth. My
brain was reeling, trying to process the information. I felt like a
backed up computer that was trying to download too many things at once.
Then it hit me. Look at the ring you goober! At this point
the ring was already on my finger, I gasped for air, for something to
say. It glistened in the bright summer sun. Yes, just as I had picked
out. I looked up at him, I could tell he was growing impatient:
"Will you say yes?" It NEVER hit me that I hadn't said yes yet. But yet that was the very word I was searching for!
"Yes! Yes, yes yes! Of course!" I'm sure he was embarrased because there were
other people in the gardens, watching, giggling at the site of a poor
fellow getting trampled by a loud, excited young woman. But she was a
young woman in love!
After things quited down (namely, me), we traveled back towards my
class, hand in hand. He said goodbye to me at the doorway inside the
Snow Fine Arts Center for my Music History class.
We then parted for one of the very last times.
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text:
Downloadsquad Pack
by filmgirl
About this Collection
Soup up your browser with selections from the Downloadsquad.com staff and some of our readers' favorite add-ons. They'll help make your browsing safer, more hassle free, and more productive!
19 Add-ons in this Collection
Adblock Plus by Wladimir Palant
14,240,226 users
Blocks annoying video ads on YouTube, Facebook ads, banners and much more.
Adblock Plus blocks all annoying ads, and supports websites by not blocking unobtrusive ads by default (configurable).
DownThemAll! by Nils Maier, Federico Parodi, others
959,759 users
The first and only download manager/accelerator built inside Firefox!
LastPass Password Manager by LastPass
638,763 users
LastPass, an award-winning password manager, saves your passwords and gives you secure access from every computer and mobile device.
NoScript Security Suite by Giorgio Maone
1,740,869 users
The best security you can get in a web browser!
Allow active content to run only from sites you trust, and protect yourself against XSS and Clickjacking attacks.
Greasemonkey by Anthony Lieuallen, Aaron Boodman, others
897,969 users
Customize the way a web page displays or behaves, by using small bits of JavaScript.
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585,599 users
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238,339 users
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text:
Posted by Jobgurus Services - VERIFIED EMPLOYER VERIFIED26 Jan 3:13 pmIkeja, Lagos
Guaranty Multiservices and Property Konsult Limited - We are a leading source of real estate investment in Nigeria, a market place for refreshingly genuine property deals and a property investment and development firm with core competence in providing authentic land and landed property deals for investment purposes and quality and luxury homes, offices, stores and shops and within a very affordable budget for residential, business and other purposes. We develop housing, business and other real estate projects to meet the seemingly insatiable real estate demand in Nigeria. Our team is constituted by experienced and veteran property consultants with well-honed skills.
Job Description/Requirements
Minimum of O'Level (WAEC, GCE, NECO) with 5 credit passes and a credit pass in English.
Competence in spoken and written English.
Within 18-25 years.
Ability to clean well with little or no supervision.
Please visit our website to apply for the position Cleaner @Guaranty Multiservices:
Stay safe!
Watch out for:


text:
$ 29.00
Juice Beauty Blemish Clearing Serum
Reduce breakouts, unclog pores, and even skin tone with this powerful serum packed with organic fruit acids and willow bark that contains natural salicylic acid; while CoQ10, Vitamin C and organic aloe renew the skin for a healthy, clear complexion. Clears While Hydrates.
Who Should Use It?
Juice Beauty's Blemish Clearing Serum is designed for oily, combination, and blemish prone-skin types.
• Reduces breakouts & clears clogged pores with beta hydroxy salicylic acid and naturally derived willow bark
• Evens skin tone and texture with organic lemon & apple fruit acids
• Helps renew the skin with Vitamin C
• Fights free radicals with antioxidant Vitamins B5 & E
• Creates a smoother looking appearance with CoQ10
• Hydrates & soothes with organic aloe and green & white tea
organic juices of vitis vinifera (organic white grape juice)*, pyrus malus (organic apple juice)*, aloe barbadensis (organic aloe juice)*, citrus medica limonum (organic lemon juice)*, vegetable glycerin, sclerotium gum, algin, salix alba (willow bark extract), allantoin, decyl glucoside, panthenol (vitamin b5), magnesium ascorbyl phosphate (vitamin c), tocopherol (vitamin e), salvia officinalis (sage leaf oil), ubiquinone (coq10), saccharum officinarum (sugar cane juice), organic botanical extracts of camellia sinensis (organic white tea leaf extract)*, camellia oleifera (organic green tea extract)*, lavandula angustifolia (organic lavender extract)*, chamomilla recutita(organic chamomile extract)*, melissa officinalis (lemon balm leaf extract), algae extract, sodium benzoate, potassium sorbate, citric acid, lavandula angustifolia (lavender)* and boswellia serrata (frankincense)* pure essential oils.
* = certified organic ingredient
After cleansing with Juice Beauty's Blemish Clearing Cleanser, apply this treatment serum to entire face or just to blemished areas. Finish with Juice Beauty's Oil-Free Moisturizer.
Use morning and night.
Sensitivity Test for a New User: Apply product sparingly to one or two small affected areas during the first 3 days. If no discomfort occurs, follow the directions stated above.