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A new picture of cell wall protein dynamics in elongating cells of Arabidopsis thaliana: Confirmed actors and newcomers
Irshad, Muhammad
Canut, Hervé
Borderies, Gisèle
Pont-Lezica, Rafael
Jamet, Elisabeth
BMC Plant Biology
Cell elongation in plants requires addition and re-arrangements of cell wall components. Even if some protein families have been shown to play roles in these events, a global picture of proteins present in cell walls of elongating cells is still missing. A proteomic study was performed on etiolated hypocotyls of Arabidopsis used as model of cells undergoing elongation followed by growth arrest within a short time.
Two developmental stages (active growth and after growth arrest) were compared. A new strategy consisting of high performance cation exchange chromatography and mono-dimensional electrophoresis was established for separation of cell wall proteins. This work allowed identification of 137 predicted secreted proteins, among which 51 had not been identified previously. Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function.
This work highlights the CWP dynamics that takes place between the two developmental stages. The presence of proteins known to be related to cell wall extension after growth arrest showed that these proteins may play other roles in cell walls. Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins.
Related Articles
Microarray Analysis of Developing Flax Hypocotyls Identifies Novel Transcripts Correlated with Specific Stages of Phloem Fibre Differentiation
Roach, Melissa J.
Deyholos, Michael K.
Annals of Botany
Background and Aims
Hypocotyls are a commonly used model to study primary growth in plants, since post-germinative hypocotyls increase in size by cell elongation rather than cell division. Flax hypocotyls produce phloem fibres in bundles one to two cell layers thick, parallel to the protoxylem poles of the stele. Cell wall deposition within these cells occurs rapidly at a well-defined stage of development. The aim was to identify transcripts associated with distinct stages of hypocotyl and phloem fibre development.
Stages of flax hypocotyl development were defined by analysing hypocotyl length in relation to fibre secondary wall deposition. Selected stages of development were used in microarray analyses to identify transcripts involved in the transition from elongation to secondary cell wall deposition in fibres. Expression of specific genes was confirmed by qRT-PCR and by enzymatic assays.
Key Results
Genes enriched in the elongation phase included transcripts related to cell-wall modification or primary-wall deposition. Transcripts specifically enriched at the transition between elongation and secondary wall deposition included β-galactosidase and arabinogalactan proteins. Later stages of wall development showed an increase in secondary metabolism-related transcripts, chitinases and glycosyl hydrolases including KORRIGAN. Microarray analysis also identified groups of transcription factors enriched at one or more stages of fibre development. Subsequent analysis of a differentially expressed β-galactosidase confirmed that the post-elongation increase in β-galactosidase enzyme activity was localized to phloem fibres.
Transcripts were identified associated with specific stages of hypocotyl development, in which phloem fibre cells were undergoing thickening of secondary walls. Temporal and spatial regulation of β-galactosidase activity suggests a role for this enzyme in remodelling of flax bast fibre cell walls during secondary cell wall deposition.
Bast; fibre; flax; hypocotyl; Linum usitatissimum; phloem; microarray; galactosidase
Related Articles
Phytosulfokine-α Controls Hypocotyl Length and Cell Expansion in Arabidopsis thaliana through Phytosulfokine Receptor 1
Stührwohldt, Nils
Dahlke, Renate I.
Steffens, Bianka
Johnson, Amanda
Sauter, Margret
Bendahmane, Mohammed
PLoS ONE
The disulfated peptide growth factor phytosulfokine-α (PSK-α) is perceived by LRR receptor kinases. In this study, a role for PSK signaling through PSK receptor PSKR1 in Arabidopsis thaliana hypocotyl cell elongation is established. Hypocotyls of etiolated pskr1-2 and pskr1-3 seedlings, but not of pskr2-1 seedlings were shorter than wt due to reduced cell elongation. Treatment with PSK-α did not promote hypocotyl growth indicating that PSK levels were saturating. Tyrosylprotein sulfotransferase (TPST) is responsible for sulfation and hence activation of the PSK precursor. The tpst-1 mutant displayed shorter hypocotyls with shorter cells than wt. Treatment of tpst-1 seedlings with PSK-α partially restored elongation growth in a dose-dependent manner. Hypocotyl elongation was significantly enhanced in tpst-1 seedlings at nanomolar PSK-α concentrations. Cell expansion was studied in hypocotyl protoplasts. WT and pskr2-1 protoplasts expanded in the presence of PSK-α in a dose-dependent manner. By contrast, pskr1-2 and pskr1-3 protoplasts were unresponsive to PSK-α. Protoplast swelling in response to PSK-α was unaffected by ortho-vanadate, which inhibits the plasma membrane H+-ATPase. In maize (Zea mays L.), coleoptile protoplast expansion was similarly induced by PSK-α in a dose-dependent manner and was dependent on the presence of K+ in the media. In conclusion, PSK-α signaling of hypocotyl elongation and protoplast expansion occurs through PSKR1 and likely involves K+ uptake, but does not require extracellular acidification by the plasma membrane H+-ATPase.
Related Articles
Hypocotyl Transcriptome Reveals Auxin Regulation of Growth-Promoting Genes through GA-Dependent and -Independent Pathways
Chapman, Elisabeth J.
Greenham, Kathleen
Castillejo, Cristina
Sartor, Ryan
Bialy, Agniezska
Sun, Tai-ping
Estelle, Mark
Blazquez, Miguel A.
PLoS ONE
Many processes critical to plant growth and development are regulated by the hormone auxin. Auxin responses are initiated through activation of a transcriptional response mediated by the TIR1/AFB family of F-box protein auxin receptors as well as the AUX/IAA and ARF families of transcriptional regulators. However, there is little information on how auxin regulates a specific cellular response. To begin to address this question, we have focused on auxin regulation of cell expansion in the Arabidopsis hypocotyl. We show that auxin-mediated hypocotyl elongation is dependent upon the TIR1/AFB family of auxin receptors and degradation of AUX/IAA repressors. We also use microarray studies of elongating hypocotyls to show that a number of growth-associated processes are activated by auxin including gibberellin biosynthesis, cell wall reorganization and biogenesis, and others. Our studies indicate that GA biosynthesis is required for normal response to auxin in the hypocotyl but that the overall transcriptional auxin output consists of PIF-dependent and -independent genes. We propose that auxin acts independently from and interdependently with PIF and GA pathways to regulate expression of growth-associated genes in cell expansion.
Related Articles
Ectopic Expression of WUS in Hypocotyl Promotes Cell Division via GRP23 in Arabidopsis
Zhang, Dajian
Wang, Xiaomin
Wang, Min
Li, Junhua
Guo, Xiaoyu
Chong, Kang
Xu, Yunyuan
Candela, Hector
PLoS ONE
WUSCHEL (WUS) is essential for preventing stem cell differentiation in Arabidopsis. Here we report that in addition to its functions in meristematic stem cell maintenance, WUS is involved in the regulation of cell division. The WUS gain-of-function mutant, stem ectopic flowers (sef), displayed elongated hypocotyls, whereas the loss-of-function wus-1 mutant had shortened hypocotyls. The long hypocotyl in sef was due to the presence of more cells, rather than increased cell elongation. Microscopic observation, flow cytometry assays, quantitative RT-PCR (qRT-PCR), and histochemical staining of CycB1;1::GUS supported the hypothesis that ectopic cell division occurred in the sef hypocotyls after germination. Both immunoblot and qRT-PCR results showed that WUS was ectopically expressed in sef hypocotyls. Luciferase activity, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) showed that GLUTAMINE-RICH PROTEIN 23 (GRP23) expression can be activated by WUS and that GRP23 is a direct target gene of WUS. The phenotypes of 35S::GRP23 plants and GRP23 knockdown lines supported the notion that GRP23 mediates the effects of WUS on hypocotyl length. Together, our data suggest that ectopic expression of WUS in hypocotyl controls cell division through its target gene GRP23.
Related Articles
A novel Arabidopsis MYB-like transcription factor, MYBH, regulates hypocotyl elongation by enhancing auxin accumulation
Kwon, Yerim
Kim, Jun Hyeok
Nguyen, Hoai Nguyen
Jikumaru, Yusuke
Kamiya, Yuji
Hong, Suk-Whan
Lee, Hojoung
Journal of Experimental Botany
Critical responses to developmental or environmental stimuli are mediated by different transcription factors, including members of the ERF, bZIP, MYB, MYC, and WRKY families. Of these, MYB genes play roles in many developmental processes. The overexpression of one MYB gene, MYBH, significantly increased hypocotyl elongation in Arabidopsis thaliana plants grown in the light, and the expression of this gene increased markedly in the dark. The MYBH protein contains a conserved motif, R/KLFGV, which was implicated in transcriptional repression. Interestingly, the gibberellin biosynthesis inhibitor paclobutrazol blocked the increase in hypocotyl elongation in seedlings that overexpressed MYBH. Moreover, the function of MYBH was dependent on phytochrome-interacting factor (PIF) proteins. Taken together, these results suggest that hypocotyl elongation is regulated by a delicate and efficient mechanism in which MYBH expression is triggered by challenging environmental conditions such as darkness, leading to an increase in PIF accumulation and subsequent enhanced auxin biosynthesis. These results indicate that MYBH is one of the molecular components that regulate hypocotyl elongation in response to darkness.
Arabidopsis; auxin; hypocotyl elongation; MYBH; PIF; photomorphogenesis.
Related Articles
Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes
Minic, Zoran
Jamet, Elisabeth
San-Clemente, Hélène
Pelletier, Sandra
Renou, Jean-Pierre
Rihouey, Christophe
Okinyo, Denis PO
Proux, Caroline
Lerouge, Patrice
Jouanin, Lise
BMC Plant Biology
Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins.
Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics.
Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins.
Related Articles
Identification of genes involved in cell wall biogenesis in grasses by differential gene expression profiling of elongating and non-elongating maize internodes
Bosch, Maurice
Mayer, Claus-Dieter
Cookson, Alan
Donnison, Iain S.
Journal of Experimental Botany
Despite the economic importance of grasses as food, feed, and energy crops, little is known about the genes that control their cell wall synthesis, assembly, and remodelling. Here a detailed transcriptome analysis that allowed the identification of genes involved in grass cell wall biogenesis is provided. Differential gene expression profiling, using maize oligonucleotide arrays, was used to identify genes differentially expressed between an elongating internode, containing cells exhibiting primary cell wall synthesis, and an internode that had just ceased elongation and in which many cells were depositing secondary cell wall material. This is one of only a few studies specifically aimed at the identification of cell wall-related genes in grasses. Analysis identified new candidate genes for a role in primary and secondary cell wall biogenesis in grasses. The results suggest that many proteins involved in cell wall processes during normal development are also recruited during defence-related cell wall remodelling events. This work provides a platform for studies in which candidate genes will be functionally tested for involvement in cell wall-related processes, increasing our knowledge of cell wall biogenesis and its regulation in grasses. Since several grasses are currently being developed as lignocellulosic feedstocks for biofuel production, this improved understanding of grass cell wall biogenesis is timely, as it will facilitate the manipulation of traits favourable for sustainable food and biofuel production.
Biofuel; cell wall; defence; grasses; lignocellulose; microarray; Zea mays
Related Articles
Restricted cell elongation in Arabidopsis hypocotyls is associated with a reduced average pectin esterification level
Derbyshire, Paul
McCann, Maureen C
Roberts, Keith
BMC Plant Biology
Cell elongation is mainly limited by the extensibility of the cell wall. Dicotyledonous primary (growing) cell walls contain cellulose, xyloglucan, pectin and proteins, but little is known about how each polymer class contributes to the cell wall mechanical properties that control extensibility.
We present evidence that the degree of pectin methyl-esterification (DE%) limits cell growth, and that a minimum level of about 60% DE is required for normal cell elongation in Arabidopsis hypocotyls. When the average DE% falls below this level, as in two gibberellic acid (GA) mutants ga1-3 and gai, and plants expressing pectin methyl-esterase (PME1) from Aspergillus aculeatus, then hypocotyl elongation is reduced.
Low average levels of pectin DE% are associated with reduced cell elongation, implicating PMEs, the enzymes that regulate DE%, in the cell elongation process and in responses to GA. At high average DE% other components of the cell wall limit GA-induced growth.
Related Articles
Identification of genes involved in the ACC-mediated control of root cell elongation in Arabidopsis thaliana
Markakis, Marios Nektarios
De Cnodder, Tinne
Lewandowski, Michal
Simon, Damien
Boron, Agnieszka
Balcerowicz, Daria
Doubbo, Thanaa
Taconnat, Ludivine
Renou, Jean-Pierre
Höfte, Herman
Verbelen, Jean-Pierre
Vissenberg, Kris
BMC Plant Biology
Along the root axis of Arabidopsis thaliana, cells pass through different developmental stages. In the apical meristem repeated cycles of division increase the numbers of cells. Upon leaving the meristem, these cells pass the transition zone where they are physiologically and mechanically prepared to undergo subsequent rapid elongation. During the process of elongation epidermal cells increase their length by 300% in a couple of hours. When elongation ceases, the cells acquire their final size, shape and functions (in the differentiation zone). Ethylene administered as its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is capable of inhibiting elongation in a concentration-dependent way. Using a microarray analysis, genes and/or processes involved in this elongation arrest are identified.
Using a CATMA-microarray analysis performed on control and 3h ACC-treated roots, 240 differentially expressed genes were identified. Quantitative Real-Time RT-PCR analysis of the 10 most up and down regulated genes combined with literature search confirmed the accurateness of the analysis. This revealed that inhibition of cell elongation is, at least partly, caused by restricting the events that under normal growth conditions initiate elongation and by increasing the processes that normally stop cellular elongation at the end of the elongation/onset of differentiation zone.
ACC interferes with cell elongation in the Arabidopsis thaliana roots by inhibiting cells from entering the elongation process and by immediately stimulating the formation of cross-links in cell wall components, diminishing the remaining elongation capacity. From the analysis of the differentially expressed genes, it becomes clear that many genes identified in this response, are also involved in several other kind of stress responses. This suggests that many responses originate from individual elicitors, but that somewhere in the downstream signaling cascade, these are converged to a 'common pathway'. Furthermore, several potential keyplayers, such as transcription factors and auxin-responsive genes, were identified by the microarray analysis. They await further analysis to reveal their exact role in the control of cell elongation.
ACC; Arabidopsis thaliana; Development; Elongation control; Ethylene; Microarray analysis; Root growth
Related Articles
Characterization of a Small Auxin-Up RNA (SAUR)-Like Gene Involved in Arabidopsis thaliana Development
Markakis, Marios Nektarios
Boron, Agnieszka Karolina
Van Loock, Bram
Saini, Kumud
Cirera, Susanna
Verbelen, Jean-Pierre
Vissenberg, Kris
Muday, Gloria
PLoS ONE
The root of Arabidopsis thaliana is used as a model system to unravel the molecular nature of cell elongation and its arrest. From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated. As it appeared as the 76th gene in the family, it was named SAUR76. Root and leaf growth of overexpression lines ectopically expressing SAUR76 indicated the possible involvement of the gene in the division process. Using promoter::GUS and GFP lines strong expression was seen in endodermal and pericycle cells at the end of the elongation zone and during several stages of lateral root primordia development. ACC and IAA/NAA were able to induce a strong up regulation of the gene and changed the expression towards cortical and even epidermal cells at the beginning of the elongation zone. Confirmation of this up regulation of expression was delivered using qPCR, which also indicated that the expression quickly returned to normal levels when the inducing IAA-stimulus was removed, a behaviour also seen in other SAUR genes. Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane. SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.
Related Articles
Circadian clock and PIF4-mediated external coincidence mechanism coordinately integrates both of the cues from seasonal changes in photoperiod and temperature to regulate plant growth in Arabidopsis thaliana
Nomoto, Yuji
Kubozono, Saori
Miyachi, Miki
Yamashino, Takafumi
Nakamichi, Norihito
Mizuno, Takeshi
Plant Signaling & Behavior
In Arabidopsis thaliana, the circadian clock regulates the photoperiodic plant growth including the elongation of hypocotyls in a short-days (SDs)-specific manner. The clock-controlled PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) gene encoding a basic helix-loop-helix (bHLH) transcription factor plays crucial roles in this regulation. The SDs-specific elongation of hypocotyls is best explained by accumulation of the active PIF4 proteins at the end of night specifically in SDs due to coincidence between internal (circadian clock) and external (photoperiod) cues. However, this external coincidence model was challenged with the recent finding that the elongation of hypocotyls is markedly promoted at high growth temperature (28˚C) even in long-days (LDs), implying that the model to explain the photoperiodic response of plant architecture appears to be conditional on ambient temperature. With regard to this problem, the results of this and previous studies showed that the model holds under a wide range of ambient temperature conditions (16˚C to 28˚C). We propose that the circadian clock and PIF4-mediated external coincidence mechanism coordinately integrates both of the cues from seasonal changes in photoperiod and temperature to regulate plant growth in natural habitats.
Arabidopsis thaliana; circadian clock; external coincidence; hormone signaling; light signaling; photomorphogenesis
Related Articles
Mutation in the Arabidopsis PASTICCINO1 Gene, Which Encodes a New FK506-Binding Protein-Like Protein, Has a Dramatic Effect on Plant Development
Vittorioso, Paola
Cowling, Rachel
Faure, Jean-Denis
Caboche, Michel
Bellini, Catherine
Molecular and Cellular Biology
The pasticcino (pas) mutants of Arabidopsis thaliana are a new class of plant developmental mutants; members of this class show ectopic cell proliferation in cotyledons, extra layers of cells in the hypocotyl, and an abnormal apical meristem. This phenotype is correlated with both cell division and cell elongation defects. There are three complementation groups of pas mutants (pas1, pas2, and pas3, with, respectively 2, 1, and 4 alleles). Here we describe in more detail the pas1-1 allele, which was obtained by insertional mutagenesis. The PAS1 gene has been cloned and characterized; it encodes an immunophilin-like protein similar to the p59 FK506-binding protein (FKBP52). PAS1 is characterized by an FKBP-like domain and three tetratricopeptide repeat units. Although the presence of immunophilins in plants has already been demonstrated, the pas1-1 mutant represents the first inactivation of an FKBP-like gene in plants. PAS1 expression is altered in pas1 mutants and in the pas2 and pas3 mutants. The expression of the PAS1 gene is increased in the presence of cytokinins, a class of phytohormones originally discovered because of their ability to stimulate cell division. These results are of particular relevance as they show for the first time that an FKBP-like protein plays an important role in the control of plant development.
Related Articles
Is there a link between protein kinase CK2 and auxin signaling?
Moreno-Romero, Jordi
Martínez, M Carmen
Plant Signaling & Behavior
Protein kinase CK2 is a pleitropic Ser/Thr kinase present in all eukaryotes. In order to study the effects of CK2 depletion on plant development, we have recently generated Arabidopsis transgenic plants expressing a CK2α-inactive mutant under the control of an inducible promoter. Our results showed that continuous expression of the transgene had a dominant negative effect and was lethal for Arabidopsis plants. Overexpression of the CK2α-inactive subunit provoked cell cycle arrest, by perturbation of both G1/S and G2 cell cycle phases. The effects on cell division were particularly strong in root meristems, causing inhibition of lateral root formation even when the mutant protein was transiently induced. Processes that rely on cell expansion, such as hypocotyl elongation in dark-grown seedlings, were also strongly affected. We propose that CK2 regulates auxin-signaling pathways.
protein kinase CK2; cell division cycle; cell expansion; lateral roots; auxin
Related Articles
Ethylene Promotes Hypocotyl Growth and HY5 Degradation by Enhancing the Movement of COP1 to the Nucleus in the Light
Yu, Yanwen
Wang, Juan
Zhang, Zhijin
Quan, Ruidang
Zhang, Haiwen
Deng, Xing Wang
Ma, Ligeng
Huang, Rongfeng
Kieber, Joseph J.
PLoS Genetics
In the dark, etiolated seedlings display a long hypocotyl, the growth of which is rapidly inhibited when the seedlings are exposed to light. In contrast, the phytohormone ethylene prevents hypocotyl elongation in the dark but enhances its growth in the light. However, the mechanism by which light and ethylene signalling oppositely affect this process at the protein level is unclear. Here, we report that ethylene enhances the movement of CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) to the nucleus where it mediates the degradation of LONG HYPOCOTYL 5 (HY5), contributing to hypocotyl growth in the light. Our results indicate that HY5 is required for ethylene-promoted hypocotyl growth in the light, but not in the dark. Using genetic and biochemical analyses, we found that HY5 functions downstream of ETHYLENE INSENSITIVE 3 (EIN3) for ethylene-promoted hypocotyl growth. Furthermore, the upstream regulation of HY5 stability by ethylene is COP1-dependent, and COP1 is genetically located downstream of EIN3, indicating that the COP1-HY5 complex integrates light and ethylene signalling downstream of EIN3. Importantly, the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) enriched the nuclear localisation of COP1; however, this effect was dependent on EIN3 only in the presence of light, strongly suggesting that ethylene promotes the effects of light on the movement of COP1 from the cytoplasm to the nucleus. Thus, our investigation demonstrates that the COP1-HY5 complex is a novel integrator that plays an essential role in ethylene-promoted hypocotyl growth in the light.
Author Summary
It is well known that light suppresses hypocotyl growth in seedlings, while the phytohormone ethylene and its precursor 1-aminocyclopropane-1-carboxylate (ACC) enhance hypocotyl growth in the light. However, the mechanism by which light and ethylene oppositely affect this process at the protein level is unclear. Here, we demonstrate that ethylene enhances the movement of CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) to the nucleus where it promotes the degradation of LONG HYPOCOTYL 5 (HY5) in the light, contributing to hypocotyl growth. Our data indicate that HY5 is required for ethylene-promoted hypocotyl growth in the light, but not in the dark. Using genetic and biochemical analyses, we found that HY5 functions downstream of ETHYLENE INSENSITIVE 3 (EIN3) during ethylene-promoted hypocotyl growth. Further, the regulation of HY5 stability by ethylene is COP1-dependent, and COP1 is genetically located downstream of EIN3, indicating that the COP1-HY5 complex integrates light and ethylene signalling downstream of EIN3. Importantly, ACC enriched the nuclear localisation of COP1 in an EIN3-dependent manner in the presence of light, suggesting that ethylene rescued the effects of light on the movement of COP1 from the cytoplasm to the nucleus. Thus, our investigation shows that the COP1-HY5 complex is a novel integrator that plays an essential role in ethylene-promoted hypocotyl growth in the light.
Related Articles
Brassinosteroid, gibberellin, and phytochrome impinge on a common transcription module in Arabidopsis
Bai, Ming-Yi
Shang, Jian-Xiu
Oh, Eunkyoo
Fan, Min
Bai, Yang
Zentella, Rodolfo
Sun, Tai-ping
Wang, Zhi-Yong
Nature cell biology
Brassinosteroid (BR) and gibberellin (GA) promote many similar developmental responses in plants; but their relationship remains unclear. Here we show that BR and GA act interdependently through a direct interaction between the BR-activated BZR1 and GA-inactivated DELLA transcription regulators. GA promotion of cell elongation required BR signaling, whereas BR or active BZR1 can suppresssed the GA-deficient dwarf phenotype. DELLAs directly interacted with BZR1 and inhibited BZR1-DNA binding both in vitro and in vivo. Genome-wide analysis defined a BZR1-dependent GA-regulated transcriptome, which is enriched with light-regulated genes and genes involved in cell wall synthesis and photosynthesis/chloroplast. GA promotion of hypocotyl elongation requires both BZR1 and the phytochrome interacting factors (PIFs), as well as their common downstream targets PREs. The results demonstrate that GA releases DELLA-mediated inhibition of BZR1, and that the DELLA-BZR1-PIF4 interaction defines a core transcription module that mediates coordinated growth regulation by GA, BR and light signals.
Related Articles
Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl
Paynel, Florence
Schaumann, Annick
Arkoun, Mustapha
Douchiche, Olfa
Morvan, Claudine
Annals of Botany
Background and Aims
In hypocotyls of flax (Linum usitatissimum) cadmium-induced reorientation of growth (i.e. an increase in expansion and a decrease in elongation) coincides with marked changes in the methylesterification and cross-linking of homogalacturonans within various cell-wall (CW) domains. The aim of the present study was to examine the involvement of pectin methylesterase (PME) and peroxidase (PER) in this cadmium-induced CW remodelling.
CW proteins were extracted from hypocotyls of 10- and 18-d-old flax that had been treated or not treated with 0·5 mm Cd(NO3)2. PME and PER expression within these extracts was detected by LC/MS, by isoelectric focusing and enzyme activity assays. Transcript expression by RT-PCR of known flax PME and PER genes was also measured in corresponding samples.
Key Results
In cadmium-treated seedlings, PME activity increased as compared with controls, particularly at day 10. The increased activity of PME was accompanied by increased abundance of both a basic protein isoform (B2) and a particular transcript (Lupme5). In contrast, induction of PER activity by cadmium was highest at day 18. Among the four reported PER genes, Flxper1 and 3 increased in abundance in the presence of cadmium at day 18.
The temporal regulation of Lupme and Flxper genes and of their respective enzyme activities fits the previously reported cadmium-induced structural changes of homogalacturonans within the CWs. After PME-catalysed de-esterification of homogalacturonans, their cross-linking would depend on the activity of PERs interacting with calcium-dimerized blocks and reinforce the cell cohesion during the cadmium-induced swelling.
Cadmium; cell wall; homogalacturonan; Linum usitatissimum; pectin methylesterase; peroxidase
Related Articles
Extracellular ATP and nitric oxide signaling pathways regulate redox-dependent responses associated to root hair growth in etiolated Arabidopsis seedlings
Terrile, María Cecilia
Tonón, Claudia Virginia
Iglesias, María José
Lamattina, Lorenzo
Casalongué, Claudia Anahí
Plant Signaling & Behavior
Extracellular ATP (eATP) and nitric oxide (NO) have emerged as crucial players in plant development, stress responses and cell viability. Glutathione (GSH) is an abundant reducing agent with proposed roles in plant growth, development and stress physiology. In a recent publication, we demonstrated that eATP and NO restore hypocotyl elongation of etiolated Arabidopsis seedlings treated with GSH. Here it is reported that exogenous ATP also restores root hair growth suggesting a role for ATP and NO in the regulation of redox balance associated to specific processes of plant morphogenesis. A tentative model integrating redox-, eATP- and NO-signaling pathways during root hair growth in Arabidopsis seedlings is presented.
Arabidopsis thaliana; extracellular ATP signaling; nitric oxide; redox system; root hair
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A Genome-Wide Association Study Identifies Variants Underlying the Arabidopsis thaliana Shade Avoidance Response
Filiault, Daniele L.
Maloof, Julin N.
Mauricio, Rodney
PLoS Genetics
Shade avoidance is an ecologically and molecularly well-understood set of plant developmental responses that occur when the ratio of red to far-red light (R∶FR) is reduced as a result of foliar shade. Here, a genome-wide association study (GWAS) in Arabidopsis thaliana was used to identify variants underlying one of these responses: increased hypocotyl elongation. Four hypocotyl phenotypes were included in the study, including height in high R∶FR conditions (simulated sun), height in low R∶FR conditions (simulated shade), and two different indices of the response of height to low R∶FR. GWAS results showed that variation in these traits is controlled by many loci of small to moderate effect. A known PHYC variant contributing to hypocotyl height variation was identified and lists of significantly associated genes were enriched in a priori candidates, suggesting that this GWAS was capable of generating meaningful results. Using metadata such as expression data, GO terms, and other annotation, we were also able to identify variants in candidate de novo genes. Patterns of significance among our four phenotypes allowed us to categorize associations into three groups: those that affected hypocotyl height without influencing shade avoidance, those that affected shade avoidance in a height-dependent fashion, and those that exerted specific control over shade avoidance. This grouping allowed for the development of explicit hypotheses about the genetics underlying shade avoidance variation. Additionally, the response to shade did not exhibit any marked geographic distribution, suggesting that variation in low R∶FR–induced hypocotyl elongation may represent a response to local conditions.
Author Summary
The goal of this work was to identify genetic variants underlying a well-characterized environmental response, the elongation of Arabidopsis thaliana hypocotyls (seedling stems) in response to shade, otherwise known as shade avoidance. We performed a genome-wide association study with four phenotypes: absolute hypocotyl height of plants grown in both simulated sun and shade and two measures of how height responded to shade. With this study, we confirmed previous findings that variants in two photoreceptors were associated with hypocotyl height variation. We also found associations with genetic variants in previously-identified shade avoidance genes, as well as with variants in genes not typically considered part of the shade avoidance pathway. By examining patterns of which of the four phenotypes were associated with each gene, we were then able to discriminate between genetic variants that have a general role in hypocotyl height variation and variants that are specifically involved in the shade avoidance response. We also found that shade avoidance was not broadly associated with geography, suggesting that variation in this trait may be due to local differences in light quality.
Related Articles
Transcriptional programming during cell wall maturation in the expanding Arabidopsis stem
Hall, Hardy
Ellis, Brian
BMC Plant Biology
Plant cell walls are complex dynamic structures that play a vital role in coordinating the directional growth of plant tissues. The rapid elongation of the inflorescence stem in the model plant Arabidopsis thaliana is accompanied by radical changes in cell wall structure and chemistry, but analysis of the underlying mechanisms and identification of the genes that are involved has been hampered by difficulties in accurately sampling discrete developmental states along the developing stem.
By creating stem growth kinematic profiles for individual expanding Arabidopsis stems we have been able to harvest and pool developmentally-matched tissue samples, and to use these for comparative analysis of global transcript profiles at four distinct phases of stem growth: the period of elongation rate increase, the point of maximum growth rate, the point of stem growth cessation and the fully matured stem. The resulting profiles identify numerous genes whose expression is affected as the stem tissues pass through these defined growth transitions, including both novel loci and genes identified in earlier studies. Of particular note is the preponderance of highly active genes associated with secondary cell wall deposition in the region of stem growth cessation, and of genes associated with defence and stress responses in the fully mature stem.
The use of growth kinematic profiling to create tissue samples that are accurately positioned along the expansion growth continuum of Arabidopsis inflorescence stems establishes a new standard for transcript profiling analyses of such tissues. The resulting expression profiles identify a substantial number of genes whose expression is correlated for the first time with rapid cell wall extension and subsequent fortification, and thus provide an important new resource for plant biologists interested in gene discovery related to plant biomass accumulation.
Cell wall; Anisotropy; Growth kinematic profiling; Transcriptome; Microarray; Arabidopsis; Inflorescence stem
Related Articles
Phytochrome-imposed oscillations in PIF3 protein abundance regulate hypocotyl growth under diurnal light/dark conditions in Arabidopsis
Soy, Judit
Leivar, Pablo
González-Schain, Nahuel
Sentandreu, Maria
Prat, Salomé
Quail, Peter H.
Monte, Elena
The Plant journal : for cell and molecular biology
Arabidopsis seedlings display rhythmic growth when grown under diurnal conditions, with maximal elongation rates occurring at the end of the night under short-day photoperiods. Current evidence indicates that this behavior involves the action of the growth-promoting bHLH factors PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) at the end of the night, through a coincidence mechanism that combines their transcriptional regulation by the circadian clock with control of protein accumulation by light. To assess the possible role of PIF3 in this process, we have analyzed hypocotyl responses and marker gene expression in pif single- and higher-order mutants. The data show that PIF3 plays a prominent role as a promoter of seedling growth under diurnal light/dark conditions, in conjunction with PIF4 and PIF5. In addition, we provide evidence that PIF3 functions in this process through its intrinsic transcriptional regulatory activity, at least in part by directly targeting growth-related genes, and independently of its ability to regulate phytochrome B (phyB) levels. Furthermore, in sharp contrast to PIF4 and PIF5, our data show that the PIF3 gene is not subject to transcriptional regulation by the clock, but that PIF3 protein abundance oscillates under diurnal conditions as a result of a progressive decline in PIF3 protein degradation mediated by photoactivated phyB, and consequent accumulation of the bHLH factor during the dark period. Collectively, the data suggest that phyB-mediated, post-translational regulation allows PIF3 accumulation to peak just before dawn, at which time it accelerates hypocotyl growth, together with PIF4 and PIF5, by directly regulating the induction of growth-related genes.
PIF3; hypocotyl elongation; short day; phytochrome-mediated degradation; transcriptional regulation; Arabidopsis
Related Articles
Space and Time in the Plant Cell Wall: Relationships between Cell Type, Cell Wall Rheology and Cell Function
Thompson, D. S.
Annals of Botany
The biomechanical behaviour of plant cells depends upon the material properties of their cell walls and, in many cases, it is necessary that these properties are quite specific. Additionally, physiological regulation may require that target cells responding to hormonal signals or environmental factors are able to modulate these characteristics.
This paper uses a rheological analysis of creep of elongating sunflower (Helianthus annuus) sunflower hypocotyls to demonstrate that the mechanical behaviour of plant cell walls is complex and involves multiple layered processes that can be distinguished from one another by the time-scale over which they lead to a change in tissue dimensions, their sensitivity to pH and temperature, and their responses to changes in spatial arrangement of the cell wall brought about by treatment with high Mr PEG. Furthermore, it appears possible to regulate individual rheological processes, with limited effect on others, in order to modulate growth without affecting tissue structural integrity. It is proposed that control of the water content of the cell wall and therefore the space between cell wall polymers may be one mechanism by which differential regulation of cell wall biomechanical properties is achieved. This hypothesis is supported by evidence showing that enzyme extracts from growing tissues can cause swelling in cell wall fragments in suspension.
The physiological implications of this complexity are then considered for growing tissues, stomatal guard cells and abscission cells. It is noted that, in each circumstance, a different combination of mechanical properties is required and that differential regulation of properties affecting behaviour over different time-scales is often necessary.
Helianthus annuus; cell wall; rheology; growth; stomata; abscission
Related Articles
A Morning-Specific Phytohormone Gene Expression Program underlying Rhythmic Plant Growth
Michael, Todd P
Breton, Ghislain
Hazen, Samuel P
Priest, Henry
Mockler, Todd C
Kay, Steve A
Chory, Joanne
Weigel, Detlef
PLoS Biology
Most organisms use daily light/dark cycles as timing cues to control many essential physiological processes. In plants, growth rates of the embryonic stem (hypocotyl) are maximal at different times of day, depending on external photoperiod and the internal circadian clock. However, the interactions between light signaling, the circadian clock, and growth-promoting hormone pathways in growth control remain poorly understood. At the molecular level, such growth rhythms could be attributed to several different layers of time-specific control such as phasing of transcription, signaling, or protein abundance. To determine the transcriptional component associated with the rhythmic control of growth, we applied temporal analysis of the Arabidopsis thaliana seedling transcriptome under multiple growth conditions and mutant backgrounds using DNA microarrays. We show that a group of plant hormone-associated genes are coexpressed at the time of day when hypocotyl growth rate is maximal. This expression correlates with overrepresentation of a cis-acting element (CACATG) in phytohormone gene promoters, which is sufficient to confer the predicted diurnal and circadian expression patterns in vivo. Using circadian clock and light signaling mutants, we show that both internal coincidence of phytohormone signaling capacity and external coincidence with darkness are required to coordinate wild-type growth. From these data, we argue that the circadian clock indirectly controls growth by permissive gating of light-mediated phytohormone transcript levels to the proper time of day. This temporal integration of hormone pathways allows plants to fine tune phytohormone responses for seasonal and shade-appropriate growth regulation.
Author Summary
In plants, stems elongate faster at dawn. This time-of-day–specific growth is controlled by integration of environmental cues and the circadian clock. The specific effectors of growth in plants are the phytohormones: auxin, ethylene, gibberellins, abscisic acid, brassinosteroids, and cytokinins. Each phytohormone plays an independent as well as an overlapping role in growth, and understanding the interactions of the phytohormones has dominated plant research over the past century. The authors present a model in which the circadian clock coordinates growth by synchronizing phytohormone gene expression at dawn, allowing a plant to control growth in a condition-specific manner. Furthermore, the results presented provide a new framework for future experiments aimed at understanding the integration and crosstalk of the phytohormones.
Why do plants grow faster at dawn? New results suggest that light and the circadian clock coordinate growth by synchronizing the expression of plant hormone genes at dawn.
Related Articles
Verification at the protein level of the PIF4-mediated external coincidence model for the temperature-adaptive photoperiodic control of plant growth in Arabidopsis thaliana
Yamashino, Takafumi
Nomoto, Yuji
Lorrain, Séverine
Miyachi, Miki
Ito, Shogo
Nakamichi, Norihito
Fankhauser, Christian
Mizuno, Takeshi
Plant Signaling & Behavior
Plant circadian clock controls a wide variety of physiological and developmental events, which include the short-days (SDs)-specific promotion of the elongation of hypocotyls during de-etiolation and also the elongation of petioles during vegetative growth. In A. thaliana, the PIF4 gene encoding a phytochrome-interacting basic helix-loop-helix (bHLH) transcription factor plays crucial roles in this photoperiodic control of plant growth. According to the proposed external coincidence model, the PIF4 gene is transcribed precociously at the end of night specifically in SDs, under which conditions the protein product is stably accumulated, while PIF4 is expressed exclusively during the daytime in long days (LDs), under which conditions the protein product is degraded by the light-activated phyB and also the residual proteins are inactivated by the DELLA family of proteins. A number of previous reports provided solid evidence to support this coincidence model mainly at the transcriptional level of the PIF4 and PIF4-traget genes. Nevertheless, the diurnal oscillation profiles of PIF4 proteins, which were postulated to be dependent on photoperiod and ambient temperature, have not yet been demonstrated. Here we present such crucial evidence on PIF4 protein level to further support the external coincidence model underlying the temperature-adaptive photoperiodic control of plant growth in A. thaliana.
arabidopsis thaliana; circadian clock; external coincidence model; light signaling; photomorphogenesis
Related Articles
Vertex-element models for anisotropic growth of elongated plant organs
Fozard, John A.
Lucas, Mikaël
King, John R.
Jensen, Oliver E.
Frontiers in Plant Science
New tools are required to address the challenge of relating plant hormone levels, hormone responses, wall biochemistry and wall mechanical properties to organ-scale growth. Current vertex-based models (applied in other contexts) can be unsuitable for simulating the growth of elongated organs such as roots because of the large aspect ratio of the cells, and these models fail to capture the mechanical properties of cell walls in sufficient detail. We describe a vertex-element model which resolves individual cells and includes anisotropic non-linear viscoelastic mechanical properties of cell walls and cell division whilst still being computationally efficient. We show that detailed consideration of the cell walls in the plane of a 2D simulation is necessary when cells have large aspect ratio, such as those in the root elongation zone of Arabidopsis thaliana, in order to avoid anomalous transverse swelling. We explore how differences in the mechanical properties of cells across an organ can result in bending and how cellulose microfibril orientation affects macroscale growth. We also demonstrate that the model can be used to simulate growth on realistic geometries, for example that of the primary root apex, using moderate computational resources. The model shows how macroscopic root shape can be sensitive to fine-scale cellular geometries.
multiscale; simulation; microfibrils; viscosity; anisotropy
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Jaguars vs. Texans: Houston's Biggest Winners and Losers from Week 11
By Ben Layman ,
The Houston Texans survived a scare from the Jacksonville Jaguars in Week 11, as one of the NFL's worst teams nearly knocked off one of the NFL's best at home in overtime.
Houston's defense knocked Blaine Gabbert out of the game just five plays into Jacksonville's first drive. Chad Henne came off the bench to give Jacksonville's offense a spark it hadn't felt all season.
The Texans were caught off guard by Henne's aggressiveness and willingness to push the ball down the field. Rookie Justin Blackmon had a breakout game and brought big plays to a Jaguars offense that desperately needed it.
Matt Schaub and Houston's offense found itself playing from behind for most of the game, which is unfamiliar territory for a unit that's become accustomed to playing with leads. The Texans were down as much as 34-20 in the second half.
Schaub was far from perfect, but kept the Texans in the game when they had to make a play. Despite nearly throwing the game away with an interception in overtime, Schaub rallied to make the game-winning throw.
Andre Johnson had a career high 273 yards and the game-winning 48-yard touchdown reception in overtime to seal the game for the Texans.
Here's a look at the biggest winners and losers for the Houston Texans from Week 11's drama-filled win over Jacksonville.
Andre Johnson should be one of the happiest players in the NFL when he wakes up Monday morning. Not only did he register a career high in yards Sunday against Jacksonville, but he also caught the game-winning touchdown in overtime.
Walk-off wins always seem more special, and Andre's game-winner was a huge sigh of relief after the Texans offense had been inconsistent all day.
Matt Schaub had a monster day largely because of his No. 1 receiver. Schaub targeted the future Hall of Fame wideout 19 times. Jacksonville's coverage was soft and showed no signs of being able to contain the Pro Bowler all game.
Andre even had a highlight-reel block in the run game on Jacksonville safety Chris Prosinski.
There's been chatter about Andre not being the player he once was, but critics might want to re-think that notion after an effort like this.
Houston Texans nose tackle Earl Mitchell has seen better days in the NFL than he did Sunday against the Jacksonville Jaguars.
Mitchell is filling in for starter Shaun Cody, whose been out with various injuries. The third-year defensive lineman was blown off the line of scrimmage routinely by double teams on Sunday.
Mitchell was frequently swallowed up by Jacksonville's man-blocking scheme. Jacksonville's center, Brad Meester, and left guard Mike Brewster had their way when lined up against Mitchell.
Newly-signed defensive lineman Terrell McClain also saw snaps at nose tackle. McClain was needed to spell Mitchell, but was painfully less effective and was thrown to the ground in one embarrassing instance.
The Texans need stouter play from their nose tackle position going forward. Shaun Cody's presence has been missed more than fans expected.
Sunday's win over the Jacksonville Jaguars was Justin Forsett's best game as a Houston Texan.
The speedy back gave the Texans much-needed chunks in the run game spelling Arian Foster. He carried the ball seven times for 59 yards and gave the running game a spark.
Defensive coordinators scheme to stop Foster, and it allows Forsett to come off the bench and catch defenses off-guard. Forsett is regarded as a receiving back, so defenses likely think pass when he's on the field. However, he has proven to be more than just a receiving back, gaining hard yards running the ball as well.
Forsett capitalized on his opportunities Sunday and continues to give fans reason to forget about the injured Ben Tate, who missed his third straight game Sunday.
Tate's absence from the offense was expected to be significant, but Forsett has emerged as a quality complement to Foster since Tate went down.
The Houston Texans pass rush as a whole has been absent for weeks now, and it finally caught up to the team against Chad Henne and the Jacksonville Jaguars.
Houston's lack of edge rush gave Henne a comfortable pocket to survey the field from all Sunday. Texans outside linebackers Connor Barwin and Brooks Reed haven't consistently delivered the edge rush the team had expected from them coming into the season.
Barwin and Reed went a second straight week without registering a sack, and the duo has combined for only 4.5 sacks this season. Sacks aren't everything, but no one expected production that low from this combo of rushers.
Rookie Whitney Mercilus hasn't been getting the increased snaps most expected, which is becoming frustrating for fans given the lack of production from the starters.
The Texans won't beat any of the elite teams in the NFL in the playoffs with the edge rush they get week in and week out. Defensive coordinator Wade Phillips can't continue to over-blitz to make up for the lack of edge rush.
The Texans need their edge rushers to make more of an impact going forward if they want to avoid near-disasters like the Jaguars delivered Sunday.
Darryl Sharpton's return to the football field was a sight for sore eyes Sunday. The linebacker opened the season on the PUP list, and hadn't played a down of football since Week 7 of last year.
Sharpton showed the pop and acceleration he possessed pre-injury, though he did it in limited snaps. Sharpton should be eased back into the lineup slowly, likely seeing more snaps as the season goes.
With the loss of Brian Cushing for the season, the Texans will take all the help they can get at inside linebacker. Sharpton now joins a rotation of inside linebackers that have done a serviceable job filling in for Cushing to this point.
Sharpton has the most upside of any of the Texans' inside linebackers. A few weeks ago there were some concerns over the Texans' inside linebacker group. Sharpton's return to the lineup will ease some of those concerns.
Kareem Jackson had been playing well through the first nine games of the season, so his drop-off in performance in Week 11 came as a surprise.
Jacksonville's first-round pick, Justin Blackmon, turned in a career day, posting 236 yards on seven catches. Unfortunately for Jackson, he was on the business end of many of those yards.
Fans likely saw shades of Kareem's rookie year when the cornerback tripped after giving up a catch at one point in the first half. The trip led to the receiver gaining another big chunk of yards and momentum for Jacksonville.
The third-year cornerback rounded off his sub-par day by dropping a poorly underthrown ball by Chad Henne. If the interception had been made, the Texans would've had the ball deep in Jacksonville's territory.
This was likely just a bad game for Kareem. In a season where the young corner has vastly improved, he had a blooper.
Kareem hadn't been toasted by a receiver to this extent this season, and fans shouldn't expect performances from him like this going forward.
The Houston Texans would not have squeaked by the Jacksonville Jaguars in Week 11 without Keshawn Martin's production.
The rookie wide receiver was a weapon for the Texans as a receiver, kick returner and punt returner. Martin caught a touchdown pass on the opening drive of the game after beating man-coverage and winning inside-leverage against the defensive back.
The fourth-round pick had five kickoff returns for 162 yards, which included a 54-yarder. His 71-yard punt return changed the momentum for the Texans in the second half.
Martin likely played his way into head coach Gary Kubiak's good graces with a huge day Sunday. His ability to bring a spark to a special teams unit that had struggled mightily all season was a major boost for the team.
Martin was the unsung hero of the win over the Jaguars.
Kickers are easy targets for blame, but Shayne Graham left six crucial points on the field when he missed two field goals in Sunday's win over the Jaguars.
Graham had only missed two field goals all season leading up to this week, so fans shouldn't exactly be calling for his head. It was a bad game for the veteran, however, and his struggles could've been magnified had the team lost (or tied).
Graham's missed 47-yard field goal at the end of regulation was particularly back-breaking. It sent the game into overtime, something the team couldn't afford due to its upcoming short week.
Graham didn't kickoff well either. One particular kickoff in overtime gave the Jaguars great field position, which could've been costly.
Fans hope Graham isn't the streaky type and shakes this rough game off before Thursday's Thanksgiving game against the Detroit Lions.

subset: commoncrawl


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Cloud walkers
Category: Sense of Place
Photo and caption by Luca Molnar
I took this photo in Letná Park, in Prague. The park gives places to runners, mothers walking with babies, kids, couples and also it is one of the biggest territories for skateboaters. People from all over Europe come to skateboard here, and after they have sampled it, they throw their shoes to a rope outspread to the so called Metronome. The Metronome is a monument which stands at the place where the famous Stalin statue used to be.
Location: Prague


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Published: 08/02/13
We sort out the truths behind organ donation and transplants from the tales that you might have heard.
Key facts
All of the UK's major faiths including Buddhism, Christianity, Hinduism, Judaism, Islam and Sikhism support organ donation and transplantation. Anyone who has doubts as to whether their religion or faith allows them to donate is encouraged to seek advice from their local religious leader.
It has been reported that recipients sometimes take on characteristics of their donors. While NHSBT is aware of the suggestion that transplant recipients take on aspects of the personality of the organ donor, the organisation is not aware of any evidence to support it.
Stories include:
The Big Reunion trio, Atomic Kitten, urge you to join the Organ Donor Register today!
Wondering why you should join the Organ Donor Register, and whom you should tell once you have?
We share the stories of organ transplant recipients, donors, and those on the donor waiting list
We sort out the truths behind organ donation and transplants from the tales that you might have heard


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Experience Walker's Bluff
Southern Illinois' breathtaking scenery forms the backdrop for gourmet cuisine, pleasurable pastimes and world-class entertainment at Walker's Bluff. What was planted as a family homestead long ago has grown into a picturesque vineyard that maintains an open-armed welcome to friends and visitors. Read more »
Weekly Hours
The General Store
Friday - Saturday: 7am-7pm
Thursday: 4pm-10pm


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For Sale
Five P 195 14 inch tires on New Yorker rims – $100
Filed under: ClassifiedsKeywords: For Sale, tires
A new California law requires that eggs sold in that state be from chickens raised in cages where birds can spread their wings. Is this a good law?
View Results


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The more I read about it, the more clearer it becomes how all of these Egyptian influences cumulated in Christian Religion (above picture from here)
It started with the ten commandments and the exact copy as in the book of Death but then I read on and on … and on.
I read about Paris, which is actually "Near the temple of Isis" … and I read that even in the 16th century people there were found offering veneration to the statues of Isis which stood in … the "Notre Dame" in Paris.
The Church about "our lady" that was build on a boat shaped island… the boat of Isis. No wonder Napoleon went mad and head for Egypt looking for the roots of Paris.
Isis was a prime Goddes in Egyptian Civilization for thousands of years. It spread all over Europe in many forms. 40 years BC Cleopatra named herself the reincarnation of Isis, at the time Caesar had a relation with her (where he studied in the Library of Alexandria (the re-imported invention of Philosophy from Greece physicalization (which was again based on the export of papyrus to Greece…))). Leading to a gigantic trend of Isis worship in the Empire. Isis conquered Europe. There was never a Maria worshipping. She is not even mentioned. Old painting of Isis are painted over, Churches arise on top of old Isis temples. I found thousands of images of Isis and Child, some were just painted over with the new meaning.
This is just a reflection of end-thoughts. Google somewhat on Isis / Maria and you will find an amazing truth.
"Many statues of Isis nursing her son Horus were renamed as Mary and Jesus. Thinly-veiled Isis remains within the Coptic Christian Church as St. Isis, still worshiped with the sound of sistra. The images of dark or black virgins frequently have Isian predecessors, and throughout Europe, churches were built on temple sites. "
It's pretty clear to me: Isis and her child Horus = Maria and her child Jesus though this is just the end-thoughts not the complete reading process which also encompasses subjects such as "the soul" which is so clearly an Egyptian concept or the "sin" which is also so very clear an Egyptian concept as is "life after death", etc.. etc.. etc… (also try )
We have been fooled luckily you will find thousand and thousands of internet pages with more reading material to read some more about this and came to the same surprise. Just a little post to get your queries started.


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What the people think, add your voice below.
protect the nhs from these savage cuts
Well done! Really innovative way of showing opposition to the NHS reforms
I am a GP working in Brixton, London.
I believe the planned government reforms shall undermine our health care service. The NHS will become localised, with postcode lotteries for care. There will be increased privatisation of services. Private business will expect to profit from their input into the NHS reducing finances available for care.
The NHS presently relies on the good will of staff and patients. Many working for the NHS put in a great deal more time and effort than they would if working for private healthcare. Erosion of the core values of the NHS shall lead to a loss of good will, increases in healthcare costs and a reduction in quality of care.
The NHS is presently as popular as it has ever been. The coalition was not voted in with a mandate to make changes in the NHS. In fact both parties promised not to make such changes.
The prime minister has correctly recognised that the cost of healthcare is increasing as new therapies become available and the population ages. Surely this means we need greater investment rather than the planned relative cut in funding.
Efficiencies and improvements can be made to the NHS, however constructive change requires planning and increased investment rather than cuts.
The planned government changes will mean that quality of care shall be eroded and as the service becomes increasingly privatised, the poor and vulnerable shall not be able to access the care they deserve.
I am scared of the planned reforms.-
I agree with everything Bruce Blackwell says. The increasing aging population need looking after better than is currently the case and this will cost more money, not less! Increased technology, new therapies and treatments have made healthcare more expensive, but this development is something to be proud of. The NHS is somthing that this country used to be very proud of and we should seek to safeguard it, not reverse all the improvements made. The planned changes will mean that the poor and vulnerable will suffer.
With a small increase in income tax, which could be ring fenced for the health service, we could return to an NHS to be proud of and which will benefit the people who should benefit from it.-
The NHS has to change. This country cannot afford to increase spending in the current climate. People demand things from the NHS that are not appropriate. What is crucial is the prevention of health problems. Cultural change can help with this. Let's take away the concept that the NHS is free, a concept that everyone thinks. Let's advertise what everything costs when people use the services so that they realise it is not really "free". Cultural change for the future is also important. Let's get kids doing more exercise, taxing fast food heavily so the prices increase to prevent obesity, heart disease, diabetes, etc, people taking responsibity for their own health rashter than relying on the NHS. The government are doing the right thing (Regardless of which government it is as they are all working to the same outcome) so let's just get on with it.
Scared, and with good reason. Privatisation will ALWAYS put profit before people – that's the essence of capitalism after all! The bankers have grown rich off the backs of ordinary people; health care consultants are carving out their planned future second and third homes in the hope that that the Tory govt will let them get away with it!
A good idea how would we link to our website?
Kill the Bill not the NHS!
The NHS is a fragile and scarce resource. We need to care for this most precious of common goods – keeping this latest brand of nasty little free-market conservative conceit away from it is the least we can do. I do however think the NHS is bigger and more expensive than it need be, if we took more care of ourselves and others I believe it would be in better shape and able to take better care of people that really need help. Again I blame the free-market people – this obsession with competition and status is making us all sick.
I worry about the reforms. As someone who has Epilepsy and other medical problems, I fear that if the Bill was to be passed, then I may be forced to stop all my treatment because I won't be able to afford it, and many other patients may feel the same way as me. Many private health insurance firms won't cover people who have pre – existing conditions, so what is the logic in that? God help us.
hello guys,to be fair,Next to peoples republic of china and indian railways,NHS has the 3rd biggest payroll,Many people in uk are working for public sectors(the govenrment has to contribute in addition to the ni contribution).how long the government can sustain this budget.it has been proposed that nhs budget will touch 230 billion in 10 years time and this country cannot afford,There should be privatisation in NHS to appreciate the talent and encourage competetiveness which is lacking in the present environment.I personally want to see hard working people getting rewarded.anyway whatever it is,Nobody can prevent nhs not being privatised in 10 years time whatever the party comes to power.i think this is reality.
How exactly do you think that privatising the NHS will save the government money? When a proportion of the cost of services is profit.. And why is it more important that we reduce expenditure than care for our people? Shall we spend the money saved on the arms trade instead?
Sad, very sad, but true. I wonder if the Govt might consider stop sending millions in aid to India any time soon – India – the land of the multi millionaires who look after themelves and spend very little on their own poor …. Sad, very sad, but true.
Of course we are fed up with the ConDem Government and here's a simple way to DO SOMETHING ABOUT IT >>>> http://tinyurl.com/3auwzvq
….Pass it on across the Internet.
Can we bring them down?
Yes we can.
Brilliant comments – now we need action! Opposition from us all has stopped the progress of the bill so far. we all need to join up as a network to share tactics expertise, leaflets and solidarity. I belong to defend Our NHS and we are on Facebook.
I believe the best way to influence the 70-80 (50 Lib Dems and say 20 Tory marginals)or so MPs that stand between the preservation of the NHS and its destruction is the voice of the constituents in these seats, challenging the kind of rubbish they talked in the Commons debate last night. They need to know they represent the views of their constituents in parliament and if they don't then they will be voted out at the next election. There are lots of ways to put pressure on:collect a supportive group of say 5 -10 people
Get a leaflet – Defend Our NHS has one you can use
Find out when your MP holds a surgery
Make appointments and go regularly
Give out leaflets in busy places – you will be amazed at the response – no one agrees with it
Ask your MP to a meeting – just a few of you in a pub is ok
And so on -
I have a list of Tory marginals if you want to know where they are. You might live in one!We've done all of this to good effect. Join us on facebook and we can all keep in touch!!
Together we can win but we need to be a loud coherent concerted voice. -
Totally, Totally against the carving up of our NHS…..They have no mandate, nothing
in the Tory Manifesto the only reference to the NHS was "No top down reorganisation of the NHS" Dismal Daves depressing poster "I will cut the deficit not the NHS", and nothing in the coalition agreement. Why does this not get TV coverage…..No documentaries on BBC or ITV showing how it will change…..Joe Public is not taking in how this will affect them….Needs to be a proper documentary on Prime Time viewing BEFORE it is too late.
I do not understand the lack of TV coverage, the changes this will cause to Britain will be shattering to many people… -
I had to agree my experience of the 'Coalition Government' was similar. Here's why:
* The Bill has no mandate.
* The complete reorganization of the NHS is in no party's manifesto.
* All major medical organizations oppose this bill.
* Has had major sections put into effect prior to becoming law.
* Competition is useful in sports — not medical care.I was born in America and lived the first forty years of my life there with their competitive, private provider, profit motivated healthcare system. It's inhumane, ineffective, it's cost os nearly twice that of the NHS. See below from an academic healthcare study:
Healthcare spending as a % of GDP:
o America … 16%
o France …. 11.2%
o Germany …10.5%
o UK ….. 8.7%In closing I offer a conclusion from another academic study by Colin Prichard (Bournemouth University contact: ) and Mark S. Wallace (Department of Economics, Latymer School, London) titled "Comparing the USA, UK and 17 Western Countries' efficiency and effectiveness in reducing morality.
A year ago today my child sustained a serious brain injury. And has, thanks to the fantastic treatment given by KCH, made a full recovery. The proposed reforms to the NHS terrify me, what if my child had another serious accident? Would I be able to afford treatment?
Glad to join in on this wondeful platform and express my utter contempt for the hacking and slashing of the welfare state by a government that I didn't vote for. keep up the good protest. It feels good to be heard.
I consider as a healthcare worker this bill/cuts are promoting inequalities of health rather than high standards of care. I work in Primary and Secondary care and I have been seeing during the last couple of years the massive impact on patients how short staff and lack of resources are leading into more hospital admissions. Ageing population increasing demands more care and individual care is going to be difficult. Long term conditions such as diabetes/COPD are considered as QOF jobs and previously were covered by District nurses.. Nowadays , a high population will be neglected with these reforms and definitively the major impact will be on people with lower incomes and who unfortunately can not afford private treatments due pre-existing conditions are not covered by private insurance. I reckon some changes are needed but no making cuts or pressurising lower class.. Staff competencies should be reviewed and standards of care can be improved in other ways and taking advantage of staff skills and abilities. I am trained assistant Practitioner working in a Surgical Ward and I reckon career progression and investment on further training will lead into professionalisation of service and best standards of care. I could be here all day giving real situations regarding how standards of care can be improved and also even saving money with the actual structure NHS … NO TO THE BILL!
The reforms are coming from the fundamental Tory/Liberal drive to claw back spending on the NHS, whilst offering no safeguards to the British people and undermining the very survival of this venerable and highly respected institution (despite its weaknesses); this is a drive that is at once unspeakable and despicable.
These proposed cuts are a disgrace. The Government, who had to scrabble to make an alliance to actually be viable, seem to be pushing for a private system. Fine for them when you consider how many of the cabinet are millionaires. BUT they do NOT represent the majority of this country. We pride ourselves on our NHS, even with its faults. We do not want a tragically flawed system like the US.
The government denies that privatisation is the aim of the Bill or that access to the NHS for patients will be restricted. My main concern is that it may mimic the system used by dentists, and that some practises may become wholly private, and that where NHS service is available, the full cost may not be met. Patients may have to make a significant contribution unless exempted due to low income.
Our NHS was the envy of the world, please don't let the politicians ruin it.
I feel the problem with the NHS is that it is heavy with managers instead of medical staff. Managers cannot treat or care for patients, they have no medical use but take a large amount of the NHS budget, perhaps bringing Matrons back on wards would be a much better use of some of that money.
This government seems to want to put everything out to private companies, yet how is it possible to pay a company to do the same amount of work plus make a profit, without cutting the service, charging more for the service or cutting the conditions of the people doing the work.
Think what has happened with dentists, it is hard to find an NHS dentist, if you do then you still have to pay a lot of money for treatment and many people who are in need of treatment do not go to the dentist because they cannot afford to, I myself am one of those. Do we want the rest of our health care to go the same way. -
How come the 'Memebers of the public' bubble is the same size on both sides? If size of circle represents the number of people included in it, it should be way bigger on the left.
At first glance it looks like members of the public support the bill – which is kind of contentious..!-
…I mean it should be bigger on the right. oops
Great work, just noticed a small issue – Paul Ireland is listed as For, but his quote is quite clearly against the Bill.
I have a GP who failed to diagnose an obvious health problem from which I suffer …. and the Govt wants to put my future care in HIS hands??
Well that's completely unbiased and fair, well done! You could of course put all your lovely circles on the left into two circles: unions, who have a vested interest, and people who work in the NHS (i.e. union members) who have a vested interest in the billions of pounds poured into the system. Everyone else is bored of union bullying, inflexiblity and waste. Still, all done now. Now for fair regionalised pay and something (anything) with the schools and the teaching unions. Really enjoying 2012 so far.
It is hard enough to get GPs to do what they should be doing without giving them extra duties!
We failed. Despite all the opposition from the public and the vast majority of healthcare professionals, the government has spat in the face of our democracy. I've written to the Queen to ask her to refuse Royal Assent of the bill, but deep down I know it's a useless gesture. We're doomed.


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Please enter your travel dates.
Standard Double Room - Meals & Beverages not inc non refundable
Standard Triple Room - Meals & Beverages not included
Deluxe Double Room - Non Refundable
Suite, 1 King Bed - Non Refundable
Buenos Aires boutique with antique, European flair
With a stay at Unique Executive Central in Buenos Aires (9 de Julio Avenue), you'll be minutes from Obelisco and Teatro Colon.
This 4-star hotel is close to Plaza de Mayo and Casa Rosada.
Make yourself at home in one of the 70 air-conditioned rooms featuring minibars. Your room comes with a pillowtop bed. Cable programming provides entertainment, and wired and wireless Internet access is available for a surcharge. Bathrooms have hair dryers and bathrobes.
Make use of convenient amenities such as complimentary wireless Internet access, concierge services, and babysitting/childcare.
Enjoy a meal at a restaurant, or stay in and take advantage of the hotel's room service (during limited hours). Quench your thirst with your favorite drink at a bar/lounge.
Featured amenities include complimentary high-speed (wired) Internet access, a business center, and express check-in. Planning an event in Buenos Aires? This hotel features 5877 square feet (546 square meters) of event facilities. Parking (subject to charges) is available onsite.
Hotel Amenities
- General
This quiet boutique Unique Executive Central Hotel is located in the heart of downtown Buenos Aires in the fashion and business district. The hotel is within walking distance of the Obelisk, Teatro Colón Opera House, and the entertainment district.
Amenities at Unique Executive Central Hotel include a business center with 5,877 square feet of meeting facilities and two meeting/conference rooms, complimentary wireless and wired high-speed Internet access, concierge services, and an on-site restaurant.
Guests receive complimentary access to an area fitness center and pool.
- Concierge services
- Restaurant
- Room service (limited hours)
- Business center
- Security guard
- Multilingual staff
- 24-hour front desk
- Safe-deposit box at front desk
- Total number of rooms - 70
- Babysitting or childcare
- Medical assistance available
- Dry cleaning/laundry service
- Air-conditioned public areas
- Elevator/lift
- Bar/lounge
- Number of floors - 9
- Business services
- Express check-in
- Express check-out
- Free wired high-speed Internet
- Luggage storage
- Laundry facilities
- Meeting facilities size (meters) - 546
- Number of meeting/conference rooms - 2
- Year Built 1998
- Meeting facilities size (feet) - 5877
- Use of nearby fitness center (complimentary)
- Free Wi-Fi
- Parking (surcharge)
- Internet
Available in all rooms: Wireless Internet access (surcharge), Wired high-speed Internet access (surcharge)
Available in some public areas: Free Wi-Fi, Free wired high-speed Internet
- Parking
Parking (surcharge)
- Room Amenities
- Climate control
- Complimentary weekday newspaper
- Air conditioning
- Minibar
- Coffee/tea maker
- Bathrobes
- Hair dryer
- In-room safe
- Iron/ironing board (on request)
- Clock radio
- Cable TV service
- Pillowtop mattress
- Wireless Internet access (surcharge)
- Wired high-speed Internet access (surcharge)
- Dining
Tango Restaurant - Offers an intimate atmosphere and a wide range of Argentine and traditional cuisine, and five o'clock tea. Room service is available (limited hours). Open for breakfast, lunch, and dinner.
Policies & Fees
- Check-in
Check-in time starts at 3 PM
Minimum check-in age is 21
- Payment types
Accepted at this hotel: American Express, Diners Club, Discover, MasterCard, Visa
- Pets
Pets allowed, Pet policy - dogs only, Maximum number of pets per room 1, Pet maximum weight in kg is 10
- General
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
- Fees
- The following fees and deposits are charged by the property at time of service, check-in, or check-out.
- Pet fee: USD 30 per day
The above list may not be comprehensive. Fees and deposits may not include tax and are subject to change.


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With all the success that she is currently garnering in the music industry, for Pink, there is nothing more amazing than her own child, referring to her daughter as the coolest thing that has ever happened to her.
The American singer-songwriter seems inseparable from her daughter as she is caught on camera from time to time with the two-year-old named Willow. Of course, her husband Carey Hart is ever present sharing the happy moments with her wife and child. On an interview, Pink shares how even through the weirdest moments, she can see the genuine and unconditional love that they have, which is practically something that every mother should be able to say about their child.
Referring to her child Pink states, "She's the coolest thing that has ever happened to me. Now I have someone to sing goofy a** songs to all day and she likes it," adding, "She's not judging me. She's probably the only person who doesn't judge me."
It was only back in June 2, 2011 when Willow Sage Hart was born and became the biggest milestone in the pop icon's life so far. Apparently, it was a pretty huge step for her personally since she was said to be terrified of being responsible for letting a baby out to the world. However, her husband Carey was there who has always wanted to have a child. According to Pink, "I had moments where I wanted a kid with Carey. You know – you first fall in love, and you're like, 'We should have kids,' and then it went away. But he always wanted kids and then we did it, and now it's like, 'F**k,' this is so rad. It's more important than anything we've done."
Now she could not have been possibly be more fulfilled than she is today. "I can use the word "happy" and not flinch," she gushed. "I'm happier than I've ever been, but I'm also still living my life."
After all, it is not only her family that is going quite well in her life but also her music career.
Just recently Pink had released a single included in her sixth studio album "The Truth about Love," "Just Give Me a Reason" featuring Nate Ruess of fun. The song became an instant hit worldwide even becoming like an anthem that has spread like wildfire. The song remains to be her most successful single in her latest album to date. It became highly successfully in the United States and Australia, but not any less in other countries around the globe. It even stayed for seven consecutive weeks at the number one spot of Canadian Hot 100.
In support of her album "The Truth about Love," Pink is currently on a tour of the same name; "The Truth about Love Tour." It is a four-legged tour that starts from North America, then Europe, Australia and back to the North America. The last tour date will take place at Atlanta's Philips Arena on December 14, 2013. You can still get your Pink tickets online at Ticket Broker today.
TicketBroker.com provides an online ticket broker offering premium event tickets for sale on the secondary market. Prices reflected on this site are determined by the buyers and sellers and may be substantially higher than the face value printed on the ticket. TicketBroker.com contains event and concert tour dates, as well as team information and game schedules for every sport. We are not affiliated with any box office, venue, sponsor, team, performer, Ticketmaster.com, or other organization. Any trademarked terms that appear on this page are used for descriptive purposes only.


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For Accommodation and information, please call:
0845 859 1006
Monday to Friday 9am - 5pm
Saturday and Sunday - closed
*Calls may be recorded
Multi-activity Centre
Discover a wealth of indoor and outdoor adventure activities that the whole family can enjoy on the stunning Galloway Coast.
Golf Courses
Cally Palace, a private course that can only be played by the residents of the hotel, is the work of well-known golf architect Tom Macaulay and opened in 1994.
Golf Courses
Set in an idyllic location in the beautiful Fleet Valley, every hole on this stunning 9-hole presents you with a unique challenge which once played is never forgotten.
Bike Tours
Enjoy bespoke cycling and mountain biking holidays through some of the most scenic countryside in Dumfries & Galloway.
Welcome to Glenquicken Farm Trout Fishery
Mountain Bike Trails
Newton Stewart
The 7stanes team has created something special. It's a couple of hours drive from the Scottish central belt but it's worth the effort if you're looking for a mountain biking experience that is at the tougher, more epic end of the scal
Swimming Pools
Enjoy the excellent swimming facilities and classes available at this safe and well-maintained pool.
Golf Courses
This golf course is Impressively perched above the Solway coastline and set within 1,200 acres of beautiful countryside.
Fitness & Leisure Centres
Situated in a superb 1,200 acre coastal location, Brighouse Bay Leisure Club is an adventure seekers' paradise.
Riding Schools & Centres
Part of the multi-activity Brighouse Bay Holiday Park, this trekking centre offers a variety of options for horse riders of all levels aged five plus.


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Orion Cooker
Orion Cooker
A very clever and unusual design, the Orion excels at keeping meat moist and cooking fast. Well, maybe it isn't that unusual. It is really a hi-tech variation on the cowboy method of cooking in a Dutch oven where the pot sits on coals and coals sit on top of the pot. It is especially good for chicken, turkey, fish, and beef brisket. It is all stainless steel, lightweight and portable, and the central cooking chamber is pretty close to airtight. Wood chips and/or water go in the bottom of the cooking chamber, but the charcoal does not. It goes in a ring around the outside and in a cup on top. About 10 pounds! They recommend Matchlight, which has an awful smell. We recommend starting charcoal in a chimney.
The result is a hot convection oven that is great at creating moist smoky meat in a hurry. Because the charcoal combustion gases are outside the cooking chamber, the smoke flavor is different than most other smokers, and because it is so moist, one does not get crispy skin on chicken or turkey or a good crust on pork. For ribs and pork butt, we prefer to cook at a lower temp. That said, this is a mighty nice smoker for the price. The manufacturer claims it can smoke six racks of ribs in 1.25 hours, about 25% of the time in a conventional smoker, or a 20 pound turkey in 2.25 hours. Includes three rib hangers to accommodate six racks of ribs, three cooking grates, a poultry stand and lifting handle that holds a 24 pound turkey.
People like it. It's easy to use, cooks fast and produces very good results. One of the only complaints is that Orion is a charcoal hog. You can easily burn through 15 pounds per cook.
$169 Manufacturer's suggested retail price (MSRP), actual price may vary.
Where To Buy (Buying from these suppliers supports this website):
Ace Hardware: Free In Store Pick Up
Primary Function:
Primary Capacity:
346 square inches ( about 17 burgers )
Manufacturer claims that all or practically all of this device is made in the USA
Review Method:
Researched it from Afar
We have researched this product from afar by gathering info from the manufacturer, owners, and other reliable sources, with our BS meters on high.
Please read this before posting a comment or question
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About this website
AmazingRibs.com is all about the science of barbecue, grilling, and outdoor cooking, with great BBQ recipes, tips on technique, and unbiased equipment reviews. Learn how to set up your grills and smokers properly, the thermodynamics of what happens when heat hits meat, as well as hundreds of excellent tested recipes including all the classics: Baby back ribs, spareribs, pulled pork, beef brisket, burgers, chicken, smoked turkey, lamb, steaks, barbecue sauces, rubs, and side dishes, with the world's best buying guide to barbecue smokers, grills, and accessories, edited by Meathead.
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OAKLAND, Calif. (KGO) -- Investors packed a federal bankruptcy court in Oakland Tuesday afternoon, with the crowd spilling into the hallway. They were there to confront the man they blame for losing their savings. Some individual families lost millions of dollars; 3,000 investors lost a total of $750 million.
It started as a trickle and became a flood. Investor after investor came to federal bankruptcy court to face Walter Ng, who with his two sons and partner Bruce Horwitz, ran several investment funds that are now in default.
"I would just ask him be honest, tell the truth," said investor John McGuire. "I think that's what people want is the truth."
"Just trying to figure out the whole mess really right now," said investor Gary Leeman. His wife, Cathy, described how she felt as "just angry."
Investor Julian Potashnick told the I-Team he lost "a little over $3 million" and it's all gone.
In his voluntary petition for bankruptcy, Ng estimates his assets between $500 million and $1 billion; his liabilities are also estimated between $500 million and $1 billion.
In a declaration, Ng writes he's filed for bankruptcy because "certain investors... have commenced or threatened to commence legal action against me based on losses." Ng says he wants to "ensure an orderly process for the resolution and payment of the claims of investors."
But Tuesday, when the judge tried to ask Ng and his bankruptcy lawyer basic questions about his investment business, Ng's criminal defense attorney cited the ongoing investigation by the FBI, the SEC and the Department of Labor. Ng refused to answer questions for fear of self-incrimination.
"I don't think you take the 5th Amendment unless you got something to hide," said Richard Brown, attorney for the class action lawsuit. "There's something going on there. We've alleged fraud and mismanagement, breach of fiduciary duty, and clearly he's been told by his lawyers to shut up."
Brown has filed a class action lawsuit accusing Ng of embezzlement, violating federal securities laws and lying to investors.
Ng and his lawyers refused to answer questions on the way out, to offer anything at all those investors who are dealing with the loss of their life savings.
The I-Team will continue to purse leads on this story and provide further coverage in the weeks and months to come.
For more background on this complex case, read the I-Team's first report from Aug. 22, 2011 and check out the I-Team Blog.
If you have a tip for the I-Team, send us an e-mail here or call 1-888-40-I-TEAM.
fraud, orinda, oakland, i-team, dan noyes
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Top News
Customer Raves
This brass brake line junction block is for the left front brake line at master cylinder. Fits 1951-54 PASS & 1/2 Ton (EXC. CONVERTIBLE) .Fix your brakes right the first time! and feel safe with a complete brake overhaul. Take a look at what we have in our brake section 180 including:BrakesBrake DrumsBrake HosesWheel Cylinder -New and Rebuild KitsMaster Cylinder -New and Rebuild KitsRubber ComponentsHardware: Return Springs, Retainers and Clips.
This part fits most Chevrolet 1951 1952 1953 1954 Cars and Trucks.; exceptions may apply. Please read the complete description to see if this part fits your specific vehicle and application.


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Mark David Gerson is the award-winning author of the popular Q'ntana Trilogy of fantasy novels, two critically acclaimed books on writing and a memoir. His screenplay adaptations of the three Q'ntana books are on their way to the big screen as a trio of epic motion pictures and he is currently adapting the Q'ntana stories for the musical stage.
Mark David is also an accomplished artist/photographer and a sought-after speaker on topics related to personal growth, spirituality and creativity. As a coach, mentor and seminar leader for more than two decades, Mark David has guided writers and non-writers alike to connect with their innate wisdom, open to their creative power and express themselves with ease.
For more on Mark David and his work, look him up on Facebook.
Find Mark David's paperbacks/ebooks on Amazon; his ebooks are also on iBook, Nook and Kobo. His recordings are on iTunes, Amazon, CD Baby and Google Play
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Fancy Dress Beginning With 'C'
Why not have a fancy dress party and invite guests to wear Letter C Costumes. Explore this section to find your favourite fancy dress starting with the letter C. We have so many ideas for this consonant you will be spoilt for choice. If you are out for an evening of merriment why not cause a whole lot of carry on dressed in convict costumes? The black and white stripes were originally worn to make any escaped prisoner stand out in a crowd as will you in this bold statement costume. You may want to ruffle some feathers dressed as a chicken or look an absolute cracker out of season in Christmas themed fancy dress. Our cocktail of letter C costumes are a fantastic mixture of creative outfits and fun ideas.
Lady Zombie Convict Costume
£17.99 was £25.99 Show me more -
Carrie Costume
£14.95 Show me more -
Clone Trooper Commander Cody Costume
£21.95 was £41.99 Show me more -
Men's Flay Face Cenobite Costume
£36.95 was £44.99 Show me more -
Halloween Chef Costume
£9.95 was £46.49 Show me more -
Commander Cody Costume Clone Trooper (Deluxe)
£31.95 was £58.99 Show me more -
Chocolate Man & Oompa Loompa Combination
£59.99 Show me more -
Chimney Sweep Costume (AND BROOM)
£39.99 Show me more -
Bad Chemistry Cook Costume
£28.99 Show me more -
Gothic 'The Crow' Avenger Costume
£44.99 Show me more -
Christmas Cracker Costume
£29.99 Show me more -
Sexy Chinese Dragon Costume
£58.99 Show me more -
Christmas Cracker & Bauble Costumes Combo
£44.99 Show me more -
Inflatable Cock Fighter Costume
£39.99 Show me more -
Cigarette Outfit
£24.99 Show me more -
Child Catcher Costume
£34.99 Show me more -
Sinister Pierrot Clown Costume
£17.99 Show me more -
The Big Cheese Costume
£24.99 Show me more -
Jamaican Bobsleigh Costume
£39.99 Show me more -
The Dark Knight Rises Catwoman Costume
£30.99 Show me more


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Seeley G. Mudd Library (aka Tech Library), located on 2233 Tech Drive, contains over 345,000 books and bound journal volumes covering fields of science and engineering and subscribes to many journals in electronic or print format. Access to online resources is provided by the Astrahan Electronic Information Center [5], which includes public workstations, printers, laptop connections, and wireless network access.
In addition there are two other science and technical libraries on the Evanston campus covering materials in specific disciplines: the Transportation Library [6] (located on 1970 Campus Drive) and the Mathematics Library [7] (located in Lunt Hall). The Northwestern University Galter Health Sciences Library [8] is located on the Chicago campus.
[1] http://muddrooms.library.northwestern.edu/GroupView
[2] http://files.library.northwestern.edu/pdfs/MuddReserveRequestForm.pdf
[3] http://www.library.northwestern.edu/muddlibrary/online-resources
[4] http://libguides.northwestern.edu/home
[5] http://www.library.northwestern.edu/node/313/
[6] http://www.library.northwestern.edu/node/432/
[7] http://www.library.northwestern.edu/node/365/
[8] http://www.galter.northwestern.edu/