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- American Society of Plant Biologists
Cell and cell wall growth are mutually dependent processes that must be tightly coordinated and controlled. LRR-extensin1 (LRX1) of Arabidopsis thaliana is a potential regulator of cell wall development, consisting of an N-terminal leucine-rich repeat domain and a C-terminal extensin-like domain typical for structural cell wall proteins. LRX1 is expressed in root hairs, and lrx1 mutant plants develop distorted root hairs that often swell, branch, or collapse. The aberrant cell wall structures found in lrx1 mutants point toward a function of LRX1 during the establishment of the extracellular matrix. To identify genes that are involved in an LRX1-dependent developmental pathway, a suppressor screen was performed on the lrx1 mutant, and two independent rol1 (for repressor of lrx1) alleles were isolated. ROL1 is allelic to Rhamnose Biosynthesis1, which codes for a protein involved in the biosynthesis of rhamnose, a major monosaccharide component of pectin. The rol1 mutations modify the pectic polysaccharide rhamnogalacturonan I and, for one allele, rhamnogalacturonan II. Furthermore, the rol1 mutations cause a change in the expression of a number of cell wall–related genes. Thus, the lrx1 mutant phenotype is likely to be suppressed by changes in pectic polysaccharides or other cell wall components.
The plant cell wall is a rigid but pliable structure that confers protection, cell cohesion, and mechanical strength but is also important for the communication between individual cells. The main components of the primary cell wall of dicotyledonous plants are a cellulose-xyloglucan network considered to be the main load-bearing structure that is embedded in a matrix of pectic polysaccharides (Carpita and Gibeaut, 1993). The pectic matrix has three major components: homogalacturonan (HGA), rhamnogalacturonan I (RG I), and rhamnogalacturonan II (RG II) (Ridley et al., 2001). HGA is a homopolymer consisting of (1→4)-α-linked galacturonic acid, which is often partially methylesterified. Upon demethylesterification, HGA can be cross-linked by Ca2+, which promotes gel formation and cell wall rigidification. RG I is a rod-like heteropolymer with a backbone of repeating (1→2)-α-l-rhamnose-(1→4)-α-galacturonic acid disaccharide units containing numerous side chains attached to the rhamnose (Rha) residues, including galactans and arabinans. RG II is a complex but conserved heteropolysaccharide consisting of an HGA backbone decorated with four characteristic side chains composed of different monosaccharides. RG II can dimerize via the formation of borate diester links through apiose residues, contributing to the tensile strength of the wall (O'Neill et al., 2001). In addition to their effect on wall strength and cell adhesion, pectins also control wall porosity (Baron-Epel et al., 1988), which in turn regulates the mobility of cell wall modifying proteins and, thus, cell wall expansion. The porosity of the cell wall is thought to be influenced by RG I, a hypothesis that is corroborated by the association of RG I with cell wall growth (Ridley et al., 2001; Willats et al., 2001a; McCartney et al., 2003).
A sophisticated sugar biosynthetic machinery is required to synthesize the monosaccharides that form the cell wall carbohydrates (Reiter and Vanzin, 2001; Seifert, 2004). Pectins are synthesized in the Golgi apparatus by glycosyltransferases using nucleotide sugars as donor substrates (Scheible and Pauly, 2004). Many glycosyltransferases have been identified, but only a few are believed to be involved in pectin biosynthesis (Bouton et al., 2002; Iwai et al., 2002; Lao et al., 2003). Rha, a major component of RG I and RG II, has been hypothesized to be synthesized in Arabidopsis thaliana by a family of three highly similar Rhamnose Biosynthesis (RHM) proteins that convert UDP-d-Glc to UDP-l-Rha (Reiter and Vanzin, 2001). RHM2 was shown to be required for the biosynthesis of the pectinaceous seed coat mucilage mainly composed of RG I (Usadel et al., 2004; Western et al., 2004). Recently, B. Link and W.-D. Reiter (unpublished data) have demonstrated the biochemical activity of RHM1, with the in vitro conversion of UDP-d-Glc to UDP-l-Rha by recombinant RHM1.
During cell wall expansion, many polysaccharides need to be rearranged. This is conducted by a number of specific hydrolytic enzymes, such as the xyloglucan endotransglucosylases/hydrolases that act on xyloglucans and the polygalacturonases that act on pectins, and by other proteins, such as the nonhydrolyzing expansins (Cosgrove, 1999). Relatively little is known about the mechanisms that control cell wall expansion and assembly. Recently, proteins were identified that potentially function in this process. Cell wall–associated kinases (WAKs) were shown to be essential for cell wall expansion, since a reduction of WAK levels results in inhibition of cell elongation (Lally et al., 2001; Wagner and Kohorn, 2001). WAKs were found to bind the cell wall through pectin, establishing a physical link between the intracellular and the extracellular compartment that might serve as a signaling conduit (Wagner and Kohorn, 2001). Glycosylphosphatidylinositol-anchored proteins, such as COBRA, localize to the cell surface where they function in cell wall matrix remodeling. Mutations in COBRA strongly affect cellulose microfibril orientation (Roudier et al., 2005), and a general reduction in glycosylphosphatidylinositol-anchored proteins in the pnt1 mutant causes changes in the cellulose and pectin content and aberrant deposition of pectin, xyloglucans, and callose (Gilmor et al., 2005). Finally, arabinogalactan proteins (AGPs) are predicted to have adhesive and signaling properties since they can bind to pectins (Majewska-Sawka and Nothnagel, 2000; Willats et al., 2001a) and might also interact with WAKs. Cell wall properties, including extensibility, are also influenced by structural cell wall proteins that are oxidatively cross-linked in the extracellular matrix. Upon pathogen attack, wounding, mechanical stress, or after termination of cell growth, these proteins can be insolubilized to reinforce the cell wall and lock it in its final shape (Carpita and Gibeaut, 1993; Showalter, 1993; Ringli et al., 2001).
In Arabidopsis, we characterized LRR-extensin1 (LRX1), a gene involved in the regulation of cell wall formation. LRX1 is a member of a family of 11 genes coding for extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a structural extensin moiety at the C terminus (Baumberger et al., 2003a; Ringli, 2005). LRR domains are involved in protein–protein interactions and are found in many proteins playing a role in disease resistance, signaling pathways, or the regulation of extracellular enzymes (Forsthoefel et al., 2005, and references therein). The C-terminal moiety of LRX proteins is composed of [Ser-Hyp4]n repeats characteristic of Hyp-rich glycoproteins, which can modify the properties of the cell wall and might also play a role in connecting the cell wall with the plasma membrane by anchoring target proteins (Knox, 1995; Cassab, 1998). LRX1 is specifically expressed in root hairs and localizes to the cell wall where it is insolubilized (Baumberger et al., 2001). Root hairs are thin, long protrusions from specialized root epidermal cells (trichoblasts) that elongate by tip growth, a polarized form of cell growth (Foreman and Dolan, 2001). lrx1 mutant root hairs are short, form branches, swell, and frequently collapse (Baumberger et al., 2001). LRX1 synergistically interacts with its paralogous gene LRX2, and the lrx1 phenotype is enhanced in lrx1 lrx2 double mutants. Ultrastructural analysis revealed severe deficiencies in the cell wall architecture of lrx1 lrx2 double mutant plants, indicating a role of LRX1 and LRX2 during cell wall formation (Baumberger et al., 2003b).
To identify genes that are likely to be involved in the same developmental process as LRX1, we performed a suppressor screen on the lrx1 mutant. Here, we report the characterization of two allelic rol1 (for repressor of lrx1) mutants that compensate for the absence of LRX1. The ROL1 locus encodes RHM1, which is involved in the formation of UDP-l-Rha, and the rol1 mutations cause a modification of RG I and, in one rol1 allele, of RG II. Furthermore, the expression of several cell wall–related genes is altered in the rol1 mutants. Because l-Rha is an abundant component of pectic polysaccharides but absent from other cell wall components, our data suggest that LRX1 might be an extracellular regulator involved in the formation of the pectin matrix.
Isolation of Suppressors of the lrx1 Mutation
The lrx1-1s allele (subsequently referred to as lrx1) used for ethyl methanesulfonate (EMS) mutagenesis was produced by the excision of the En-1 transposon from the original lrx1-1 allele (Baumberger et al., 2001). The excision resulted in a deletion of 6 bp or, on the protein level, the deletion of two amino acids and a change in a third residue (Diet et al., 2004). This has a strong impact on LRX1 function and results in a mutant phenotype that is very similar to the initially isolated lrx1-1 allele containing the En-1 insertion (Baumberger et al., 2001). Twenty-three individual plants were isolated from the EMS-mutagenized M2 population that displayed a wild type–like phenotype. These mutants were called rol (for repressor of lrx1). Mapping and allelism tests revealed the presence of two allelic, recessive mutants, rol1-1 and rol1-2, that were selected for detailed analysis. The subsequently cloned ROL1 gene (see below) was sequenced in the remaining 21 rol mutants, but none of them showed a nucleotide change. Accordingly, we assume that no additional rol1 alleles are present in the rol mutant collection.
rol1 Mutations Suppress the lrx1 Root Hair Formation Phenotype
While wild-type seedlings display regular, thin, and long root hairs, lrx1 mutants are affected in root hair formation, with many short, collapsed, distorted, and sometimes branched root hairs (Baumberger et al., 2001; Figures 1A and 1B). By contrast, root hairs of lrx1 rol1-1 double mutants had a restored wild-type phenotype. Root hair length was comparable to the wild type, and branching or collapsing of root hairs was suppressed (Figure 1C). lrx1 rol1-2 seedlings were characterized by somewhat shorter root hairs compared with wild-type or lrx1 rol1-1 plants (Figure 1D). The length of root hairs was compared between the lrx1 rol1 mutants and wild-type plants. In lrx1 rol1-1 seedlings, root hairs were not significantly different from the wild type, but in lrx1 rol1-2, they were 32% shorter (Table 1 ). In addition, primary roots of lrx1 rol1-2 plants were shorter (Table 2 ), with an apparently higher root hair density compared with the wild type. To determine whether the latter phenotype is due to the formation of ectopic root hairs or to reduced cell expansion of trichoblasts of the root epidermis, we measured the length of wild-type and lrx1 rol1-2 trichoblasts. There was a 41% reduction in trichoblast length in lrx1 rol1-2 seedlings (0.115 ± 0.015 mm in lrx1 rol1-2 versus 0.195 ± 0.042 mm in the wild type), while the pattern of root hair formation appeared to be normal in the mutant. Thus, the higher density of root hairs in lrx1 rol1-2 plants can be explained by the reduced length of the trichoblasts.
To investigate the effect of the rol1 mutations on root hair development in the absence of the lrx1 mutation, the two lrx1 rol1 lines were backcrossed to wild-type Columbia (LRX1/LRX1) plants. For lrx1 rol1-1, only lrx1 and wild-type phenotypes were found in the F2 population derived from the backcross, whereas for lrx1 rol1-2, one-quarter of the F2 plants displayed the mutant phenotype of the lrx1 rol1-2 double mutant. This was confirmed in the F3 population of individual rol1-1 and rol1-2 single mutant lines. Hence, the rol1-1 mutation does not have an obvious effect on root hair development in the LRX1/LRX1 wild-type background, whereas the rol1-2 phenotype develops also in the absence of the lrx1 mutation and is thus epistatic to lrx1 (Figures 1E and 1F). In addition to the stunted root phenotype of rol1-2, both rol1 mutants revealed a mutant phenotype in cotyledons. Instead of a smooth cotyledon surface observed in wild-type seedlings, both rol1 mutants develop a rough surface. This phenotype is more pronounced in rol1-2 seedlings and is limited to cotyledons (see Supplemental Figure 1 online).
The rol1 Mutations Also Suppress the lrx1 lrx2 Double Mutant Root Hair Phenotype
LRX2 is the paralog of and synergistically interacts with LRX1 during root hair cell wall formation. Its overexpression can suppress the lrx1 mutant phenotype (Baumberger et al., 2003b). This prompted us to investigate the role of LRX2 in the suppression of the lrx1 mutant phenotype by constructing lrx1 lrx2 rol1 triple mutants. The lrx1 rol1 mutants were crossed with an lrx1 lrx2 double mutant to create triple mutants. While lrx2 single mutants are indistinguishable from the wild type, lrx1 lrx2 double mutants develop an enhanced lrx1 phenotype with very few root hairs (Baumberger et al., 2003b; Figures 2A to 2C). The rol1 mutations suppressed the lrx1 lrx2 double mutant phenotype to different extents. lrx1 lrx2 rol1-1 triple mutants displayed a phenotype that was intermediate between the lrx1 mutant and the lrx1 rol1-1 double mutant (Figures 2B, 2D, and 2E). The lrx1 lrx2 rol1-2 triple mutants developed root hairs that are characteristic of the rol1-2 mutant, even though not all root hairs were formed properly (Figures 2F and 2G). Although the addition of the lrx2 mutation decreased the effectiveness of rol1 suppression, the effect of the rol1 mutations is not dependent on LRX2.
Map-Based Cloning of the rol1 Gene
The rol1-1 and rol1-2 mutations were mapped to chromosome 1 south of nga111. The region containing the rol1 gene was delimited by the two markers uzu63 (BAC F3F9; position 38,200) and uzu58 (BAC F9K20; position 60,500) for rol1-1 and uzu67 and uzu68 (BAC T30F21; positions 29,700 and 58,300, respectively) for rol1-2 (Figure 3A ). The genes encoded in this region were sequenced, and for both rol1-1 and rol1-2, a point mutation was identified in the RHM1 gene (At1g78570) (Figure 3B). To demonstrate that the mutations found in the rol1 alleles are responsible for the suppression of the lrx1 phenotype, both lrx1 rol1-1 and lrx1 rol1-2 plants were transformed with a 4.2-kb genomic fragment of the RHM1 gene containing the coding region, 1.5 kb of upstream promoter sequence, and 600 bp of 3′ noncoding sequence. Of each lrx1 rol1 mutant, 10 independent primary transformants (T1) were propagated to the T2 generation. The T2 populations segregated in a 3:1 ratio for the lrx1 to lrx1 rol1 phenotype. A DNA gel blot experiment demonstrated that the lrx1 phenotype cosegregated with the presence of the T-DNA (data not shown). The T2 seedlings developing the lrx1 root hair phenotype also displayed a reversion of the cotyledon and, in transgenic lines of the lrx1 rol1-2 genetic background, restoration of root length to that of lrx1 and wild-type seedlings (Table 2). This demonstrates that the presence of a wild-type copy of RHM1 in either lrx1 rol1-1 or lrx1 rol1-2 mutants restores the original lrx1 phenotype (abnormal root hair development, wild-type cotyledons, and wild-type root length) and, hence, that the suppression of the lrx1 phenotype in the lrx1 rol1 mutants is caused by the mutant RHM1 gene.
RHM1 belongs to a subclass of the short chain dehydrogenase/reductase family of enzymes and, together with the highly similar RHM2 and RHM3, has been hypothesized to catalyze the conversion of UDP-d-Glc to UDP-l-Rha (Reiter and Vanzin, 2001). RHM1 is predicted to consist of an N-terminal dehydratase and a C-terminal epimerase/reductase domain, and its proposed activity has recently been demonstrated by assaying the recombinant enzyme in vitro (B. Link and W.-D. Reiter, unpublished data). rol1-1 contains a G-to-A mutation that results in the introduction of a stop codon near the end of the dehydratase domain at Trp-318. The rol1-2 mutant harbours a G-to-A mutation that leads to the replacement of Arg-283 by Lys (Figure 3B).
To investigate the effect of the rol1 mutations on RHM1 expression, quantitative real-time RT-PCR analysis was performed on wild-type, lrx1, lrx1 rol1-1, and lrx1 rol1-2 seedlings. Root RNA was used as starting material, and expression levels were normalized against ACTIN2 expression as an internal standard. Compared with the wild type and lrx1, which have comparable expression levels, the expression of the RHM1 gene was found to be barely detectable in lrx1 rol1-1 plants (RHM1 message was only detected in one of three experiments) but was present at wild-type levels in lrx1 rol1-2 (Figure 4A ).
To determine the tissue specificity of RHM1 expression in Arabidopsis seedlings, 1.5 kb of the RHM1 promoter was fused to the β-glucuronidase reporter gene (uidA, subsequently referred to as GUS) and transformed into wild-type Columbia plants. Eight individual T1 transgenic plants were obtained and grown to the T2 generation. Whole seedlings were stained for GUS activity, which was found in all tissues (i.e., root including root hairs, hypocotyl, and cotyledons). In weakly expressing lines, mainly the vascular tissue showed activity, whereas in plants with higher GUS expression levels, whole cotyledons, the hypocotyl, and the root were stained (Figures 4B to 4D). The RHM1 expression pattern deduced from the promoter:GUS fusion experiment is consistent with the mutant phenotypes found in the roots and cotyledons of both rol1 alleles.
rol1 Mutations Have a Deleterious Effect on the Dehydratase Activity of RHM1
The effect of the point mutations in the rol1 mutants on the enzymatic activity of RHM1 was tested by an in vitro enzyme assay. Because expression of full-length wild-type RHM1 in Escherichia coli yielded very little soluble protein, the dehydratase domain of RHM1 (RHM1-D) was expressed separately and shown to catalyze the conversion of UDP-d-Glc to the reaction intermediate UDP-4-keto-6-deoxy-d-Glc, which was quantified by gas chromatography–mass spectrometry (GC-MS) (B. Link and W.-D. Reiter, unpublished data). Subsequently, the dehydratase domain of RHM1 encoded by rol1-1 and rol1-2, referred to as ROL1-1-D and ROL1-2-D, were expressed in E. coli and tested for their in vitro activity. Since rol1-1 contains a stop codon at position 318, ROL1-1-D was 43 amino acids shorter than RHM1-D. Although both ROL1-D proteins were successfully expressed, they failed to produce detectable amounts of UDP-4-keto-6-deoxy-d-Glc, whereas a wild-type control yielded the expected intermediate (data not shown). Thus, both rol1-1 and rol1-2 encode proteins with a dehydratase domain that is inactive in vitro, suggesting that both proteins fail to convert UDP-d-Glc to UDP-l-Rha in vivo.
Monosaccharide Composition of the Extracellular Matrix
Because the rol1 mutations affect the biosynthesis of Rha, the abundance of Rha in cell wall material may be reduced. To test this possibility, the composition of the cell wall of root tissue of wild-type and mutant seedlings was determined. Although we did not find any significant differences (P < 0.1) in the amounts of the major sugars of cell wall material, including Rha (Figure 5A ), rol1-2 and lrx1 rol1-2 showed an ∼30% reduction in the RG II–specific monosaccharides 2-O-methyl-d-xylose and 2-O-methyl-l-fucose (Figure 5B). These methylated sugars are diagnostic components of the two large Rha-containing side chains of RG II, and their reduced abundance may reflect a defect in the synthesis of RG II. The relative amount of both 2-O-methyl sugars was not significantly different between rol1-1 and wild-type lines.
rol1-1 and rol1-2 Mutants Form Aberrant RG I
Although no significant alteration in the major sugars of cell wall material was detected in the rol1 mutants, it is possible that the structure of individual components of the cell wall is modified or that only particular tissues are affected. To test this, monoclonal antibodies against specific cell wall components were used to provide information about cell wall composition in planta. The absence of immunolabeling can indicate either the absence or masking of the epitope. Therefore, root transverse sections of wild-type, lrx1, lrx1 rol1, and rol1 single mutant seedlings were immunolabeled with antibodies that specifically detect different carbohydrate epitopes, mainly of pectin. The antibodies CCRC-M1 (Fuc-Gal side chains of xyloglucan; Puhlmann et al., 1994), JIM5 and JIM7 (highly and partially methylesterified epitope of HGA, respectively; Knox et al., 1990), LM2 (AGP side chains with GlcA; Yates et al., 1996), CCRC-M7 (AGPs; Steffan et al., 1995), and LM6 (arabinan side chains of RG I; Willats et al., 1998) showed no difference in labeling intensity (data not shown). A strong reduction in labeling was observed in lrx1 rol1-1 and lrx1 rol1-2 plants compared with the wild type or lrx1 with the LM5 antibody that detects the (1→4)-β-d-galactan side chains of RG I (Jones et al., 1997) (Figure 6 ). No difference in staining was observed between the wild type and lrx1. A comparison of wild-type and rol1 single mutants confirmed this observation (data not shown). In wild-type root tissue of the root hair–containing region, LM5 bound to walls of the cortex, endodermis, and stele but not to epidermal cells, including root hair forming cells (indicated by arrows in Figure 6). This is in line with previous findings that LM5 does not bind epidermal cells in root hair developing zones of the root (Willats et al., 2001b; McCartney et al., 2003).
rol1-1 and rol1-2 Have Different Effects on the Genome-Wide Gene Expression Profile
A microarray analysis of gene expression in roots of wild-type, lrx1, and the two lrx1 rol1 suppressor mutants was performed to assess the gene expression profiles (see Supplemental Table 1 online). ATH1 Arabidopsis whole-genome chips from Affymetrix containing ∼23,000 genes were used for hybridization with total root RNA. The microarray experiments were performed in biological triplicates, the data were analyzed as described in Methods, and induction/repression by a factor of 2 was chosen as the threshold value. First, the lrx1 mutant was compared with wild-type Columbia. No significant change in gene expression between these two lines could be found. Thus, the dramatic morphological difference between the wild type and lrx1 is not reflected by an extensive change in the gene expression profile.
In the next step, lrx1 and the two lrx1 rol1 mutants were compared. Considering the ubiquitous expression of RHM1 in roots, it was surprising not to find a single significant change in gene expression between lrx1 and lrx1 rol1-1. By contrast, the rol1-2 mutation produced a number of changes, with 38 induced and 19 repressed genes in lrx1 rol1-2 compared with lrx1 (Table 3 ).
Interestingly, genes with known or predicted functions in cell wall development (indicated in bold in Table 3) were highly abundant in this collection (23 out of 57, or 40%). We further investigated this set of 23 cell wall–related genes in the lrx1 rol1-1 line using gene set enrichment analysis (GSEA; see Methods). This analysis determines whether the coordinate differential regulation of this set of genes was also found in lrx1 compared with lrx1 rol1-1 and whether the change in expression was significantly more consistent than the differences in expression seen in a randomly selected gene set of similar size. This subset was found significantly enriched in the set of regulated genes between lrx1 and lrx1 rol1-1 mutants (enrichment score = 0.89; P value < 0.005). This finding indicates that the cell wall–related genes modified in their expression by the rol1-2 mutation are also affected in the rol1-1 mutant, although in a subtler way. This shows that rol1-1 and rol1-2 have overlapping effects on gene expression.
Suppressor screens are valuable for identifying genes that are involved in the same process and can reveal relationships between genes that would not have been established by other methods (Huang and Sternberg, 1995; Prelich, 1999). They also allow for the identification of mutants such as rol1-1 that would not have been detected in normal forward genetic screens due to the absence of a visible phenotype. The lrx1 mutant, which is defective in root hair cell wall formation (Baumberger et al., 2001, 2003b), was EMS mutagenized, and rol mutants were identified that revert the lrx1 phenotype to the wild type and are thus likely to encode proteins that are related to LRX1 function.
Modification of Rha Biosynthesis Suppresses the lrx1 Phenotype
RHM1 codes for a 668–amino acid Rha biosynthetic protein that is part of the sugar interconversion pathway required for the synthesis of cell wall monosaccharides (Reiter and Vanzin, 2001; Seifert, 2004). RHM1 belongs to a subclass of the NAD(P)-dependent short chain dehydrogenase/reductase family of enzymes. Short chain dehydrogenase/reductase proteins share motifs such as the Rossmann-fold for cofactor binding and a highly conserved sequence motif (YxxxK), which is part of the catalytic triad (Kleiger and Eisenberg, 2002; Allard et al., 2004). In Arabidopsis, the three highly similar isoforms RHM1-RHM3 were identified as likely UDP-l-Rha synthases (Reiter and Vanzin, 2001), and RHM1 was shown to convert UDP-d-Glc to UDP-l-Rha (B. Link and W.-D. Reiter, unpublished data). Further evidence for a function of RHM proteins as UDP-l-Rha synthases is provided by rhm2 mutants that have a reduced amount of the pectinaceous seed coat mucilage mainly consisting of RG I (Usadel et al., 2004; Western et al., 2004). Since the cell wall composition of other tissues appears not to be affected in rhm2 mutants, the three RHM genes seem to be redundant in most parts of the plant. This conclusion is corroborated by the expression of all RHM genes in stems, roots, leaves, inflorescences, siliques, and seedlings as determined by RT-PCR (Usadel et al., 2004; Western et al., 2004). The RHM1 promoter:GUS analysis indicates that RHM1 is expressed in all tissues of seedlings. This expression pattern is supported by the observed rol1 mutant phenotypes in seedling roots, root hairs, and cotyledons.
The mutant version of RHM1 encoded by rol1-1 is truncated, and both rol1-1 and rol1-2 mutations lead to an inactive dehydratase domain in vitro. Furthermore, the expression level of rhm1 in rol1-1 mutants is very low, presumably due to nonsense-mediated mRNA decay. Thus, the lack of Rha biosynthesis through RHM1 causes the suppression of lrx1. Both rol1 mutants, however, show wild-type Rha levels in total cell wall material from roots, indicating that RHM2 and RHM3 are sufficient to compensate for the lack of functional RHM1. Nevertheless, RG I and RG II are affected in the rol1 mutants, suggesting a link between individual RHM isozymes and particular glycosidic linkages formed by Rha with other sugars, which could explain the modification of RG I and RG II in rol1 plants despite normal Rha levels.
rol1-1 and rol1-2 Display Different Phenotypes
Although both rol1 mutations suppress the lrx1 mutant phenotype, the two mutations have different effects on plant development. rol1-2 single mutants develop shorter root hairs and roots compared with the wild type, a phenotype that is not observed in rol1-1 plants. The microarray experiments revealed 57 genes with changes in the expression level by a factor of ≥2 in the lrx1 rol1-2 mutant compared with lrx1, while the effect of the rol1-1 mutation on gene expression is much more subtle. Thus, rol1-1 and rol1-2 are phenotypically distinct both on the morphological and the molecular level. The moderate influence of rol1-1 suggests that this mutation mainly affects the cell wall structure, whereas the effect of rol1-2 is more dramatic and might also include other processes besides the biosynthesis of Rha. In the rol1-1 mutant, only a truncated protein can be produced, which is enzymatically inactive at least in vitro. By contrast, the rol1-2 mutant encodes a full-length protein with an Arg-to-Lys substitution in the dehydratase domain. The mutated Arg residue is completely conserved between the RHM proteins of plants and dTDP-glucose 4,6-dehydratases from prokaryotes and has been shown to interact with the diphospho group of dTDP-glucose (Allard et al., 2004). Accordingly, this residue may be essential for substrate binding. The dehydratase domain encoded by rol1-2 is inactive in vitro, but the mutant protein is likely to be stable. It is possible that it binds to potential in vivo interaction partners of wild-type RHM1 and locks the protein complex in an inactive state, which has a stronger impact on plant development and thus explains the phenotypic differences between the two rol1 alleles.
Among the 38 genes induced in lrx1 rol1-2 compared with lrx1, 20 genes (>50%) are presumed to be involved in cell wall–related processes. This is >5 times more than the estimated 2000 genes corresponding to <10% of the Arabidopsis genome that are predicted to have such a function (Yong et al., 2005). Among the 19 genes repressed in lrx1 rol1-2, three genes (16%) are presumed to be cell wall related. The cell wall alterations induced by the rol1-2 mutation seem to preferentially affect the expression of cell wall–related genes, which presumably reflects the ability of the plants to respond to alterations in the extracellular matrix. Although the changes in gene expression are more subtle in the lrx1 rol1-1 mutant, they affect the same cell wall–related genes that are differentially regulated in the lrx1 rol1-2 line.
Of the 20 cell wall–related genes induced in lrx1 rol1-2 compared with lrx1, seven are involved in the modification of xyloglucan or pectic polysaccharides. Four genes encode structural cell wall proteins (one Pro-rich protein and three extensins), which may strengthen the cell wall (Carpita and Gibeaut, 1993; Cassab, 1998). It remains to be shown whether the changes in gene expression are the cause of the visible phenotypes of rol1-2 plants. Since these modifications in gene expression are also induced by the rol1-1 mutation, even though to a lesser extent, they may contribute to the suppression of the lrx1 root hair phenotype. Higher expression of extensin genes might help to stabilize the aberrant and weakened root hair cell wall of the lrx1 mutant and thus prevent bulging and collapsing of the root hair structure. LRX6, a member of the LRX gene family, is also upregulated in the lrx1 rol1-2 line compared with lrx1. LRX6 is specifically expressed during lateral root development in wild-type plants (Baumberger et al., 2003a) and is not known to influence root hair formation. By contrast, LRX2, the paralog of LRX1, is not induced in the rol1 mutants and does not appear to play a role in the suppression of the lrx1 phenotype by the rol1 mutations. We were surprised to find no changes in gene expression between wild-type and lrx1 plants; however, trichoblasts account for only a small fraction of the total number of cells in the root (Dolan et al., 1994). For this reason, changes in gene expression in only this cell type might not be detectable. Alternatively, small changes in expression levels might be masked by the biological variance in gene expression (Hudson et al., 2003).
rol1 Suppressors Indicate a Possible Role of LRX1 during Pectin Matrix Formation
The involvement of the RHM proteins in pectin formation (Usadel et al., 2004; Western et al., 2004), the modifications of pectin in rol1 mutants, and the altered expression of a number of cell wall–related genes in the rol1 mutants suggest that structural changes in the cell wall cause suppression of the lrx1 mutant phenotype. Our data indicate a reduction in the two large Rha-containing side chains of RG II in the rol1-2 allele. This modification might account for the stunted root phenotype of the rol1-2 mutant, as changes in the RG II structure can affect plant growth (O'Neill et al., 2001). Our immunolocalization data indicate that both rol1 alleles contain modified RG I. Even though the effects of the rol1 mutations on root hair formation suggest an alteration of pectin in this cell type, the molecular basis of this modification is unknown. The LM5 antibody does not bind to root hair cells, possibly due to masking of the epitope. Alternatively, an unrecognized structure of pectin could be affected. Suppression of the lrx1 phenotype through modifications of pectin points toward a possible function of LRX1 in a pectin-related process. In future experiments, it will be useful to investigate whether the rol1 and lrx1 mutants change the pectin structure of root hairs and whether, for example, pore sizes might be affected in these lines. A method to measure porosity of pectin has been established for Chenopodium album (Fleischer et al., 1998) and can most likely be adapted for Arabidopsis to investigate this point. In a complementary approach, Fourier transform infrared spectroscopy (McCann and Carpita, 2005) might reveal changes in the molecular structure of cell walls of the different mutants. Another possibility is that the changes in gene expression caused by the rol1 mutations are the basis of suppression of the lrx1 phenotype. For example, the increased expression of structural cell wall proteins may lead to a stabilization of the weakened root hair cell wall of the lrx1 mutant. This hypothesis can be tested by analyzing plant lines that are mutated in these genes or that overexpress the respective proteins.
Plant Material, EMS Mutagenesis, and Mapping
The lrx1-1s allele and the EMS mutagenesis procedure used for this mutant line are described by Diet et al. (2004). The lrx1 single and lrx1 lrx2 double mutants are in the Columbia genetic background. For vertical growth on plates, seeds were surface sterilized with a solution of 1% sodium hypochlorite and 0.03% Triton X-100, stratified 3 to 4 d at 4°C, and grown in a vertical orientation on the surface of half-strength MS medium containing 0.6% phytagel and 2% sucrose (Sigma-Aldrich) with a 16-h-light/8-h-dark cycle at 22°C. For crosses and propagation of the plants, seedlings were transferred to soil and grown in growth chambers with a 16-h-light/8-h-dark cycle at 22°C. Light microscopy observations were done on 4-d-old vertically grown seedlings with a Leica LZ M125 stereomicroscope. For measurements of the lengths of trichoblasts and root hairs, 50 cells of the mature root of more than five different seedlings per plant line were used. For the root length measurement, >30 roots per plant line were measured.
Molecular markers for all mutations were established to confirm the genotype of the different lines. The marker for lrx1 is described by Diet et al. (2004). The lrx2 mutation used in this study is a footprint allele (lrx2-2) caused by the excision of the En-1 transposon initially inserted at position 1478 of the coding region. The excision of En-1 resulted in the insertion of 4 bp (GTAC) and a frame shift in the beginning of the extensin coding region. On the DNA level, an RsaI restriction site polymorphism was introduced, which allows for the detection of the lrx2-2 mutation. For rol1-1 and rol1-2, CAPS markers were established using the primers 5′-ACTCCGGGTTCTGTGGGTAC-3′/5′-GATGTTGCCAAAGACATCTGC-3′ for rol1-1 and 5′-GTACCTCTGATCGTTAAAACGT-3′/5′-TTGTTCTTCACAAGGGAGAAG-3′ for rol1-2. The mismatches in the primers (underlined positions) introduce a KpnI site in wild-type DNA but not rol1-1 and an AclI site in rol1-2 but not wild-type DNA.
For mapping, the lrx1 rol1 mutants were crossed with Landsberg erecta (Ler) and propagated to the F2 generation. The F2 population containing the rol1-2 allele was selected for seedlings displaying the rol1-2 phenotype. Nine hundred mutant F2 seedlings were subsequently used for mapping the rol1-2 locus to <40 kb. For rol1-1, 500 F2 seedlings displaying a wild-type root hair phenotype were selected and screened by PCR for homozygous lrx1 mutant plants. These plants were assumed to be homozygous mutant for rol1-1 and were thus used for initial mapping. Once the approximate map position of rol1-1 was identified, F2 plants displaying an lrx1 mutant phenotype (i.e., being homozygous mutant lrx1) were selected, and those heterozygous Columbia/Ler in the region containing the rol1-1 locus were propagated to the F3 generation. As expected, seedlings of the F3 population segregated 3:1 for lrx1 versus wild-type root hairs. Five hundred wild type–like F3 seedlings were selected for detailed mapping. Mapping was performed using standard SSLP and CAPS markers developed based on the Columbia/Ler polymorphism databank (Jander et al., 2002).
Constructs and Plant Transformation
For the RHM1 promoter:GUS fusion construct, 1.5 kb of the promoter region was amplified by PCR using the primers RHM1GUSF 5′-GGATGTCGACGTATGAGTCTGTTG-3′ and RHM1GUSR 5′-TCGAAGTCGACGTGGAGTGAGTCT-3′, and the resulting fragment was digested with SalI for cloning into the pGPTV-Kan plant transformation vector cut with the same enzyme (Becker et al., 1992). For the RHM1 genomic clone used for the complementation test, 4.2 kb containing 1.5 kb of the promoter region, the coding region, and 600 bp of 3′ region were amplified by long-range PCR using the oligos RHM1genoF 5′-TCATGCGGCCGCGACCGAAGACCACCT-3′ and RHM1genofR 5′-ACGAGCGGCCGCAACGAGGAACGAA-3′. The PCR product was cloned into the TOPO 10 blunt end cloning vector (Invitrogen) for control sequencing and then cloned into the pART27 plant transformation vector (Gleave, 1992) by digesting with NotI. The binary vectors were transformed by electroporation into Agrobacterium tumefaciens GV3101. Plants were transformed by the floral dip method described by Clough and Bent (1998), and transgenic T1 plants were selected on 50 μg/mL kanamycin. The presence of the transgene was confirmed using primers specific for the kanamycin resistance gene NPTII. GUS staining was performed in 50 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 0.1% Triton X-100, and 1 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid for 3 h at 37°C.
RNA Isolation and RT-PCR Analysis
Two hundred seedlings per plant line were grown in a vertical orientation on half-strength MS plates, and root total RNA was extracted using the TRIzol method (Gibco BRL). Ten micrograms of each RNA sample were reverse transcribed using oligo(dT) and a Superscript II RNase H reverse transcriptase kit (Invitrogen) following the manufacturer's recommendations. Quantitative real-time PCR was performed with an ABI PRISM 7700 sequence detection system (Applied Biosystems) using a SYBR Green PCR kit from Applied Biosystems and primers specific for the RHM1 gene. Relative mRNA abundance was calculated using the comparative Δ-Ct method and normalized to the corresponding ACTIN2 transcript levels.
Expression of Recombinant Protein and Activity Assay
The RHM1-D constructs were amplified by the primers 5′ Nhe I, 5′-TAAGTGCTAGCATGGCTTCGTACACTCCCAAGAACATTC-3′, and 3′ Bam HI, 5′-GATAAGGATCCTCAGTGGTGGTGGTGGTGGTGAGATAAAGTAGCTGCCAGCGAATTGTCCTC-3′, and cloned into the vector pET11a (Novagen) digested with NheI and BamHI. A His tag was added by the 3′ Bam HI primer, allowing the protein to be partially purified using standard nickel-nitrilotriacetic acid agarose columns. For protein expression, the constructs were transformed into Rosetta DE3 cells (Novagen). Protein expression and enzymatic analysis were performed as described by B. Link and W.-D. Reiter (unpublished data). In summary, bacterial cultures were grown to OD600 ≥ 0.6, and protein expression was induced with 0.3 mM isopropylthio-β-galactoside. The protein extract was purified over a Ni-NTA column (Qiagen), and protein activity was assayed in 20 mM glycine buffer, pH 8.5, 1 mM NAD+, and 1.6 mM UDP-glucose using 0.5 to 1 μg protein. After stopping the reactions, nucleotide sugars were hydrolyzed by adding an equal volume (100 μL) of 4 M trifluoroacetic acid and incubating them at 90°C for 30 min. The hydrolysate was mixed with 0.25 mL of 2 M ammonia and 4% NaBH4 and incubated at 40°C for 1 to 2 h. Next, 0.25 mL of acetic acid, 1 mL DMSO, 0.25 mL 1-methylimidazole, and 4 mL of acetic anhydride were added to the reactions to convert the reduced sugars to alditol acetates. The alditol acetates were extracted from the aqueous solutions using 1 mL dichloromethane, and the latter was used for injection into a 30-m SP-2330 column with 0.20-μm film thickness (Supelco) for analysis by GC-MS in selected ion monitoring mode. RHM1-D protein produced robust signals (>3 μM of product) in 20 min. Since the solubility of the truncated RHM1-D protein encoded by rol1-1 was very low, the protein was also expressed without a His tag, and bacterial extracts were used for the analysis. The result was the same for both approaches.
Preparation of Cell Wall Material and Sugar Composition Analysis
Cell wall material was prepared from roots of 1-week-old seedlings. Plant tissue was frozen in liquid nitrogen and macerated in 70% ethanol (aqueous) using a Retschmill (Retsch). The ground tissue was pelleted by centrifugation, and the resulting pellet washed with a 1:1 (v/v) mixture of chloroform and methanol. The pellet was then washed twice with acetone and dried. The cell wall material was hydrolyzed using 2 M trifluoroacetic acid, and the solubilized monosaccharides were converted into their corresponding alditol acetates followed by GC-MS analysis, and the uronic acid content was determined using the m-hydroxy-biphenyl assay as described by Usadel et al. (2004). The 2-O-methyl-sugars were quantified by selective ion monitoring at a mass-to-charge ratio of 117 with hydrolyzed 2-O-methyl-β-d-galactopyranosyl-(1→4)- d-glucose (Sigma-Aldrich) as an internal standard.
Reflection microscopy of silver-enhanced, immunogold-labeled, resin-embedded tissue sections was performed as described previously (Bush and McCann, 1999). All treatments of the wild type and mutants, including image acquisition and processing, were performed in parallel.
Microarray Analysis
To obtain total RNA, Columbia, lrx1, and the rol mutants (in the lrx1/lrx1 mutant background) were grown in a vertical orientation on half-strength MS medium, and root tissue was collected. RNA was extracted by the TRIzol method (Gibco BRL), further purified using the RNeasy kit (Qiagen), and tested for degradation by a lab on a chip analysis (Agilent Technologies). Double-stranded cDNA was synthesized with the SuperScript kit (Invitrogen) using 20 μg total RNA as starting material. Purification was done with the Affymetrix GeneChip sample cleanup module (Affymetrix). The probe was labeled with the ENZO-BioArray labeling kit (Loxo) and finally purified again with the Affymetrix GeneChip sample cleanup module. Hydrolysis of the labeled RNA and chip hybridization were performed as recommended by Affymetrix. All the experiments were performed in biological triplicates. After hybridization and scanning, probe cell intensities were calculated with Affymetrix MAS5 software (Affymetrix). Summarization and normalization for the respective probe sets were performed using dCHIP software (Li and Wong, 2003). Genes were filtered out from the resulting lists of normalized expression values in Genespring 7.2 when not showing present calls in all replicate measurements of at least one condition. Subsequently, the significance of changes in expression was tested in Genespring by an equal-variance t test, applying a Benjamini-Hochberg correction (Benjamini and Hochberg, 1995) with a false discovery rate of 0.05.
GSEA was performed as described by Subramanian et al. (2005). GSEA provides an enrichment score that measures the degree of enrichment of the gene set at the top (upregulated in lrx1 versus lrx1 rol1-1) or bottom (downregulated in lrx1 versus lrx1 rol1-1) of a rank-ordered gene list derived from the data set. The nominal P value is used to assess the significance of the enrichment score.
Accession Numbers
Sequence data from this article can be found in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MEXP-722. The locus identifiers are At1g12040 (LRX1) and At1g78570 (RHM1).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Cotyledon Phenotype of rol1 Mutants.
Supplemental Table 1. Effect of lrx1 and lrx1 rol1 Mutations on Gene Expression.
We thank Andreas Patrigniani for technical assistance with the microarray analysis, Julia Schönfeld for the monosaccharide quantification, Kim Findlay and Susan Bunnewell for technical help with tissue sectioning, and Beat Keller for critical reading of the manuscript. This work was supported by Swiss National Science Foundation Grants 31-61419.00 and 3100A0-103891, by a Nachwuchsforscher-Stipendium of the University of Zürich (560027), and by the Functional Genomics Center (A.D. and C.R.). It was also supported by Biotechnology and Biological Science Research Council Grant 208/D10332 and European Union Grant QLK5-CT-2001-00443 (G.J.S.), by U.S. Department of Energy Grant DE-FG02-95ER20203 (W.-D.R.), and by the German Ministry for Science and Education GABI Grant 0312277D (M.P.).
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Christoph Ringli (chringli{at}botinst.unizh.ch).
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.038653.
↵[W] Online version contains Web-only data.
- Received October 12, 2005.
- Revised May 4, 2006.
- Accepted May 15, 2006.
- Published June 9, 2006.


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Do'nt forget that the french system is different than the anglo-saxon one, where Federations are managing their clubs but are also bringing money at home...
So there is a tough battle between the FFR (you know, the historical shadowmen and ex-players, also called the "old dusty overcoats") who lives and depends for the most of the money of the clubs, but got the executive power (even to spoil LNR ballot paper on the ERC election) and which is close to the celts and to old amateur world and their special privilege of "rugby values" or "highest interests of rugby"...
And on the other side, the LNR that brings ALL the money on the table, divided between a minority of pro-FFR (Serge and Clermont representatives essentially) and a fragile majority of modern CEO (Django Boudjellal and Calamity Jacky for example) and businessmen that would like to be unchained of the FFR's slavery and would like little more money in rugby (but not as much as football, just x2 ou x3) and less amateur rules and management.
I understood the english battle between clubs and RFU are quite the same than the french, but final interests are a bit different, I think ?
The celts problem is they'd like to survive in a Grand Circus, but the actual system made it impossible without special privileges that are unfair for english and french.
I think the present problem, even for the Celts, is the ERC/IRB old Cartel and godfathers who still want to live on their thrones with 19th century rules.
But that the french and english clubs should be released of the cowshed to the corral. With fences. But no ropes anymore.

subset: commoncrawl


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Shalimar Paints Ltd
Shalimar Paints Ltd, established in 1902, one of the first paint company in India and South East Asia, embarked on an initiative to paint smiles by taking up the task to transform Sanjay camp, Chanakyapuri into Delhi`s first artistic slum. In association with Button Mushrooms, students of fine art and social service societies from Delhi University and volunteers not only made the Sanjay Camp colourful but also artistic with their creativity.
There is so much a colour can do, it gives black and white a new life, adds happiness to sorrow. We at Shalimar Paints believe a little colour definitely makes a lot of difference may it be a feeling of joy, an identity and sense of change, clean and vividness. Kachi Gali – 'Rang Desh ke', was an attempt to galvanize today's youth to bring alive this thought using their talent and passion to bring a change in the society- adding an artsy colourful touch to Swachh Bharat Campaign. As a part of the initiative, primary school`s walls were also covered in artistic murals so that more and more kids get encouraged to come to school. Happy with the transformation of the camp few residents remarked, "It feels Diwali festivities have begun and we just need to light up crackers," while others who were returning from their work chores said, "It feels we have come back to a different place."
The two-day activity held on 17th-18th March 2018 at Sanjay Camp saw a large-scale participation from the student and societies of Delhi University, where along with students even local dwellers came together to beautify their own vicinity. Speaking about the initiative, Mr. Anmol Gagroo, Head – Marketing, Shalimar Paints Ltd, said – "We strongly believe today's youth has an infinite potential and Kachi Gali is an example of that. In 48 hrs, 350+ volunteers painted over 750 households covering over 1000+ walls and a primary school. The artists created over 200+ graffiti and countless smiles. In future, you are definitely going to see more of such activities."
Ayushi Mukherjee Brave,
|Image Caption : Beautifying the walls of Kachigali
|Click for Real Image
|Image Caption : Graffiti in making
|Click for Real Image
|Image Caption : Graffiti on women safety
|Click for Real Image
|Image Caption : Painting relevant graffiti inside the school's wall
|Click for Real Image


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Mark you calendars now for FIESTA: Celebrating 10 years of Exploring the Other Side of Nowhere at Big Bend Ranch State Park! CDRG is proud to host this celebration in honor of the 10th year anniversary of the official public opening of Big Bend Ranch State Park and our 10th year as the official friends group for the largest state park in Texas. Fun, Fun, Fun is in store for guests with guided hikes, bike rides, horseback rides, jeep tours, birding activities, vendors, entertainment, barbecue, and so much more. The Fiesta event is open to everyone, but registering now sure helps with the planning!
Bring your friends and family to enjoy the day because Life's Better Outside!


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First, let's get this out of the way: I am accident prone. Seriously.
So that said, and with the acknowledgement that the following post really IS TMI in content and is not for the faint of heart, please keep reading at your own risk.
I started my period the other day. I was happy when it was on time (because it hasn't been, ever, since I've moved back to Kentucky and I was beginning to wonder if Florida was some special place where my period was always regular and the sun was always shining). I put in a tampon, like I do, and went about my day. But, see, the thing about periods, is that I've been having them and using tampons so regularly throughout my life that I don't really think about it anymore. I just put them in, change them, get on with my life, no problem. So with this one, I could remember putting one in, I could remember taking one out, and I thought I could remember not replacing the last one with another one because the flow was so light that I didn't think it was needed.
But I wasn't certain about that. I was fairly certain. But not 100 percent certain. So when Lord Ormsby and I had sex that evening, I just took it for granted that I hadn't replaced the tampon and that the only thing that would be keeping company in my vagina that evening was Lord Ormsby himself. I wasn't worried. Not at all. I mean it's hard to worry when you're having multiple orgasms and can't remember your own name.
Until we were finished. Because when I looked at the trash can in the bathroom, I saw TWO tampon wrappers. I didn't remember putting two in. I only remembered putting one in and taking one out. Frantically I dug through the trash can looking for two tampons to match the two wrappers. I found only one.
Not gonna lie. I panicked a little bit. Checked all the trash cans in the house. Still didn't find it. I panicked more… because if a second used tampon was not in the trash can, in my mind, that could only mean that it had gotten pushed way, way, way back in my vagina. I dug around in there as far as I could reach. I couldn't feel anything, but you know, that's still not the most comforting thing in the world because it's not like I can just pry it open and look in there myself.
We don't have a bathtub, but Lord Ormsby, who is kind of a MacGyver in that he can rig up pretty much anything, created a system in the shower stall so that it would fill up enough to maybe help me saturate that thing with enough water to make it expand so I could fish it out. It sort of worked, but I still couldn't find it. He offered to create a speculum out of spatulas from the kitchen and his flashlight and look for it himself, but I declined. I'm cool with him MacGyvering a lot of things, and he's good at MacGyvering up a lot of things… but I was NOT going to let him MacGyver my vagina.
This was a big fucking deal. Because Toxic Shock Syndrome is a BIG FUCKING DEAL. It's a rare bacterial infection that can be caused by the extended wear of a tampon. It can be fatal, quickly, and losing a tampon in my vagina is just the sort of thing that would cause it. And I'm just accident prone enough to get it. And look… while I've accepted the idea that I'm going to die, eventually, of something, death by tampon is NOT what I had in mind. So I told him I wanted to go to the ER or Urgent Care the next day to have them look for it.
Except that night it snowed. A lot. And the next day the urgent care center that we normally go to was not open because of the snow. I had already slept very little the night before and I was sitting on pins and needles the entire day, obsessively reading about and watching for TSS symptoms. I kept pushing, I kept feeling around, I couldn't find it, and I was convinced that it was stuck behind my cervix somewhere, compacted because of the fucking, and at the same time flagellating myself for not just pausing everything and double checking for it before I sat on top of Ormsby.
Now, the other thing you have to remember, is that Ormsby is a photographer. So the fact that it was a full moon with clear skies and there had been a big snow was a pretty big deal to him. He decided he wanted to go out and photograph it. He asked if I wanted to stay home, but I elected to go with him (despite the cold), because I figured if I was going to go into shock and die from this stuck tampon, I'd be better off doing it while I was in the car with him instead of home alone with my cat (See?? This is how my brain works.). That's every recovering cat lady's worst fear. So I got in the car with my gloves, three layers of clothes, and a blanket, and we went out.
While he worked, I sat in the car and searched for 24-hour urgent care centers that might be open. I found one and during a break while we waited for the clouds to clear, we decided to find it so I could get this taken care of. We stopped for dinner at Steak and Shake and then traveled out to the location to see if they could take care of the problem (and if there was no problem, at least put my mind at ease by telling me for sure that there was nothing in there).
Once we got to the center, I somehow had to find the words to explain to the woman at the desk what had happened. Not my proudest moment, because at 32, you know, I'm supposed to know how this shit works, but there you go. My stomach was also beginning to rumble. Like not in a, "I'm hungry" way, but more in a, "Hey, I'm thinking that I might have to sit on the toilet soon" kind of way, and you know, shitting on the doctor that has a speculum in your vagina while he searches for a lost tampon is not an experience I have had (nor one that I want to have). But I also knew that I could sit on the toilet right then and nothing would come out… not shit… not a lost tampon… nothing. So, feeling particularly secure in this knowledge, I filled out the paperwork and waited.
Long story short: they looked, I did not shit on the doctor, the doctor did not find evidence of a lost tampon, there had been nothing to worry about all along. Hooray. Relieved after a full twenty-four hours of worry, I was ready to go home. Rest. Watch TV. Be near a toilet. Maybe have sex again.
Not Ormsby. Nope. After we got out of the clinic, the skies had cleared, the moon was visible, and "going home" was a long way off. But what did I have to worry about? There was no lost tampon in my vagina. I was not going to get TSS. I could stop pushing. I could seize the day… or… er… night. Or something.
To be continued:
Tagged: Lord Ormsby, Lost Tampon, Louisville, Snow, Tampons, Toxic Shock Syndrome, TSS


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An IRA bomb explodes in the Bishopsgate area of London.
The Bishopsgate bombing occurred on 24 April 1993, when the Provisional Irish Republican Army detonated a powerful truck bomb on Bishopsgate, a major thoroughfare in London's financial district, the City of London. Telephoned warnings were sent about an hour beforehand, but a news photographer was killed in the blast and 44 people were injured, with fatalities only minimised due to it occurring on a Saturday. The blast destroyed the nearby St Ethelburga's church and wrecked Liverpool Street station and the NatWest Tower. The financial cost was severe, estimated at the time to be over £1 billion of damage, making it the costliest terrorist attack at the time.
As a result of the bombing, which happened just over a year after the bombing of the nearby Baltic Exchange, a "ring of steel" was implemented to protect the City, and many firms introduced disaster recovery plans in case of further attacks or similar disasters. £350 million was spent on repairing damage. In 1994 detectives believed they know the identities of the IRA bombers, but lacked sufficient evidence to arrest them.
In March 1993, an Iveco tipper truck was stolen in Newcastle-under-Lyme, Staffordshire, and repainted from white to dark blue. A 1 tonne ANFO bomb made by the IRA's South Armagh Brigade had been smuggled into England and was placed in the truck, hidden underneath a layer of tarmac. At about 9 am on 24 April, two volunteers from an IRA active service unit drove the truck containing the bomb onto Bishopsgate. They parked the truck outside 99 Bishopsgate, which was then the Hongkong and Shanghai Bank, located by the junction with Wormwood Street and Camomile Street, and left the area in a car driven by an accomplice. A series of telephone warnings were then sent from a phonebox in Forkhill, County Armagh, Northern Ireland, with the caller using a recognised IRA codeword and stating "a massive bomb… clear a wide area". The first warnings were sent about one hour before the bomb detonated. Two police officers were already making inquiries into the truck when the warnings were received, and police began evacuating the area.
An Iveco tipper truck, the type used to carry the bomb
The bomb exploded at 10:27 am, causing extensive damage to buildings along a significant stretch of Bishopsgate. The blast raised a mushroom cloud that could be seen across much of London and gouged a 15-foot wide crater in the street. Buildings up to 500 metres away were damaged, with 1,500,000 sq ft of office space being affected and over 500 tonnes of glass broken. The NatWest Tower — at the time the City's tallest skyscraper – was amongst the structures badly damaged, with many windows on the east side of the tower destroyed; the Daily Mail said "black gaps punched its fifty-two floors like a mouth full of bad teeth". Damage extended as far north as Liverpool Street station and south beyond Threadneedle Street. St Ethelburga's church, seven metres away from the bomb, collapsed as a result of the explosion. The cost of repair was estimated at the time at £1 billion. Civilian casualties were low as it was a Saturday morning and the City was typically occupied by only a small number of residents, office workers, security guards, builders, and maintenance staff. Forty-four people were injured by the bomb and News of the World photographer Ed Henty was killed after ignoring police warnings and rushing to the scene. The truck-bomb produced the explosive power of 1,200 kg of TNT.


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My second of 3 days on the Carlogie beat, River Dee. Today the water was dropping away nicely which opened up more pools for us to fish. The gauge at Potarch Bridge read 2' 6" first thing in the morning but it finished the day about 1' 9". I started my day on the Boat and Village pools and in the afternoon I headed up to another new pool called Alan's. This pool is named after long serving Ballogie tennant Alan Lankshear. He has fished on the estate for an amazing 50 years. Well done! The wind was a howling gale all day and it must have been gusting to 40mph. Not the easiest wind to cast into and made it really difficult to get a line out straight. You had to wait until the gust past and cast as quick as you could! Nothing landed for the day and not much from the whole river. Only 5 fish from the Dee! Last day on Carlogie tomorrow so hopefully the wind will have died down a good bit. Here are some pictures from today.
|An up-stream look of Alan's pool. It is very deep just feet from the bank and you can see why the fish would rest here after pushing up through the fast water of the Long Haugh pool.
|The hut on the Pitslug pool. I like taking pictures of all the fishing huts as it makes you think of the history and stories that have been told by anglers.
|Rossach Pool. The last pool I fished today. Another nice bit of water at this height. Again was not easy to cast a line out due to the wind. Did see a couple of fish showing near the tail of the pool though.


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Back to Business Management Basics to Survive in Today's Stock Market
Reboot to the 1990s. That is when Peter M. Senge first published his writings of the Art & Practice of The Learning Organization. The stock market scene of today makes better sense when seen in the light of the key Business Management skills that Senge first advocated some two decades ago. Senge is of MIT and Sloan School vintage, so obviously he did not use astrology to prophecy what could happen to the current US economy!
Sub-prime crime by banks, gross errors in bond insurance, and credit rating manipulations, are generally beyond the reaches of small stock investors. That does not mean we have to exit buying and holding stock altogether. There is all this talk of gold and going back to gold old 401(k) alone, but such investing fears are exaggerated. Classic Business Management teaching can make sound stock investing profitable and fun as well. A top benefit of the Internet is that online dialog is possible-even for those who have forgotten what they learned in Business Management schools!
Gambling luck is all day trading in stock needs. However, stock investing is a different story. No stock can be evaluated professionally for its true future worth without Business Management skills. We have started a thorough revisit of The Fifth Discipline as put forth by Peter M. Senge. Watch this web site for our upcoming premium publication.
Do you use Business Management practices to analyze stock? Is an informal method better? Does the current stock market turbulence worry you? Join our community and dialog to your heart's content. Start posting today!


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Welcome to the thinkmiracle online course
Our site provides many free spiritual tools, from meditations to healthy recipe ideas, and these will always be offered free of charge.
However, the online course has been developed to move your life to a whole new level.
It is a seven week program that will require you to commit seriously to one thing: the willingness to change your life. You start it when you're ready and then move through the program at your own pace. This course is not about going through the motions, it will require your dedication and desire to implement lasting change in your life.


text: It's not about me! It's about you! Your goals, concerns and your interests about real estate are what matter, not mine! I understand that. My focus is on my clients' interests and goals before my own - that way we all are more likely to succeed in meeting your goals as well as my goals. Whether you already own your own home, are a seasoned investor, or are a first-time buyer, my focus must be on your best interests. You may not want to work with someone who thinks otherwise. What you need to know is that I am equipped in many ways to do that. I left a full time law practice to sell real estate because I really enjoy it so much. And as a consequence of my law background, I am very well prepared to be your advocate in real estate and I take my fiduciary duties very seriously. I am on your side. If you are now an owner of real estate you may be just fine and looking for a long-term strategy, or you may be facing disappointment, confusion, fear, and worse. I am well equipped to help you understand your options. If you are an owner in distress, I can walk you through your options from sitting tight, to possible loan modification to short sale, from cash for keys to foreclosure, even how bankruptcy might need to be in your range of options. But don't wait to find out your options until they disappear before you can use them. Get connected with the information you need sooner rather than later. You will have more options to explore earlier on. And it is much easier to hear and understand them when you are not living in the fear of the immediate unknown. I've had my real estate license since 1983 and enjoy all aspects of real estate. I enjoy selling all over Santa Clara County, from Saratoga to Morgan Hill, and in the Santa Cruz Mountains, from Boulder Creek to Santa Cruz. I enjoy referrals from my satisfied clients. I enjoy meeting new clients and I look forward to working with you. Feel free to ask me as many questions as you need. And I will look forward to discovering what it is that you want to do with real estate. You may come to see that more is possible than you imagined. I will look forward to talking with you.


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"First time I tried Vicodin, it was laying around my mom's house and I kept taking them whenever I can get them," narrates a voice-over for a White House ad on opioid addiction. "I didn't know they'd be this addictive. I didn't know how far I would go to get more."
Seconds later, a young man deliberately slams a door on his arm to break it. Nonlinear noises play as distorted images show him screaming.
This is one of four advertisements the Trump administration officially launched on Thursday. They're all graphic, telling true stories of young people hurting themselves to get more prescription opioids.
The campaign release is a highly anticipated move, following the president's comments in March announcing that the White House will be "spending a lot of money on great commercials to show how bad it is… you scare them from ending up like the people in the commercials."
And his ads are just that, scary. But they aren't very helpful.
"Who does not know at this moment, after 22 years of an epidemic, that these drugs are addictive?" said Keith Humphreys, a drug policy expert at Stanford University and former senior policy adviser for the White House Office of National Drug Control Policy. "They don't give information. They are just trying to terrify everyone, and I think people are already terrified."
In addition to learning nothing new from these advertisements, Humphreys says the videos further stigmatize people who struggle with addiction.
"They're awful. Can you imagine being addicted to opioids and then seeing these ads — which basically say you are a freak and inhuman monster? Would you go into care and say, 'Yeah I'm one of those people?'" he said. "We've learned nothing."
The ads do emphasize that "opioid dependence can happen after just five days." But they focus only on the lengths people will go to to feed addiction. The campaign, dubbed the "Truth About Opioids," could have told audiences something new, advising viewers on how to properly dispose of leftover opioids, or where to purchase the life-saving overdose drug naloxone.
The White House Office of National Drug Control Policy partnered with two nonprofits, the Truth Initiative and the Ad Council, in order to create the advertisements. That ad space will cost $30 million according to an official with the Ad Council, and the White House told reporters on Thursday they will only spend $384,000 on the campaign. The rest will be aided by donations. Organizers reportedly tested over 150 different images before settling on the four advertisements chosen.
The White House says the advertisements released Thursday are the first of many campaign ads to come and mark "another critical step" in addressing opioid epidemic. These videos are specifically focused on preventing drug misuse among young adults, ages 18 to 24.
By virtually all measures, the opioid crisis is devastating young people. Last week, researchers released a study showing one in five young people who died in 2016 suffered from opioid-related side-effects. But public health experts say drug awareness campaigns aren't the best way to address this.
"Historically, programs that have sought to scare people into avoiding drugs have either been ineffective or had the opposite effect," said Omar Manejwala, chief medical officer of Catasys and an expert on addiction. "Effective approaches to adolescent prevention generally focus on skill building, developmental factors, community, and environmental contributors and policies."
The "Just Say No" campaign unveiled in the 1980s is a major example. The campaign's advertisements are largely seen in retrospect as unhelpful and many ads backfired. Experts say contemporary White House efforts will likely be more of the same.
"The problem with the '80s ads was, they were aimed to please Congress, and when you are trying to please an audience of people who are about 50 years old on average and wear a suit to work and they think kids these days don't listen — they're going to love it. Trump will love these commercials," said Humphreys. "But that's not who these commercials are for. What is a teenager going to think? What did teenagers think when people smashed up a kitchen after smashing an egg in the 1980s? They thought it was ridiculous."
The most infamous anti-drug ad is likely one in which popular 1990s television star Rachael Leigh Cook smashes an egg to dramatize the effects of heroin. Cook later starred in a reprise of the ad, highlighting the connection between the War on Drugs and mass incarceration.
The Trump administration first declared the opioid crisis an emergency in October and has taken a few steps to address the crisis, which is now considered deadlier than the Vietnam War. The president has said that his focus is on enforcement, not treatment. Congress is working its way through legislation, but many have expressed concern that lawmakers are not addressing the underlying issues of addiction, let alone dedicating enough funds to the crisis.


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The UCAM Murcia is close to passing to the next stage of the Eurocup, after beating Unicaja 83-75
The UCAM Murcia received at the Palacio de los Deportes Unicaja Malaga with the opportunity to position itself as leader of group C of the Eurocup and Oscar Quintana did not miss the opportunity. After a first half of honours shared Murcia broke away in the third quarter and conserved their lead in the last period. Despite the loss of Facundo Campazzo, performances by Billy Baron (22 points), Vitor Benite (21) and Sadiel Rojas (25 rating) were keys to this victory that puts the UCAM Murcia with 3 wins and 1 loss at the head of its group.
Starting the fourth quarter the score of the two teams slowed during the first few minutes which favored UCAM Murcia at it had the advantage. After this Billy Baron scored 5 points which out did the 4 point in the other ring by Ndiaye. The university team arrived at the mid point of the quarter with a lead of 5 points, while the visitors missed from distaince, first Pocius and then Faverani moved the gap to +10. Unicaja did not throw the towel with 3 minutes left but Malaga only scored from 4.60 (80-74). At the last minute and 5 ahead UCAM Murcia knew how to manage its advantage and a triple by Billy Baron finally sentenced the match for the locals. The best players for UCAM Murcia were Billy Baron with 22 points and Sadiel Rojas with 25 points and assists.


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Black Bean Corn Avocado Salad - CIAProChef.com
BLACK BEAN, TOMATO, FRESH CORN, BIBB LEAF SALAD WITH AVOCADO AND SHERRY VINAIGRETTE Yield: 8 portions Ingredients Amounts Bean Salad Black beans, cooked 3 cups ... Read Here
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Level 1 Certification: Specialist
This 16-week program grounds participants in the RROHC system philosophy and set of practices. Hansten Healthcare PLLC will send you the syllabus and portfolio, you purchase the text books, and we guide your staff in completing the program and applying the concepts to their own clinical areas and through ongoing conferences and online support.
Each participant receives approximately 8 hours of coaching (in a group format) from RROHC Master Coaches Ruth Hansten and Kimberly McNally.
At the end of Level 1, participants gain:
- An understanding of the RROHC philosophy and practices for immediate application on the job
- Practical information on how to delegate, supervise & lead a clinical team
- Experience applying critical thinking & problem solving skills to clinical scenarios
- Methods to communicate effectively & resolve conflict
- Understanding of the coaching relationship.
Upon certification at the Specialist level, participants may progress through the Facilitator & Master Coach levels. Download our brochure.
We offer this program twice a year beginning in January and June. The Specialist Program is more successful when offered after the one-day Foundations Seminar. The seminar promotes staff buy-in and provides an excellent context for those who will be participating in the Specialist Program.
Includes Foundations Seminar
Without the Foundations Seminar
Cancellations and Discounts
All team members must register together to be eligible for the team rate discount.
Refunds for cancellations, less a $50.00 processing fee, will be made if Hansten Healthcare is notified in writing (email or fax) 10 days before the program.
For cancellations less than 10 days before the program, fees may be applied to future programs (less a $150.00 processing fee).
Please complete all sections of the registration form. The information will be used for all program correspondence and name badges.
All registrations must include the appropriate fees.


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Fizazi K, Greco FA, Pavlidis N, et al. Cancers of unknown primary site: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. (2011). Annals of Oncology. Oxford University Press. 22(Supplement 5): vi64-vi68 doi: 10.1093/annoc/mdr389.
Greco FA & Hainsworth JD. Cancer of unknown primary site. DeVita VT Jr, Lawrence TS, & Rosenberg SA. (2015). Cancer: Principles & Practice of Oncology. (10th Edition). Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins. 113:1720-1737.
Kim KW, Krajewski, KM, Jagannathan JP, et al. Cancer of unknown primary sites: what radiologists need to know and what oncologists want to know. (2013). AJR: American Journal of Roentgenology. The American Roentgen Ray Society. 200(3): 484-492 doi: 10.2241/AJR.12.9363.
National Comprehensive Cancer Network. Occult Primary (Cancer of Unknown Primary [CUP]) (Version 3.2014). Retrieved on 7/7/2014. Retrieved from: http://www.nccn.org/professionals/physician_gls/pdf/occult.pdf.×


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Texas No Money Down – Texas Home Loans – VA Loans – The method of purchasing a home with no down payment that most people are familiar with is through the Department of Veterans Affairs, or VA. This benefit is available to active and retired members of the military service, veterans, POW's and MIA's and their unmarried widows.
Construction loans – Home Construction loans – Owner-Builder Construction Loans Build your new Home with Instant Home Equity and pay No Money Down! If you have tried to obtain a construction loan from a bank to build a new home, you probably found it to be very difficult. Most banks that offer construction loans require up to 15% down payment!
Loans and Home Down Payment Assistance | Texas State. – TSAHC provides mortgage loans and down payment assistance grants and second liens to eligible home buyers through the following programs: Homes for Texas Heroes Home Loan Program: for teachers, fire fighters and EMS personnel, police and correctional officers, and veterans.; Homes Sweet Texas Home Loan Program: for Texas home buyers with low and moderate incomes.
Fha House Qualifications FHA cash out refinance guidelines and mortgage rates for 2019 – FHA loans require a DTI 43 percent or less unless significant compensating factors are present, such as high credit scores or lots of equity in the house. In these cases, a DTI of up to 50 is.
Texas Home Loans With No Down Payment – Contents maximum home loan Finance corporation (seth) offers texas residents Texas heroes home loan program: Congress. fees Competing subprime loans max home Loan Amount The maximum home loan amount you can get depends on your credit worthiness and borrowing potential determined in terms of the norms and standards of the bank.
Texas (TX) First-Time Home Buyer Programs for 2019 – SmartAsset – Both the Texas state government and the federal government offer special programs for first-time home buyers. These programs may allow you to get a loan with low down payment and credit score requirements.
5 STAR Texas Advantage Program – SETHFC – seth 5 star texas Advantage Program provides eligible homebuyers Down Payment and Closing cost Assistance when purchasing a home. There are several options of assistance available. Borrowers will select the amount of assistance and loan option that best meets their needs.
How Long Does It Take To Get An Fha Loan
Home Loans – Best Home Loan Rates – Reviews & Requirements – Home loans: explore today's refinance and mortgage rates for a variety of mortgage products, and learn how we can help you reach your home financing goals.
The Zero Program! No Down Payment! No Closing Costs? Help. – FHA Home Loans can work with the Zero Program too! FHA requires a minimum down payment of 3.5 percent of the sales price. Through the usage of down payment assistance programs, if your credit score is 620 or higher, you can use one of many down payment assistance options available to you.