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- © 2008 American Society of Plant Biologists
In the absence of sulfur (S), Chlamydomonas reinhardtii increases the abundance of several transcripts encoding proteins associated with S acquisition and assimilation, conserves S amino acids, and acclimates to suboptimal growth conditions. A positive regulator, SAC1 (for sulfur acclimation protein 1), and a negative regulator, SAC3, were shown to participate in the control of these processes. In this study, we investigated two allelic mutants (ars11 and ars44) affected in a gene encoding a SNRK2 (for SNF1-related protein kinase 2) kinase designated SNRK2.1. Like the sac1 mutant, both snrk2.1 mutants were deficient in the expression of S-responsive genes. Furthermore, the mutant cells bleached more rapidly than wild-type cells during S deprivation, although the phenotypes of ars11 and ars44 were not identical: ars11 exhibited a more severe phenotype than either ars44 or sac1. The phenotypic differences between the ars11 and ars44 mutants reflected distinct alterations of SNRK2.1 mRNA splicing caused by insertion of the marker gene. The ars11 phenotype could be rescued by complementation with SNRK2.1 cDNA. In contrast to the nonepistatic relationship between SAC3 and SAC1, characterization of the sac3 ars11 double mutant showed that SNRK2.1 is epistatic to SAC3. These data reveal the crucial regulatory role of SNRK2.1 in the signaling cascade critical for eliciting S deprivation responses in Chlamydomonas. The phylogenetic relationships and structures of the eight members of the SNRK2 family in Chlamydomonas are discussed.
Sulfur (S) is an essential element present in proteins, lipids, carbohydrates, electron carriers, various metabolites (some involved in the detoxification of heavy metals and xenobiotics), and signaling molecules (Meister and Anderson, 1983; Gupta et al., 1990; Schultze et al., 1992; Marrs, 1996; Grossman and Takahashi, 2001). The preferred S source for most organisms is sulfate (SO42−), which can be limiting in the environment; limitations for SO42− may result in reduced quality and yield of seeds and cause stunted plant growth (Mahler and Maples, 1986, 1987; Warman and Sampson, 1994). Most organisms are unable to efficiently store S; therefore, they are dependent on a continuous supply of this nutrient.
When organisms become S limited, they exhibit a suite of responses that have been described as either specific or general. The specific responses to nutrient limitation are those associated with deprivation for a single nutrient and are often involved in scavenging or conserving that specific nutrient. For example, during S deprivation, Chlamydomonas reinhardtii synthesizes periplasmic arylsulfatases (ARS) that catalyze the hydrolysis of organic SO42− esters (de Hostos et al., 1989), develops more efficient SO42− transport (Yildiz et al., 1994) and assimilation (Yildiz et al., 1996; Ravina et al., 2002), and maximizes S utilization efficiency, which could involve significant changes in cell architecture (Grossman and Takahashi, 2001; Zhang et al., 2004). General responses are those associated with deprivation for any essential nutrient and include the cessation of cell growth and division, the accumulation of storage carbohydrates, and the modulation of metabolic processes, including a decrease in photosynthetic activity. The coordination of metabolic processes in the cell during nutrient limitation is critical because an imbalance between the generation of fixed carbon and reducing equivalents with the potential for the cell to grow and divide can stimulate the production of damaging reactive oxygen species. Managing photosynthetic activities and the accumulation of fixed carbon is particularly important, and a number of investigators have characterized the ways in which photosynthesis is controlled by conditions that limit cell growth (Peltier and Schmidt, 1991; Wykoff et al., 1998).
Very little is known about regulatory mechanisms that control S nutrition in vascular plants and other photosynthetic eukaryotes. The generation of insertional mutants of Chlamydomonas has resulted in the identification of two specific regulators of S deprivation responses, SAC1 (sulfur acclimation protein 1) and SAC3. SAC1 plays a central role in controlling S deprivation responses (Davies et al., 1996; Zhang et al., 2004), and sac1 mutants are unable to synthesize ARS and to induce many genes that are normally up-regulated when the cells are starved for S (Yildiz et al., 1996; Takahashi et al., 2001; Ravina et al., 2002; Zhang et al., 2004). In addition, the general responses in sac1 mutants are impaired, and the cells rapidly bleach and die during S deprivation (Davies et al., 1996). This death is light dependent and has been linked to a failure of the cells to down-regulate photosynthetic electron flow out of PSII, suggesting that the modification of photosynthetic electron transport during S deprivation is critical for cell survival.
SAC1 has significant sequence similarity to the animal S transporters (Na+/SO42− transporters). Three additional genes that encode proteins with strong sequence similarity to mammalian Na+/SO42− transporters (SLT1–SLT3) have been identified on the Chlamydomonas genome. The physiological effects of the sac1 mutation strongly imply that SAC1 is a hierarchical regulator critical for the acclimation of cells to S deprivation and raise the possibility that transporter-like proteins might have evolved into sensor proteins that are critical for acclimation processes (Davies et al., 1996).
The SAC3 S deprivation regulator of Chlamydomonas is a putative Ser/Thr kinase in the plant-specific SNF1-related protein kinase 2 (SNRK2) family. The sac3 mutants exhibit low-level constitutive ARS activity in S-replete medium, but like wild-type cells, they accumulate high levels of ARS following exposure of the cells to S deprivation. Other S-responsive genes (in addition to ARS) are also negatively regulated by SAC3 (Ravina et al., 2002). Furthermore, the sac3 mutant does not show the dramatic decrease in chloroplast transcriptional activity that is observed in wild-type cells during S starvation, and the SAC3 kinase may be required to inactivate chloroplast RNA polymerase sigma factor SIG1 under S deprivation conditions (Irihimovitch and Stern, 2006). The relationship between SAC1 and SAC3 is nonepistatic, since the sac1 sac3 double mutant maintains the phenotype of both of the parental strains (Davies et al., 1994).
Recently, a collection of mutants that exhibit low ARS activity and that are potentially affected in S deprivation responses was generated by insertional mutagenesis (Pollock et al., 2005). Among this mutant population are two strains, ars11 and ars44, that harbor an interruption in the same gene. This gene, designated SNRK2.1, encodes a SNRK2 kinase with sequence similarity to SAC3. In this work, we demonstrate that SNRK2.1 plays a crucial role in the control of S deprivation responses. The ars11 lesion dramatically affects S deprivation-responsive gene expression, and the mutant cells rapidly bleach following transfer to medium lacking S. Furthermore, unlike sac1, ars11 is epistatic to sac3, reflecting a key position of the SNRK2.1 kinase in the control of S deprivation responses.
Isolation of the ars Mutants
Insertional mutants (disrupted with the AphVIII marker gene) of Chlamydomonas were screened for abnormal levels of extracellular ARS activity after transferring cells to TAP-S medium (for Tris-acetate phosphate medium without S). Two kinds of mutants were identified: those with no or low ARS activity (ars− mutants) and those that exhibited more ARS activity than the parental strain (ars+ mutants). Approximately 30,000 transformants were screened, and from those 50 strains exhibited an ars− phenotype and two exhibited an ars+ phenotype (Pollock et al., 2005).
An adaptor-mediated PCR technique (Padegirnas and Reichert, 1998) was successfully used to determine the genomics regions flanking the AphVIII marker for a number of the ars mutants. Data showing ARS activities and the flanking regions of some of these mutants were reported previously (Pollock et al., 2005). Among the ars− mutants for which flanking sequences were obtained, both ars11 and ars44 had an AphVIII insertion in the same gene, but at different sites within that gene. The interrupted gene encodes a putative Ser/Thr protein kinase belonging to the SNRK2 plant kinase family (Halford and Hardie, 1998). This gene has been designated SNRK2.1.
Cosegregation of Paromomycin Resistance with the ars Phenotype
To determine whether the ars− phenotype was linked to the AphVIII insertion, the ars11 and ars44 mutants were crossed with the wild-type 21gr strain, which is sensitive to paromomycin. For both mutants, the paro+ progeny (approximately 30 of a total of 60 analyzed) cosegregated with the ars− phenotype, strongly supporting the conclusion suggested by the occurrence of two alleles with related phenotypes that the insertion was the cause of the mutant phenotype.
Physiological Characterization of ars11 and ars44 Mutants
The ars11 and ars44 mutants exhibited no or low ARS activity when the cells were deprived of S, although the phenotypes of the strains were not identical. Like sac1, ars11 had no detectable ARS activity on agar plates, while ars44 exhibited reduced ARS activity; the ars44 strain accumulated substantial levels of ARS activity if the cells were maintained on TAP-S plates for a long enough period of time (Fig. 1A ). In liquid cultures, ars44 showed essentially no ARS activity after 24 h of S starvation (Fig. 1B), and even after several days of S starvation, very low-level activity was observed (data not shown).
Davies et al. (1996) showed that the sac1 strain is defective for the specific and general S deprivation responses. One feature of sac1 is its rapid loss of chlorophyll and death, relative to wild-type cells, following the imposition of S deprivation; this death response may be a consequence of the inability of the mutant to decrease the rate of photosynthetic electron flow through PSII (Davies et al., 1996). The ars11 and ars44 mutants also bleached (Fig. 1C) and died much more rapidly than wild-type cells when transferred to TAP-S medium, suggesting that, like sac1, these strains are aberrant for both the specific and general S deprivation responses. Interestingly, the ars11 strain bleached and died significantly more rapidly than either ars44 or sac1.
To further analyze the responses of the ars11 and ars44 strains to S deprivation, the levels of transcripts from several genes that were already known to be controlled by the S status of the medium were analyzed; these genes included ARS1 for the arylsulfatase (de Hostos et al., 1989), SLT1 for the Na+/SO42−-like transporter (Zhang et al., 2004), SULTR2 for the H+/SO42−-like transporter (W. Pootakham and A.R. Grossman, unpublished data), ECP76 for an extracellular protein that is probably associated with the cell wall (Takahashi et al., 2001), and SBDP for a putative selenium-binding protein (Zhang et al., 2004; Fig. 2, A–C ). Both the ars11 and ars44 mutants had lower basal levels for most of these transcripts, with little or no increase (relative to wild-type cells) following the imposition of S deprivation (with some exceptions for ars44). However, both the ARS activity assay and the kinetics of the bleaching response (Fig. 1) demonstrated that the phenotype of ars44 is significantly less severe than that of ars11. This difference in severity is apparent based on the analysis of transcript levels from most of the genes examined and exemplified by the changes in SLT1 transcript abundance following S deprivation; while no increase in the transcript level was observed in ars11, a strong increase (somewhat lower than that of the parental D66 strain) was observed for ars44. The pattern of expression for these same genes was also analyzed in sac1 (Fig. 2D). As in ars11, the sac1 mutant showed no or only a small increase in the levels of known S deprivation-responsive transcripts when the cells were starved for S, although the basal transcript levels in TAP medium in the sac1 strain were generally higher than in either ars11 or ars44. In contrast, the sac3 mutant showed relatively high levels of transcripts for a number of the genes tested, but especially for ARS1, under S-replete conditions, with increased accumulation when the cells were starved for S (Fig. 2E). These results demonstrate that the SNRK2.1 protein is critical for the accumulation of transcripts from the S deprivation-responsive genes when cells are starved for S. The inability of the mutant cells to respond to S deprivation at the level of transcript accumulation (and likely gene activation) may also lead to the rapid bleaching and loss of viability following the transfer of the mutant strains to TAP-S medium.
The cDNAs and Sites of Insertion in ars11 and ars44
There were no EST sequences to help identify the complete coding sequence (CDS) and intron-exon boundaries of the SNRK2.1 genomic sequence. Therefore, a 2,129-bp cDNA was amplified by reverse transcription (RT)-PCR from the parental strain RNA. Based on the sequence of the cDNA, the gene contains a 1,173-bp CDS with an 810-bp 3′ untranslated region (UTR) and a 146-bp 5′ UTR. Other cDNA sequences that could represent alternative splice forms of the SNRK2.1 transcript were also amplified (see below). The cDNA sequence was different from the predicted sequence of the ab initio model for the gene that was generated by the Joint Genome Institute (JGI; http://genome.jgi-psf.org/Chlre3/Chlre3.home.html). We recently updated this model to take into account cDNA sequence information. The genomic sequence corresponding to the cDNA was 5.46 kb in length, consisting of 11 exons and 10 introns (Fig. 3A ), encoding a predicted polypeptide of 390 amino acids.
Adaptor-mediated PCR was used to identify one of the genomic regions flanking the AphVIII marker gene for both ars11 and ars44 (the border close to the 3′ end of AphVIII; Pollock et al., 2005). In ars11, the insertion was positioned within exon VII, whereas in ars44, the insertion was within intron 6 (Fig. 3B). To identify the flanking regions on both sides of the insertions, specific primer pairs were designed to anneal to the SNRK2.1 genomic sequence close to the site of insertion and to the marker gene. Amplifications using these primers and analysis of the flanking sequences showed that neither of the insertion events was accompanied by deletions or by a reorganization of the genome around the site of the insertion (Kindle, 1990). Using RT-PCR, we demonstrated that neither ars11 nor ars44 was completely lacking SNRK2.1 transcripts; however, the mRNA from SNRK2.1 in ars11 was missing the sequence encoded by exon VII, while ars44 retained a full-length SNRK2.1 transcript that was indistinguishable from that of the parental strain (Fig. 3C). In ars11, splicing resulted in a fusion of exon VI to exon VIII, which eliminated exon VII (the site of the insertion) from the processed transcript (Fig. 3C). Furthermore, the translated protein of the ars11 mutant would have a shift in the reading frame generated as a consequence of the fusion of exons VI to VIII. When SNRK2.1 transcript accumulation was determined using primers specific for the 3′ UTR, no significant difference between the mutant and wild-type transcript levels was observed (data not shown). However, when SNRK2.1 transcript accumulation in ars11 was determined using primers specific for exons VI and VII, no transcript was detected. In contrast, the same primers did reveal the presence of a transcript with the proper fusion of exon VI and VII in ars44, although the transcript was reduced by approximately two orders of magnitude in this mutant strain (with the greatest reduction observed in cells exposed to 4 h of −S conditions) relative to parental cells (Fig. 4 ). Thus, the ars44 strain is likely able to synthesize normal SNRK2.1 protein, but the level of this protein may be significantly lower than in parental cells because of the low-level accumulation of the mRNA. The molecular differences associated with mRNA processing are likely responsible for the different mutant phenotypes. Furthermore, the data demonstrate that splicing of intron 6 of SNRK2.1 can occur even if the intron is interrupted with the 1.7-kb PSAD∷AphVIII marker gene.
Potential Alternative Splicing of SNRK2.1
As mentioned above, SNRK2.1 cDNA generation led to multiple cDNAs of different lengths. At least five different SNRK2.1 cDNAs were identified using the same set of specific primer pairs for RT-PCR (the RNA template was from D66 cells grown in TAP and TAP-S); these cDNAs were designated SNRK2.1 v1, v2, v3, v4, and v5, and the differences among them are shown in Supplemental Figure S1. SNRK2.1 v1 was the longest cDNA, encoding a polypeptide of 390 amino acids, and is the isoform discussed above (referred to as SNRK2.1). All of these potential transcripts encoded putative polypeptides that used the same start codon, with SNRK2.1 v1, v2, and v3 maintaining the same coding frame and stop codon. In contrast, the SNRK2.1 v4 and v5 reading frames changed as a result of the alternative splicing, which also generated a different stop codon. For SNRK2.1 v2 to v4, the potential alternative 5′ splice sites were positioned inside a 54-bp GC-rich region of the transcript. The deduced SNRK2.1 v2 protein maintained the kinase catalytic domain and the end of the C-terminal domain, whereas v3 lacked 20 amino acids from the kinase domain and part of the C-terminal region. The SNRK2.1 v4 and v5 predicted polypeptides lacked the kinase domain and the entire C terminus. An alignment of all of the potential splice variants is shown in Supplemental Figure S2. None of the potential splice variants was associated with a specific culture condition (e.g. TAP compared with TAP-S), and the only variant reproducibly observed was SNRK2.1 v1; the others were less abundant and in some reactions were not detected. Finally, only in SNRK2.1 v1 did all of the splice junctions match the consensus junctions associated with Chlamydomonas genes (Silflow, 1998), which raised the possibility that the variants were a consequence of artifacts associated with cDNA synthesis and amplification. In addition, two alternative 3′ polyadenylation sites were identified by 3′ RACE from the SNRK2.1 cDNA sequences (Supplemental Fig. S3); these sites were not associated with specific recombinant libraries or splice variants.
To help establish if the small SNRK2.1 transcripts were potentially artifacts, we used specific primers to generate PCR products from a pGEM-T clone harboring the SNRK2.1 v1 cDNA. A multiband amplification pattern was observed, and at least one of the products had a gap in the sequence starting within the same GC-rich region that was identified as the 5′ splice junction for putative splice variants. However, the products generated by PCR from the plasmid did not perfectly match any of the putative SNRK2.1 splice versions (data not shown). These observations raise the possibility that putative alternative splice forms can arise from aberrant cDNA synthesis and amplification in vitro, and not from in vivo splicing of precursor mRNA.
Complementation of the snrk2.1 Mutation
Four of the SNRK2.1 cDNA potential splice variants (v1, v2, v3, and v4) were introduced into the ars11 mutant strain. Each cDNA version was inserted between the PSAD promoter and the 3′ UTR terminator and cloned into a vector containing the ble gene as a selectable marker. Fifty ble-resistant colonies for each splice variant were tested for ARS activity following exposure to TAP-S. Four colonies transformed with the SNRK2.1 v1 cDNA restored the wild-type ARS phenotype, based on ARS plate assays. An analysis of ARS activity in liquid for one of the potentially complemented mutant strains (tars11#34) showed that the strain was rescued for the ars− phenotype; it produced high levels of ARS when deprived of S (Fig. 1B), with no ARS activity detectable when grown in TAP (data not shown). No complemented colonies were obtained that harbored v2, v3, or v4 cDNA variants. Analysis of chlorosis of the tars11#34 strain in TAP-S also revealed restoration of the wild-type phenotype (Fig. 1C); transformants expressing ARS survived S deprivation for an extended period of time, like the D66 parental strain. Finally, the levels of ARS1, SLT1, and SULTR2 mRNAs measured in tars11#34 were similar to those of the parental strain (Fig. 2G). These results clearly demonstrated that the mutant phenotype can be complemented by the full-length SNRK2.1 gene.
ars11 sac3 Double Mutant Analysis
SNRK2.1 is required for the activation of genes associated with S deprivation responses, while SAC3 acts as a negative regulator of at least some of these same genes (Davies et al., 1999). To help define the role of the SAC3 and SNRK2.1 kinases in the regulation of S deprivation responses, the epistatic relationships between the sac3 and ars11 lesions were examined by constructing strains harboring both lesions. Four double mutants were identified, and none exhibited detectable ARS activity when grown on TAP or following transfer of the cells to TAP-S (data not shown), indicating that the sac3 lesion is hypostatic to the snrk2.1 lesion; SNRK2.1 is needed in a sac3 mutant to enable the constitutive expression of ARS. Expression of ARS1, SLT1, SULTR2, ECP76, and SBDP was analyzed in the randomly selected double mutant 11-3#16 (Fig. 2F). The double mutant, like the ars11 single mutant, exhibited extremely low levels of transcript accumulation for all tested transcripts in both TAP and TAP-S (Fig. 2, compare B with F).
The SNRK2 Family
SNRK2.1 belongs to the plant-specific Ser/Thr protein kinase family SNRK2. Like other SNRK2 family members, SNRK2.1 has an N-terminal conserved catalytic domain similar to those of SNF1/AMP kinases and a short C-terminal regulatory domain that is not highly conserved. SAC3 (Davies et al., 1999; Irihimovitch and Stern, 2006) is also a member of the SNRK2 family, with 43% sequence identity with SNRK2.1. In addition to SNRK2.1 and SAC3, there are six other SNRK2 family members encoded on the Chlamydomonas genome. The complete eight-member family, SNRK2.1 to SNRK2.8, with SAC3 now being designated SNRK2.2, is presented in Table I . The deduced proteins for SNRK2.4, SNRK2.5, SNRK2.6, and SNRK2.8 should be taken as provisional, since the corresponding JGI gene models are impossible to accurately predict because of gaps in the genomic sequences and incomplete EST sequence coverage. Although we have improved on these gene models based on EST and genomic sequences and similarities to other SNRK2 kinases, they are still incomplete, mainly in their C-terminal regions. SNRK2.1 and SNRK2.3 are clustered in the same genomic region (scaffold 7, linkage group II) in a tail-to-head orientation and separated by less than 500 bp. SNRK2.3 and SNRK2.4 are the most closely related to SNRK2.1. These three family members form a Chlamydomonas SNRK2 subgroup that is most different from the plant SNRK2 homologs. Members of this subgroup also have unique sequence features that are not present in other SNRK2 proteins; one major feature is the presence of an extra loop of 17 to 22 amino acids immediately preceding the SNF1 kinase activation domain (Hardie and Carling, 1997), which is shown in the alignment in Figure 5 (broken line). Although this extra loop is not conserved at the sequence level among the subfamily members, it does contain a NLH motif (asterisks above the broken line in Fig. 5) and several residues that might serve as phosphorylation sites. An alignment of the entire Chlamydomonas SNRK2 family of proteins is shown as Supplemental Figure S4.
In this study, we identified SNRK2.1, a member of the plant-specific SNRK2 kinase family and the larger SNF1 superfamily, as a key regulator of the pathway that governs S deprivation responses in Chlamydomonas. Two independent mutant strains in which the SNRK2.1 gene was interrupted, ars11 and ars44, were identified. The insertions did not cause reorganization or deletions of the genomic region at the insertion site, and the mutant phenotypes were linked to the insertions. Even though the mutants had a sequence of 1.7 kb (AphVIII under the control of the PSAD promoter and the 3′ UTR) integrated into the SNRK2.1 gene, the splicing machinery of Chlamydomonas was able to excise the inserted sequence and generate a mature mRNA; this mRNA was aberrant in the case of ars11. Insertion of the AphVIII marker gene into exon VII of SNRK2.1 in ars11 caused the loss of this exon in the final splice product and a change in the reading frame of the C-terminal region of the protein. These modifications of the SNRK2.1 protein are likely the reason for the severe mutant phenotype (the lesion probably represents a null mutation). In contrast, in ars44, the marker gene is integrated into intron 6. This interrupted intron (with the inserted marker DNA) was spliced out of the nascent transcript, generating a mature transcript that appeared to be identical to the transcript in the parental strain, although the level of accumulation of this mature transcript was more than 10 times lower than in the parental strain (Fig. 4). Therefore, the leaky phenotype of ars44 is probably a consequence of inefficient splicing of intron 6, which in turn could generate less protein.
Interestingly, in spite of a clear difference in ARS activity between ars11 and ars44 mutants based on agar plate assays (Fig. 1A), both strains showed no ARS activity when they were transferred to liquid TAP-S medium for 24 h (Fig. 1B); there was little or no ARS activity measured in either of the mutants, even after several days of S starvation (data not shown). Several hypotheses might explain these findings. (1) There may be a difference in some crucial condition (e.g. oxygen availability) that results in elevated induction/activation of ARS activity on solid medium. This idea is supported by the finding that cells growing under the agar, at lower oxygen levels, exhibit higher ARS activity than cells growing on top of the agar (data not shown). (2) The splicing machinery may be somewhat different under different growth conditions, which might change the efficiency at which some transcripts are spliced. (3) ARS stability could be lower in cells growing in a liquid environment. Further experimentation should distinguish these possibilities.
The analysis of multiple potential splice variants of the SNRK2.1 transcript has raised critical issues concerning the in vivo synthesis of these variants and whether the procedure for cDNA synthesis could lead to their artifactual generation. The likelihood of an artifact generated during the synthesis of the cDNA is supported by the finding that the sequences of the splice junction sites for the potential variants identified differ markedly from the Chlamydomonas consensus splice junction sequences (Silflow, 1998). Also, the levels of splice variants observed following RT-PCR were highly variable, and truncated transcripts were also observed if the full-length SNRK2.1 cDNA (as plasmid) was used for PCR amplification. In addition, no complementation of the ars11 mutant was observed when mutant cells were transformed with the SNRK2.1 v2 to v4 cDNAs. Others recent works have demonstrated that PCR synthesis of cDNAs could generate artifactual alternative splice forms (Hampl et al., 1998; Oh et al., 2005; Cocquet et al., 2006). In most cases, the shortened transcripts were deleted for part of the internal coding sequence, and it was suggested that these transcripts formed as a consequence of sequence identity between the 3′ and 5′ splice sites among putative introns, allowing for the formation of heteroduplexes. All of the putative splice variants of SNRK2.1 have sequence identity between the 3′ and 5′ splice sites (Supplemental Fig. S1C). During PCR, full-length and partially elongated products would be generated. If the partial products have 3′ sequences that can pair with multiple sites within the complementary strand, they can anneal to the complementary full-length strands and serve as primers for elongation. This would lead to the generation of a product that contains internal deletions. Furthermore, since these products would contain the primer sites for both the 5′ and 3′ PCR primers, they would be efficiently amplified in subsequent PCR cycles. The GC-rich region at which all 5′ splice sites are localized could elicit the formation of partial elongation products able to form heteroduplexes with the complementary cDNA strand. Although the existence of multiple splice variants cannot be completely excluded based on this work, our results do suggest that caution should be used when considering the generation of splice variants. Additionally, the existence of variability in the placement of the poly(A) tail (Supplemental Fig. S3) suggests some complexity in the regulation of SNRK2.1 transcripts.
The SNRK2, SNRK1, and SNRK3 kinase families are plant specific and belong to the SNF1 superfamily. SNRK2 kinases constitute a large protein family in plants with an N-terminal conserved catalytic domain and a short regulatory C-terminal region. The regulatory region is not highly conserved but usually has a characteristic stretch of the acidic amino acids Asp and Glu, which define subfamilies SNRK2a and SNRK2b, respectively (Halford and Hardie, 1998). Furthermore, SNRK2 proteins have multiple phosphorylation states that are critical for activity, but in most cases the specific phosphorylated residues have not been identified (Kobayashi et al., 2004; Boudsocq et al., 2007). Members of the SNRK2 protein family play a role in the acclimation of plants to environmental stresses; a number of the plant enzymes are involved in controlling osmotic stress responses and are activated by abscisic acid (ABA; Li and Assmann, 1996; Mikolajczyk et al., 2000; Boudsocq et al., 2004; Kobayashi et al., 2004). Some downstream target genes that are activated by plant SNRK2 kinases are SSHLP, a phosphatidylinositol transfer-like protein (Monks et al., 2001), AKIP1, a protein regulating RNA stability (Li et al., 2002), and a basic Leu zipper transcription factor required for ABA gene regulation.
Interestingly, some members of the Arabidopsis (Arabidopsis thaliana) SNRK2 kinase family may function in controlling S limitation responses. Arabidopsis mutants defective for SNRK2.3 exhibit a slight decrease in the level of SULTR2;2 mRNA (encoding the low-affinity SO42− transporter) and elevated O-acetyl-Ser (precursor to Cys) accumulation (Kimura et al., 2006). However, snrk2.3 mutant plants did not show alterations in the expression of other S-responsive genes (APR and SAT), the accumulation of SO42−, or differences in their growth phenotype relative to the wild-type strain when placed in medium devoid of S. These findings suggest a much more moderate role of AtSNRK2.3 than CrSNRK2.1 in controlling S starvation responses. Curiously, five of the 10 AtSNRK2 genes showed increased expression following S starvation of plants (Kimura et al., 2006), which raises the possibility that the activity of other SNRK2 proteins from Arabidopsis may affect S starvation responses. Overall, these results suggest that the SNRK2 family in plants and algae may have some conserved functionalities with respect to controlling nutrient deprivation responses.
The Arabidopsis kinase SNRK2.6 can be activated by two independent mechanisms, one that is ABA dependent and another that is ABA independent and stress dependent (Yoshida et al., 2006). The C-terminal domain, required for both mechanisms, can be functionally divided into domains I and II (Fig. 5). Domain I functions in ABA-independent activation, while domain II, which contains the acidic residues, functions in ABA-dependent activation. Chlamydomonas SNRK2 members have the conserved C-terminal domain I and have retained an acidic stretch of amino acids that characterizes domain II, although the number of acidic amino acids in this region is fewer than in most SNRK2 proteins of plants.
Domain II of Arabidopsis SNRK2.6 is needed to elicit full stomatal closure and for the interaction with the PP2C-type phosphatase ABI1, and deletion of the rice (Oryza sativa) OSRK1 C terminus destroys the functionality of the protein (Chae et al., 2007). Similarly, the full-length SNRK2.1 v1 cDNA can fully complement the ars11 mutant, while the SNRK2.1 cDNA v2, which lacks 45 amino acids of the C terminus (part of both domains I and II), appears to be unable to rescue the mutant phenotype.
Some of the Chlamydomonas SNRK2 proteins (SNRK2.1, SNRK2.3, and SNRK2.4) have a specific feature that is not present in plant SNRK2 proteins: an inserted, nonconserved loop of 17 to 22 amino acids located immediately to the N-terminal side of the activation domain (Fig. 5). The activation domain contains the conserved Ser phosphorylation site (Johnson et al., 1996) that is crucial for kinase activity and/or activation of the enzyme (Kobayashi et al., 2004) upon imposition of a stimulus (Boudsocq et al., 2007). The extra loop present in a subset of the Chlamydomonas SNRK2 proteins (1, 3, and 4) has several potential phosphorylation sites that may participate in regulation/activation (Supplemental Fig. S4).
SNRK2.1, SAC1, and SAC3 are the three genes that have been shown to have a regulatory role in the S deprivation responses of Chlamydomonas. While other mutants have been isolated that exhibit aberrant S deprivation responses, in most cases the gene responsible for the mutant phenotype has not been identified (Davies et al., 1994; Pollock et al., 2005). It is also intriguing that the regulators encoded by SNRK2.1 and SAC3 (SNRK2.2) both belong to the plant-specific SNRK2 family. This strongly indicates the importance of phosphorelays in the control of S deprivation-triggered responses. Curiously, SNRK2.1 and SAC3 have opposite regulatory effects, with the former required for activation of the S-responsive genes, including those encoding ARS and the sulfate transporters, and SAC3 required for full suppression of the same genes during growth under S-replete conditions.
Based on the phenotypes of the sac1 and ars11 mutants, SAC1 and SNRK2.1 have some similar regulatory features. However, SAC1 resembles animal Na+/SO4− transporters, such as SLT1 and SLT2 in Chlamydomonas (Davies and Grossman, 1998). Both SAC1 and SNRK2.1 are required for the appearance of ARS activity and the accumulation of transcripts that normally become abundant during S deprivation (Fig. 2, B and D). However, the regulatory aberrations are more extreme in ars11 than in the sac1 mutant; this is most evident in a comparison of the levels of transcripts for the sulfate transporters SULTR2 and SLT1 in the two strains and in the finding that ars11 bleaches significantly more rapidly than sac1 following the imposition of S deprivation. Furthermore, while the sac1 mutant shows no up-regulation of S deprivation-regulated genes, the levels of transcripts from these genes during S-replete growth are similar to those of the parental D66 strain. In contrast, ars11 has extremely low levels of the SLT1 and SULTR2 transcripts, even under nutrient-replete conditions, relative to the parental strain. As deduced from the mixed phenotype of the sac1 sac3 double mutant, there is no epistatic relationship between SAC1 and SAC3 (the double mutant exhibits low constitutive ARS activity in TAP and TAP-S media; Davies et al., 1994). In contrast, SNRK2.1 is epistatic to SAC3; the ars11 sac3 double mutant does not have any detectable ARS activity in TAP-S (Fig. 1B) or TAP (data not shown), and the patterns of ARS1, SLT1, SULTR2, ECP76, and SDBP transcript accumulation are similar to those of the ars11 single mutant (Fig. 2F). Hence, SAC3 and SAC1 appear to be dependent on SNRK2.1 for their phenotypic features. These genetic and phenotypic results suggest that SNRK2.1 is central to the pathway regulating S-responsive gene expression in Chlamydomonas. SAC1 could be a membrane-bound regulator that senses the S status of the environment and that initiates a signaling cascade that enhances SNRK2.1 activation, which is downstream in this pathway and more directly involved in the activation of a specific transcriptional regulator(s). A possible model that accounts for many of the responses observed in Chlamydomonas during S deprivation, and the ways in which the various mutant strains are affected in these responses, is depicted in Figure 6 .
Strains, Culture Conditions, and Mating
Chlamydomonas reinhardtii strains used in this work were D66 (nit2− cw15 mt+; Pollock et al., 2003), 21gr (nit5− mt−; Harris, 1989), sac1 (sac1− mt+), and sac3 (sac3− mt+; Davies et al., 1994). Cells were cultured under continuous light at 23°C in liquid and solid TAP medium (Harris, 1989). To impose S starvation, cells in midlogarithmic growth were washed twice with liquid TAP-S (Harris, 1989) and then split and resuspended in either TAP or TAP-S. Paromomycin and bleomycin were used at 10 and 3 μg mL−1, respectively. Genetic analyses were performed with the various strains according to a previously described protocol (Harris, 1989).
Generation of the ars11 sac3 Double Mutant
The sac3 mt+ mutant was crossed with the parental strain, 21gr (mt−), to obtain a sac3 mt− strain. The mating type of the ars constitutive progeny (sac3 phenotype) was determined by a PCR method (Werner and Mergenhagen, 1998), and one of the sac3 mt− progeny was crossed with ars11 mt+. The double mutant was identified by first selecting for paromomycin resistance (ars11 marker) and then determining which of the resistant isolates contained the pBK-arg2 chimeric sequence (sac3 marker) using the primers 5′-CGTACAAGGCCCATGCGTGAGTC-3′ and 5′-TCGCCGAAAATGACCCAGAGC-3′.
Transformation of Chlamydomonas
Cell wall-less strains D66 and ars11 were transformed by electroporation (Shimogawara et al., 1998) using a modifications of the procedure reported by Colombo et al. (2002).
Generation of Insertional Mutants in Chlamydomonas
The selectable marker gene used for mutant generation was AphVIII under the control of the PSAD promoter and terminator; this gene confers paromomycin resistance to transformants (Sizova et al., 2001). A 1.7-kb PCR fragment containing this construction was used to transform Chlamydomonas strain D66. After transformation, cells were incubated in TAP for 6 to 12 h under continuous light to allow for the accumulation of the AphVIII protein. Transformants were then selected on TAP supplemented with 5 μg mL−1 paromomycin.
ARS Activity and Chlorophyll Determinations
ARS activity was visualized directly on agar plates (Davies et al., 1994) or quantified from liquid medium (de Hostos et al., 1988) as absorbance units per cell number or per milligram of chlorophyll. The data presented correspond to mean values of at least three independent experiments. The concentrations of chlorophylls a and b, extracted from cells in methanol, were estimated using the equations of Porra (2002).
DNA and RNA Isolation
Genomic DNA and total RNA were isolated according to previously described methods (Schloss et al., 1984; Sambrook et al., 1989).
Synthesis of cDNA, RT-PCR, and 3′ RACE Amplifications
RNA samples were treated with DNase (Qiagen; catalog no. 79254) and further cleaned using RNeasy columns (Qiagen) following the manufacturer's directions. Single-stranded cDNAs were synthesized from total RNA using an N-(polyT)20mer primer according to the SuperScript III RNaseH reverse transcriptase manual (Invitrogen). This cDNA population was used as a substrate for real-time PCR with gene-specific primer pairs; the single-stranded cDNAs were diluted 5-fold prior to inclusion in the reaction mixture. The CDS of SNRK2.1 was obtained by RT-PCR using the specific primers 5′-GCCTTCTTGCGACTGCCATACG-3′ and 5′-CCTCAGTCGTTCATGCCGAA-3′. 3′ RACE for SNRK2.1 transcripts was performed using the specific 5′ primer 5′-GTTCCGTGAGGACCTACCCGAG-3′ and a previously described polyT-Qt primer that allowed semi-nested PCR amplifications (Frohman, 1990). All PCRs were performed in the presence of 2% to 5% dimethyl sulfoxide.
Complementation of the ars11 Mutant
The ble gene from pSP124S (Lumbreras et al., 1998) was introduced (at the XhoI-XhoI site) into the pJM43 plasmid (provided by J. Moseley) containing the PSAD promoter; the plasmid generated was designated pBleJM43. SNRK2.1 single-stranded cDNA was amplified using the modified primers 5′-ATCGATTAACGTTGGGGAACGATTCAATG-3′ and 5′-TGGATCCAACGTTGGGGAACGATTCAATG-3′, which introduced a ClaI and a BamHI site at the 5′ and 3′ end, respectively, of the amplified sequences. Four different amplicons were obtained: v1, 1,204 bp; v2, 1,068 bp; v3, 952 bp; and v4, 672 bp. These amplification products were introduced between the PSAD promoter and terminator in the pBleJM43 vector using the ClaI and BamHI sites. The new plasmids containing the differently sized sequences were designated pDV1, pDV2, pDV3, and pDV4. Sequencing of the insert in each of these vectors confirmed the absence of PCR-introduced sequence errors. The pDV1 to -4 plasmids were introduced into the ars11 mutant, and transformants were assayed for rescue of the mutant phenotytpe.
Quantitative PCR
Real-time PCR was performed using the Chromo4 thermocycler (Bio-Rad). Individual reactions had 25 μL final volume, consisting of 10 μL of DyNAMO SYBR Green quantitative PCR (qPCR) reagent (Finnzymes), 3.7 pmol of each primer, 1 to 2 μL of single-stranded cDNA (5-fold diluted from the reverse transcriptase reaction), and distilled water to 25 μL. The Chromo4 run protocol was as follows: denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 10 s, annealing at 60°C for 30 s, and amplification at 72°C for 30 s, and fluorescence measurement after 80°C for 15 s. This last step avoids background signals that can result from the formation of primer dimers. The specificity of the PCR amplification was evaluated by a melting curve program (60°C–100°C, with a heating rate of 0.5°C s−1 and continuous fluorescence measurements) and electrophoretic analysis on 4% agarose gels. We used the CBLP gene as a housekeeping gene control (Chang et al., 2005). Threshold cycle (Ct) values were determined in three independent experiments, with three replicates for each experiment. Relative fold differences were calculated based on the relative ΔCt method [2−(Ctsample − Ctcontrol gene)] using the CBLP amplification product as an internal standard. The primer pairs used for qPCR were as follows: 5′-CTTCTCGCCCATGACCAC-3′ and 5′-CCCACCAGGTTGTTCTTCAG-3′ for CBLP, 5′-CGCGCCGTCACTTGTTTGTTG-3′ and 5′-GCCCACTTCTTTACCCAGCACCTC-3′ for ARS1, 5′-ACGACGCCATGGACAACATGTAC-3′ and 5′-ACCCAGTGCGCTCCGTTCAG-3′ for SAC3, 5′-TCGGTACTTGCAGCTGAGGTTAGG-3′ and 5′-ACACCGTCCAGCCCATGTATCTT-3′ for the SNRK2.1 3′ UTR, 5′-TATGGAGCGTGGGCGTTATCTTG-3′ (underlined sequence binds to the beginning of exon VII, whereas the remaining 5′ sequence of the primer binds to the end of exon VI) and 5′-CCTCCATGGTGATGCGCTT-3′ for SNRK2.1 exons VI and VII, 5′-ACGTGGCATGCAGCTCAT-3′ and 5′-CTTGCCACTTTGCCAGGT-3′ for SULTR2, 5′-ACGGGTCTTCGAGCGAATTGC-3′ and 5′-CGACTGCTTACGCAACAATCTTGG-3′ for SLT1, 5′-CCTCGCTCTCCTCGCTGCTG-3′ and 5′-CGGCCGACTTGGGTAATTGC-3′ for ECP76, and 5′-GGACGGCAGCATCATGGTGAGC-3′ and 5′-TCCACACGCCCTTGACCTTGAG-3′ for SBDP. The sizes of the amplification products were between 100 and 300 bp.
Analysis of Sequences
Sequences were analyzed using DNAstar software version 4.05 (Lasergene Navigator), BioEdit Sequence Alignment Editor version 5.0.9 (Department of Microbiology, North Carolina State University), the National Center for Biotechnology Information BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/), and Chlre3 (version 3) of the Chlamydomonas genome generated by the JGI (http://genome.jgi-psf.org/Chlre3/Chlre3.home.html).
Sequence data for SNRK2.1 cDNA v1 has been deposited with the EMBL database under accession number AM900768.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Putative cDNA splice variants and proposed mechanism that could lead to the PCR generation of artifactual splice variants.
Supplemental Figure S2. Alignment of SNRK2.1 splice variants.
Supplemental Figure S3. Detail of exon XI of SNRK2.1 v1 showing the alternative 5′ splice region and the 3′ UTR.
Supplemental Figure S4. Alignment of the Chlamydomonas SNRK2 protein family.
We thank Dr. Jeffrey Moseley for critical reading of the manuscript and for providing pJM43 vector and the JGI for generating a draft Chlamydomonas genome sequence, which has been invaluable for both gene identification and mutant analyses. We would also to dedicate this manuscript to Winslow Briggs in honor of his 80th birthday; Winslow has been both an enormously supportive colleague and an inspiration to all members of the Grossman laboratory.
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.plantphysiol.org) is: David Gonzalez-Ballester (davidg3{at}stanford.edu).
↵1 This work was supported by National Science Foundation Grant MCB 0235878 awarded to A.R.G. and by the Carnegie Institution, the Ministerio de Educación y Ciencia (Spain), and the Marie Curie OIF-6 (European Union).
↵[OA] Open Access articles can be viewed online without a subscription.
↵[W] The online version of this article contains Web-only data.
- Received January 9, 2008.
- Accepted March 2, 2008.
- Published March 7, 2008.


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Is it too early for winter engagement session inspiration? If your DC-area engagement session is in the coming months, then you're certainly ready for some wintertime loveliness! For their Washington DC wedding engagement, Carolyn and Graham took a romantic ride on their bikes to Meridian Hill Park near where they live. Even though it was a rainy and foggy day, they let their love shine through in front of the camera with Caitlin Dennis Photography.
Congratulations Carolyn and Graham!
Looking for more on engagements? Check out other local DC-area engagement photography sessions, or find out what to wear for your engagement and how to do your make-up for your engagement session!
Photo Credits: Caitlin Dennis Photography

subset: commoncrawl


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How to Beat the Goalie
As a coach and player I see this scenario all too often. A player gets a clean break, one on one with the goalie. They look down at their stick and shoot, or completely freeze up and then get too close to the goalie to do anything and jam the puck right into his pads or chest. Don't let this happen ...READ MORE +


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Chillaxed Denver Apartments
Sometimes it's hard to take a break, whether you're rushing to get a project in an office done before the looming due date, you're struggling through rush hour traffic to get to where you need to go, or you're simply just exhausted from stress. Our luxury Denver apartments at Alexan Cherry Creek offers no miracle cure, but our great amenities allow you the chance to find some peace right in the comfort of home, giving you more energy to enjoy the people and things you really care for.
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Take a deep breath and enjoy those fleeting moments of comfort here at Alexan Cherry Creek. Explore our soothing luxury Denver apartments and amenities today.


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As part of the Unified Community Platform project, your wiki will be migrated to the new platform in the next few weeks. Read more here.
Landing Party
Mission Chain
Landing Party is a level 2 mission available to Smugglers. It is obtained on Ord Mantell, immediately after the player arrives on the planet following character creation. This mission is the first part of the Smuggler class story line, and takes place in the area of Drelliad Village.
Background[edit | edit source]
Skavak has been waiting at Drelliad Village's spaceport in order to receive and pay for a shipment of blasters. Once the shipment arrives, however, separatist activity in the area increases; several air defense cannons are captured, preventing vessels from entering or leaving the area. Should the smuggler that delivered the weapons wish to leave the planet, the separatists' control of those cannons must be disrupted.
Summary[edit | edit source]
|After delivering a shipment of blasters to Republic forces, you find yourself trapped on Ord Mantell. Separatists have seized control of the local air defense cannon and are shooting down any starships flying in or out.
|~ Star Wars: The Old Republic, Landing Party mission description
Objectives[edit | edit source]
- Speak to Skavak
- Destroy the Remote Control Stations: 0/3
- Disable the Targeting Computer
- Return to the Hangar
- Defeat the Separatist Ambush
- Speak to Corso Riggs
Rewards[edit | edit source]
- 20
- Title: Captain
- Codex Entries: Captain, Skavak
- Drelliad Jacket
Notable quotes[edit | edit source]
|Skavak helped those separatists get in here. He stabbed us in the back!"
"Last thing I remember was Skavak sucker-punching me. Looks like he and his separatist buddies stole everything!
|~ Corso Riggs
|Hang on... where's Torchy? I don't believe this! Skavak stole my blaster! Torchy's a genuine BlasTech ALT-25 with magnatomic adhesion grip and side-mounted rangefinder. She's too good for Skavak.
|~ Corso Riggs
|Not to worry. The Republic Army runs Fort Garnik, but they let the boss do as he pleases. He keeps their supply lines open. Viidu's a smart guy with connections everywhere. If anybody can find your ship, it's him.
|~ Corso Riggs


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13 December 2018
FY18 Full-Year Results
13 December 2018
Online Annual Report
Online Annual Report 2018
13 December 2018
Annual Report
Annual Report 2018
100% of the donation goes to educational programs in holiday destinations.
Fourth consecutive year of double-digit earnings growth post-merger, with 10.9% increase in underlying EBITA1 and continued strong ROIC performance.
1 Based on constant currency growth


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The Wombat boxes
The Wombat Art Boxes are published in limited edition and hand-assembled in our Parisian workshops.
No. 39 - Betina du Toit
Betina draws inspiration from childhood, from an ideal of freedom, from autumnal colours and winter skies, upon which elegance is delicately frozen, revealing a hypersensitive nakedness.
No. 38 - Stanley Kubrick
Before his celebrated career as one of the 20th century's great artists, Kubrick was a photographer. His photographs are visually stunning, with dramatic compositions and shadowy, chiaroscuro lighting.
Our artists
World's most celebrated photographers or ermerging talents share their vision of the world with you.Discover


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Major food groups like carbohydrates, protein and fat are essential for survival, but vitamins nourish your body on a deeper level. Vitamins assist crucial bodily functions like digestion, immunity and growth. Here's a list of important vitamins you are probably missing out on.
(Also read: 13 Vitamins and Minerals That Help You Become Slimmer, Stronger and More Energetic)
All photos: 123rf.com


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Linda Buquet
- Joined
- Jun 28, 2012
- Messages
- 13,313
There is a really good discussion over at YouMoz / SEOMoz about the ethics of SEO especially as it relates to SMBs… and it's about Google and Spammers and SMBs that want quick easy rankings and marketing responsibility and… it's worth a read.
It was written by Marie Haynes, a member here at the Local Search Forum and the very 1st person I ever did Google Places Training for.
It's sort of a made up story about a Plumber, but a story everyone here should be able to relate to. She summarizes at the end with the following...
What do you think?
Comment here or there and share your thoughts.
It was written by Marie Haynes, a member here at the Local Search Forum and the very 1st person I ever did Google Places Training for.
It's sort of a made up story about a Plumber, but a story everyone here should be able to relate to. She summarizes at the end with the following...
I could really relate to many of Dana's comments - snippets below.Are SEOs Destroying Small Businesses? A Penguin Story - YouMoz | SEOmoz
The world of SEO is changing! I believe that the day is coming when there will be no easy ways to get backlinks. A good SEO knows how to network and where to cultivate links. And better yet, a good SEO can help with content creation that is going to attract natural links...and then promote that content!
But what do you think? Will today's linkbuilding strategies last? Are SEOs who use these tactics likely to cause their clients to get penalized? And more importantly, are poor quality SEOs destroying small businesses?
Following are a couple of my commentsdanatanseo
Still, we are all so susceptible to the idea of making it big overnight. We are so easily seduced because we are greedy. Good marketers know how to captialize on that greed, and SEOs marketing SEO are apt to tap into that just like anyone else selling stuff.
So, I'd argue, that the blame really should be shared by business owners looking for an easy, fast, cheap way to get what they want and SEO's who are irresponsibly giving it to them.
I do think that Google's lack of transparency and being very difficult to use for small business owners is a huge part of the problem. Google expects small businesses to learn how to submit sitemaps, shopping feeds, and schema.org markup. Big companies can hire people to do this for them, while the little guys just get further and further behind.
and later I said...Even though I specialize in local now, local IS currently mainly about the organic algo. So when SMBs come for help at the Google and your Business forum or my Local Search Forum asking why their ranking dropped - it's no longer as simple as checking to see if they have any G+ Local violations or dupes. You have to look at what they've done on the organic SEO side of the house and sometimes, as with Joe, it ain't pretty.
True of any business, but I think SMBs often want to take the quick, cheap, easy route to ranking and often the companies that make the biggest promises for the lowest buck are the link building SEOs. And even though I think business owners need to be accountable for their own online campaigns, I just think many have no clue what type of marketing is being done for them or what type of negative impact it can have.
Anyway, head over and read the whole discussion. Are SEOs Destroying Small Businesses? A Penguin Story - YouMoz | SEOmozDoug said: "Google created SEO as the cat and mouse game that it is. Personally, I'd be happy if Google made SEO easy and honest & open. The system they've allowed to flourish is one that hurts many small businesses as much as it helps others."
Actually I think the problem is the damn spammers! That's why Google can't make things transparent and easy. As soon as spammers find a way to game the system, Google has to find a way to thwart them. It's been that way for years. (Think old old days, tiny text or whatever.) Spammers do something, Google cracks down. Innocent webmaster does the same thing for the right reasons not trying to be spammy, not knowing it's a problem and the baby gets thrown out with the bath water.
I know this from being a Top Contributor at the Google Business forum and dealing with the Google Places guidelines all the time. There too, Google can't come right out and say "always do this and never do this" because that just gives the spammers and fake Place page builders a road map to follow. So guidelines are intentionally vague and SMBs break the rules all the time not even knowing it's a problem - because it's not clearly spelled out and can't be.
Google and the spammers - that's the biggest cat and mouse game of all IMO.
Not to negate what you are saying. I totally get it from your viewpoint, just explaining why Google can't make it easy and transparent.
What do you think?
Comment here or there and share your thoughts.


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hey everyone! happy wednesday! link up with jamie!
I'm loving that I ordered P90x a couple of days ago, and it is suppose to arrive tomorrow! Yay, then maybe I can start working off my muffin top!
I'm loving that today and tomorrow are my days off! I really needed a couple days just to catch up on sleep and decompress!
I'm loving that I'm starting to figure out my kindle! Love it!
I'm loving my man. Not going to lie, I am feeling so good in our relationship. The best I ever have. So crazy about my bearded love.
I'm loving sweet Dallas! Love her!


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Take It or Leave It? Ethics & Faith at Work
By Becca Broaddus
Integrity (n.): 1) firm adherence to a code of especially moral or artistic values, 2) an unimpaired condition, 3) the quality or state of being complete or undivided.
(Definition from Merriam-Webster Dictionary)
"What does it mean to have integrity? Most of my students say it's when you do what you say you're going to do. But I add to that, it should have some kind of moral good too," Chavanne Chair of Christian Ethics in Business Mitch Neubert said. "To have integrity is to be the same person Monday as you are Sunday. And yet, a lot of people live separate lives. Some of my research explores how people integrate their faith in the workplace, and what happens when they do."
Work and worship are often compartmentalized into secular and spiritual spheres for Christians. According to the Baylor Religion Survey (BRS), a national study of beliefs and values in the United States administered by Gallup, less than half of employed adults who attend religious services regularly indicated they often or always see connections between faith and work. Essentially, half of those who regularly attend church don't see significant connections between their work and spiritual life.
"Business schools don't teach about faith at work, but most churches don't either," Neubert said. "Many churches focus on being a good steward of the money you make at work, but overlook how you can be a light with your ethics and how your work can honor God and bless other people."
Director of the Princeton University Faith & Work Initiative David Miller created a framework on faith integration at work. According to the framework, when someone brings their faith to work, it manifests in one of four ways:
- Ethics—Faith motiving ethical behavior and excellence within the workplace. This could manifest itself as individual or collective ethics.
- Experience—Faith offering meaning to work as a place to live out one's calling and a context for utilizing one's unique gifts and talents in serving others.
- Enrichment—Faith assisting in work by providing strength, guidance and the capability to cope with difficulties or suffering.
- Expression—Faith being shared in word and deed as an example or witness to others.
Sixty-three percent of working adults who attend church regularly agreed or strongly agreed that their congregation promoted the ethical manifestation of "considering what is morally right when facing a tough decision at work," according to the BRS. The ethical framework is the most common form of faith integration at work, and it's particularly important for those people of faith who lead and shape the ethical culture of their organizations.
In fact, according to the Ethics Resource Center's 2013 National Business Ethics Survey of the U.S. Workforce, only 20 percent of workers reported seeing misconduct in companies where the ethical culture was perceived as "strong," as compared with 88 percent of workers who witnessed wrongdoing in ethically weak work environments.
In addition to the moral and faith-based benefits, an ethical workplace pays off. The Society for Human Resource Management suggested that organizations with positive, virtuous ethical cultures enjoy bottom-line and top-line benefits, including: higher employee job satisfaction, increased legal compliance and rule following, increased organizational commitment, increased cooperation, increased change management success, increased attraction of high-potential talent, lower turnover, lower healthcare costs and lower legal risk.
"You've got a resource to bring to bear in the workplace," Neubert said. "It's your deepest values, and in most cases, it's rooted in your faith."
Baylor Business Review, Spring 2018
3 Comments
What are the specific sources for the surveys mentioned in this article? I cannot find them through a general search
Thank you to Becca and Dr. Neubert's surveys pointing to the fact roughly 50% of "religious service attendees" do not see a work connection to their faith. It is a sad truth. I have seen it with my own eyes in work and investing over the last 18 years. Whereas a "Christian" who can quote Bible verses on Sunday, can take advantage of and financially hurt investors, employees, customers by Tuesday.
I think integrity is the wrong word to use. I would like to see whether future surveys can show whether the Christian business leader sees himself as a true servant leader, OR feels entitled and privileged. Sadly, I think surveys would show pride beats humility with too many Christian business leaders.


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Articles and Publications - More
Top Ten New Years Resolutions for Runners©
by Ken Mierke
The new year is finally here and it is time to start preparing for races. So many athletes are telling themselves, "This year will be different." Before you get too hyped up, remember that there are many little steps that contribute to your success on race day. Make plans right now to incorporate the following strategies into your preparation.
Resolution #1: I will train easy when I should this year.
Almost every runner conducts their long workouts and easy workouts too hard. This is very ineffective. Smart runners train at a very easy pace when working on endurance and very hard when it is time to go hard. Doing easy workouts at the proper intensity leaves an athlete well-rested and enables the hard workouts to be most effective. When working on speed, work on speed. When working on endurance, work only on endurance and slow way down.
Resolution #2: This year I will work hard on running technique and efficiency.
Every runner trains hard to become stronger and fitter, but few pay enough attention to efficiency. Fitness is limited by genetics, available training time, and several other factors, but efficiency is an area that holds huge potential gains. Runners can improve efficiency by 4 to 8 percent by working specifically on technique. This equals a minimum of 14.4 seconds per mile at 6 minute pace or 24 seconds per mile at 10 minute pace. Those are huge gains that are unlikely through hard training alone.
Resolution #3: I will engage in strength training to improve my running.
Serious strength training has many benefits for runners, including reducing injuries, improving efficiency, and raising the resting metabolic rate. Few runners take full advantage of it. Done properly, strength training is an extremely time efficient workout that has great value.
Resolution #4: I will consume much more protein this year.
Giving the body the right nutrients during training, racing and recovery is critical. Protein is the building block of the muscles, required to increase strength and for simple recovery from workouts. Few athletes consume enough protein. Recent research has determined that hard-training athletes need significantly more than previously thought. We recommend about 0.8 grams of protein per pound of bodyweight per day. Also, since your GI tract can only digest and absorb a certain amount of protein in one sitting, consume significant servings at least 4 times per day. Breakfast is frequently the biggest culprit, with few lean sources of protein. Be sure to get protein at breakfast.
Resolution #5: I will focus as much on mental preparation as on physical.
Working on mental skills is one of the most neglected aspects of run training. Most athletes work out to make their legs stronger, but many ignore mental strength. Focusing on mental skills is not a sign of a weak mind any more than focusing on long runs is a sign of weak legs. We train, physically or mentally, to make what is already strong even better. Developing mental skills improves an athlete's ability to focus on the task at hand and get the most out of the body despite the pain. Many athletes think about anything but the effort and the pain of racing at the red line (disassociation). Top athletes stay intensely tuned in to the pain and effort in order to constantly monitor the body's condition, pace appropriately, and maintain efficient technique (association).
Resolution #6: I will follow an injury prevention program.
Injuries are an ever-present risk for runners. Smart runners follow a comprehensive injury-prevention program that includes stretching, strength training, technique correction, and functional exercises to minimize the risk and severity of injuries.
Resolution #7: I will develop and maintain a process orientation instead of a product orientation this year.
All runners want to race fast, but there is so much more to the sport than that. We see many athletes get so caught up in getting faster so that they can have a good race that they fail to enjoy the workouts which comprise 99% of their involvement in the sport. Plan and train hard for your priority races, and enjoy them, but this year don't forget to smell the roses along the way. You might find that this is the road to that long sought PR.
Resolution #8: This year I will get guidance from a qualified coach.
Someone who spent four years studying exercise physiology knows more about how your body will respond than you do. In addition to a deeper knowledge base, a coach can be objective about your training and racing, which is impossible for the racer himself. Help from a qualified expert will help you not only race faster, but enjoy your running experience more.
Resolution #9: I will get leaner the right way this year.
In January so many of us begin to think about how we can lose a couple of pounds of fat and be leaner than ever before. Many rely on simply eating less, which not only does not work, but actually has the opposite effect. This year, eat enough food, be patient and make moderate changes. Reduce the amount of fat in your diet. Eat concentrated carbohydrates only after workouts. Eat a significant serving of protein at every meal. Eat large servings of lean meats, fruits, and vegetables.
Resolution #10: I will enjoy my racing this year!!!
Isn't this really the most important of all? Research shows that athletes perform their best when having fun. Even if winning races is more important to you than having fun, science says that having fun will make you faster. Putting a smile on your face may help as much as putting that disk wheel on your bike.
Ken Mierke, developer of Evolution Running®, author of Lean for a Lifetime: An Athlete's Guide to Losing the Last Ten Pounds and The Triathlete's Guide to Run Training, is a two-time world champion triathlete and a top coach. Ken can be reached at email@example.com


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11 years after cheating 31 job-seekers, senior citizen extradited from Singapore,…
A 60-year-old man was extradited from Singapore for allegedly cheating 31 job-seekers of Rs40 lakh in 2006. The Mumbai police arrested him on Sunday morning.
The Economics Offences Wing (EOW) of the Mumbai police had issued a red corner against Mohammed Sultan Kadir, after which he was arrested by the Singapore police where he had been living. Kadir's wife Majeeda was arrested in 2007 for her involvement in the case while Kadir was wanted.
According to an officer, Kadir had promised jobs to 31 people in shipping companies and took around Rs 40 lakh from them in 2006. "A month later, he took the people to Singapore and accommodated them into in a hotel. The next morning, he left for a meeting with the officials of the shipping company. He returned and promised that the firm would hire next month," the officer said.
He then returned to India with the victims, said the official.
"After this, the victims claimed that Kadir had disappeared and did not attend their phone calls. They gave a written application after which a case was registered with the EOW," added the official.
In 2007, the police arrested Majeeda as she had also allegedly accepted money from a few victims. The officials then issued a red corner notice against Kadir.
In 2017, the EOW received a tip off that Kadir had been living in Singapore. In May, based on the red corner notice, the Singapore police arrested him. A team of four officials from EOW left for Singapore last week to complete the formalities to bring him back to India.
On Sunday, the police arrested Kadir and presented him before the Esplanade Court where he was remanded in police custody.


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Return from Bombay without excess baggage - check
Get upgraded to Business Class on the flight - check
Smuggle common cold viruses into Dubai which torment for weeks - check
Mark blog anniversary - check
Start the gym - check
Get back in touch with a friend I'd always want to get in touch with - check
Go on a somewhat interesting date - check
Fix and service car - check
Get lost when dropping and picking up car from service station - check
Try out two new recipes - check
Neglect blog - check
Write out goals and plans - check
There, always helps to make a checklist. Haven't given up on the diary, Parmanu, it was just taking a while and encouraging procrastination. One of these days...


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Clay soils respond to fertiliser biocatalyst injected in-furrow
Strengthened root structure in barley and improved wheat yields have reinforced the Hannafords' commitment to liquid injection in South Australia's North East.
Injecting a liquid fertiliser biocatalyst in-furrow is helping South Australian cropping farmer Peter Hannaford unlock nutrients in his property's heavy clay soils.
- WHO: Peter Hannaford
- WHERE: Frances, SA – 32km north east of Naracoorte, South Australia
- CROP: 1400ha of wheat, barley, beans and canola
- EQUIPMENT: Liquid Systems (SA) PR-1 Module and Stacker Distribution Kit with Terminal Assembly
- Increased root structure
- Barley plants with signs of better phosphorous uptake
- Approx 300 – 500kg/ha wheat yield increases in 2016
Guided by Landmark senior agronomist Craig Hole, Peter purchased a Liquid Systems (SA) PR-1 Module in 2015 to improve soil nutrition and structure.
"The basis of any good cropping program is how well your soil is performing," said Craig, an agronomist for the past 30 years. "The challenge on Peter's property is balancing soil structure and nutrition. Some of his soils are sodic, so when it's wet they turn to slush and when it's dry they set like concrete. "You have trouble making nutrients available to plants in those conditions."
Using his Liquid Systems (SA) unit last season, Peter applied the Loveland liquid product, Foundation Liquid Manure (LM), a fertiliser biocatalyst that activates microbial activity in the soil, freeing nutrients for plant use.
"It helps speed up the breakdown process of any applied nutrients, by making the good microbes that are already in the soil far more active," said Craig.
"That means background levels of essential nutrients – especially phosphorus that is not mobile and can get tied up easily – become available to the plant. In addition, it has a very positive effect on soil structure. For Peter, that means reducing soil sodicity."
Craig says the impact from applying Foundation LM in-furrow was immediate, with a noticeable change to root structure in Peter's barley crops.
"The first thing we noticed was a more strongly developed root system, with more secondary roots and root hairs," said Craig. "The fine plant roots intercept more nutrition and moisture, so the more you can develop those, the more the crop can grow.
"Plus, waterlogging is common in the North East, so if you get a plant up and going more rapidly with a stronger root system, you've got more chance of getting it through a wet period."
The stem colour of Peter's barley plants was another interesting outcome. Barley that had received a liquid injection of Foundation LM had green stalks whereas those without it appeared purple in colour. "The purply stalks showed that those plants were struggling to take up phosphorus," said Peter.
Although Peter says exact yield numbers from trials conducted on his farm last year are yet to be verified, it is clear there were wheat yield increases.
"It does look good. We're waiting for the results to be analysed, but from what I saw in the header, there were increases of between 300 and 500 kilograms per hectare across my wheat crops," said Peter. "Based on that, I'll certainly inject liquids again this year."
Applied at a low rate ranging between 2.5L/ha and 5L/ha, ensuring every seed in every row receives the same amount of liquid is critical for the success of the application. "Five litres of Foundation LM over 10,000 square metres is not a lot of product, so you want to make sure it's applied accurately," said Craig. "It's crucial that it's not going out at 10L/ha in some areas and none in others. You want an even 5L/ha and it needs to be a constant stream, so you know that every plant can access it. If it's starting and stopping, then you've lost your advantage straight away.
"The accuracy of the Liquid Systems (SA) equipment, right across the bar, is second to none. The good thing with Peter Burgess' systems is they are built to a standard. And because he's a perfectionist, nothing goes out of his factory unless it's accurate and it lasts."
Consistency of application proved an early challenge for the Hannafords, due to their comparatively small seeder bar, at 9.75m wide. "In the beginning, we tried to put out a 25L/ha mix of water and Foundation LM but we couldn't maintain the pressure across my small seeder bar at such a low rate. But Peter Burgess offered us a special pressure regulator that was able to maintain lower rates and that solved the problem. Our application of Foundation LM mixed in water now ranges from 30L/ha to 33L/ha and the system is holding that rate well."
For the Hannafords, equipment reliability is also key at one of the most critical times in their farming program.
"You don't want issues when you're in the middle of seeding," said Peter. "While the Liquid Systems (SA) equipment might not be cheap, it is a very well built machine with no issues. And that's what Peter Burgess is all about. He doesn't want a machine out there that's going to create issues."
Another adjustment the Hannafords made on their Liquid System (SA) unit was the distance between the end of the injection tubing and the end of the stainless steel protective pipes. Initially, the pipes and the tubing were level with one another, but they found this set-up was prone to blockages, so adjusted the tubes to sit 3mm to 5mm below the pipes. "The tubes now flicker, which prevents any blockages. Peter didn't get a blockage at all last year," said Craig.
This year, the Hannafords plan to inject trace elements and a fungicide in-furrow, including copper, zinc and Flutriafol, which will be added to their 30L/ha mix of water and Foundation LM. Last year they applied granular fertiliser coated with trace elements. "Some of the soils on the Hannaford's property are quite trace element deficient – especially from a copper perspective," said Craig. "We hope to maximise their availability by applying them as a liquid at seeding. The issue with coated granular applications of trace elements is that they are not continuously available. By nature, granular fertilisers are interspersed along the plant line, so it is often only pure luck that plant roots access an adequate amount of product. An injected liquid product is readily available to the growing plant in a continuous stream. Perfect."


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Hi Everyone,
Today's inspiration is a CAS (Clean and Simple) card I created at camp.
I love the way it turned out and am thinking I might use this as a template for my Christmas cards this year.
I hope you like it.
Stamp: Hanglar Stanglar
Sentiment: Occassions (Darkroom Door)
Copics Used:
Skin: E000, E00, E21, E11, R11, Molotow
Clothes: B01, B02, B04, B05, C0, C1, C3
Heart: R12, R14, R17, R27
Glitter (Martha Stewart)
Foam Dots (American Crafts)
Until next time


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Happy as you could be!
Mira, MultiCh MultiW Tejarpsdalens Marion My Precious, BOB and BIS-4 veteran and 3rd best bitch on Malmö Int. today! Her brother Wilmer, T. Mandeville The Strong BOS veteran!
Thank's to our daughter Malin Lindahl for taking your valuable time to show her! Showed lovely as always!
Thank's to Judge in breed Tom Hehir IE. In veteran final Sanja Vretencic Serbia.


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Detailed financials available on request
The business was established in 2005, and comprises a team of PSIRA (Private Security Regulatory Authority) registered staff members who combine their talents as highly skilled technicians and who have become leaders in the installation and management of Access Control, Biometric and Closed Circuit Television (CCTV) security cameras in the Eastern Cape and Garden Route areas. Extensive work has been done for clients such as Algoa FM, McDonalds and Social Housing Project. The business is owner operated. All financial information can be verified with validated documentation. The business trading entity is a Sole Proprietorship and is not registered for VAT. Up to date Management Accounts are available for inspection as are the Annual Financial Statements to Dec 2017. Earnings Before Proprietors Income, Interest, Tax & Depreciation (EBPITD) R 789,784 | ROI 36% | Payback 25 months. Excellent opportunity for new owner to grow further or to acquire as a strategic fit to an existing business. Total price R 1,75 mil including stock. For more information contact the listing broker


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The Horror Writers Association (HWA) has announced Bruce Boston as Poet Guest of Honor for the Bram Stoker Awards™ Weekend 2013, to be held in New Orleans from June 13-16, 2013.
Bruce Boston is the author of fifty books and chapbooks, including the novels The Guardener's Tale and Stained Glass Rain. His poetry and fiction have appeared in hundreds of publications, including Asimov's SF Magazine, Amazing Stories, Weird Tales, Strange Horizons, Year's Best Fantasy and Horror, and The Nebula Awards Showcase. One of the leading genre poets for more than a quarter century, Bruce has won a record four Bram Stoker Awards for Poetry, a record six Asimov's Readers Awards for Poetry, and a record seven Rhysling Awards from the Science Fiction Poetry Association (SFPA). He received the first Grandmaster Award of the SFPA in 1999. His fiction has received a Pushcart Prize and been a finalist for the Bram Stoker Award for Superior Achievement in a Novel and the Micro Award for flash fiction. Bruce lives in Ocala, Florida, once known as the City of Trees, with his wife, writer-artist Marge Simon, and the ghosts of two cats.
HWA President Rocky Wood said, "Bruce is one of the most respected genre poets of recent decades. His work is original, compelling and challenging. We are pleased he has agreed to share his knowledge and experience as a poet with the Convention and I know attendees will be looking forward to his Guest of Honor panel and his readings. It will be interesting to hear him expound on how he composes across genres and also writes prose, including award nominated novels. As well as being highly awarded Bruce is well known for giving back to the genre through his active participation in the Horror Writers Association and tirelessly working to promote the poetic form."
"We are proud to have a Poet Guest of Honor, which recognizes the importance poetry has always played in the horror genre and undoubtedly always will," Wood added.
Bruce joins Ramsey Campbell, John Joseph Adams, Jeff Strand, Caitlin R Kiernan, Jonathan Maberry and Glenn Chadbourne in a stellar lineup of author, editor, poet and artist Guests.
The Bram Stoker Awards Weekend incorporating the World Horror Convention 2013 will be held at the Hotel Monteleone in New Orleans, Louisiana. The Horror Writers Association, the peak group for horror writers, is also hosting World Horror Convention as part of our regular Weekend. The HWA is a worldwide organization promoting dark literature and its creators. Started in 1985, it has over 700 members writing professionally in fiction, nonfiction, videogames, film, poetry, comics, and other media.


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At Monsanto, we're all about sustainable solutions. That's why we're committed to getting the public involved in a global conversation about the methods we're using in order to help grow enough food for a growing world. Ask your own questions regarding anything from honey bee health, GMOs, to sustainability in agriculture and more.
Whether it's in soybean or cotton, the Roundup Ready® Xtend Crop System will utilize herbicide-tolerant trait technologies to help maximize yield potential, weed control and quality.
In December 2015, Monsanto announced a first-of-its-kind program to introduce a model for carbon neutral crop production that will help reduce the carbon footprint of crop production.
Sustainability is a journey that presents a constant but welcome challenge: how can we push ourselves to achieve even more? How do we feed a growing planet in a changing climate?
We work to help farmers produce food in a sustainable way. We think holistically about how our food is grown so farmers have the tools they need to have better harvests - to make a plate of meats, grains, fruits and vegetables within reach for every family.
Update Sept. 26, 2014: USDA's report is welcome news for U.S. wheat industry
Today's report by the USDA affirms that no genetically modified wheat is in commerce and that the commercial seed and grain supply does not contain genetically modified wheat. This finding is supported by extensive testing by USDA, state universities, Monsanto and others. The USDA report also concludes that the unexpected detection of genetically modified wheat in Oregon last May was an isolated event on one farm, one field. Completion of the USDA investigation provides closure for the wheat industry as U.S. wheat growers continue to grow their global business.
Monsanto has been performing field trials with GM crops for over 20 years. "As a company, product stewardship and regulatory compliance are at the heart of everything we do," said Phil Miller, Monsanto's global regulatory affairs lead. "While we believe our compliance program is best in class, we continuously review our processes and procedures to improve them, including site selection, field trial isolation and verification and auditing of field trial locations."
Monsanto is committed to working with USDA to identify innovative stewardship practices, technologies and other opportunities to enable continuous improvement. Full compliance is our top priority. To ensure continuous improvement, Monsanto participates in both the USDA's Biotechnology Quality Management System Program and the industry's Excellence Through Stewardship Initiative.
As the report also notes, USDA is reviewing an unrelated situation involving closed-out genetically modified wheat field trials at Montana State University, and Monsanto is cooperating fully with that investigation. In addition, the agency noted there is no evidence that genetically modified wheat is in commerce. For more information, click here.
USDA has published a Q&A summarizing its investigation and emphasizing that there is no evidence that GM wheat is in commerce. That document is available here.
Monsanto has established this site as a resource for those interested in learning about ongoing developments related to the U.S. Department of Agriculture (USDA) investigation on the reported detection of Roundup Ready Wheat by one farmer in one field in Oregon. This section provides Monsanto's history with GM wheat; answers to questions you may have; and additional resources.
Monsanto is taking the matter seriously and is cooperating with the USDA fully. We are conducting a rigorous investigation to validate the scope of and to address any presence of a Monsanto Roundup Ready trait in wheat.
We are confident about the safety of Roundup Ready wheat and all of our products. The USDA has also underscored the Food and Drug Administration's position that there is no public health concern related to this report.
We are committed to working with the USDA, wheat growers, and other members of the wheat value chain to address the questions raised by the USDA's recent announcement so that the U.S. wheat industry remains strong.
Monsanto's Statement on Montana State University and GM WheatSeptember 26, 2014
Monsanto Statement On Test Results Of Wheat VarietiesJune 10, 2013
Monsanto Provides Validated Test Method for Roundup Ready WheatJune 3, 2013
Updated: Monsanto Statement on USDA GM Wheat ReportMay 31, 2013
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