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- © 2010 American Society of Plant Biologists
The genome of Thellungiella parvula, a halophytic relative of Arabidopsis (Arabidopsis thaliana), is being assembled using Roche-454 sequencing. Analyses of a 10-Mb scaffold revealed synteny with Arabidopsis, with recombination and inversion and an uneven distribution of repeat sequences. T. parvula genome structure and DNA sequences were compared with orthologous regions from Arabidopsis and publicly available bacterial artificial chromosome sequences from Thellungiella salsuginea (previously Thellungiella halophila). The three-way comparison of sequences, from one abiotic stress-sensitive species and two tolerant species, revealed extensive sequence conservation and microcolinearity, but grouping Thellungiella species separately from Arabidopsis. However, the T. parvula segments are distinguished from their T. salsuginea counterparts by a pronounced paucity of repeat sequences, resulting in a 30% shorter DNA segment with essentially the same gene content in T. parvula. Among the genes is SALT OVERLY SENSITIVE1 (SOS1), a sodium/proton antiporter, which represents an essential component of plant salinity stress tolerance. Although the SOS1 coding region is highly conserved among all three species, the promoter regions show conservation only between the two Thellungiella species. Comparative transcript analyses revealed higher levels of basal as well as salt-induced SOS1 expression in both Thellungiella species as compared with Arabidopsis. The Thellungiella species and other halophytes share conserved pyrimidine-rich 5′ untranslated region proximal regions of SOS1 that are missing in Arabidopsis. Completion of the genome structure of T. parvula is expected to highlight distinctive genetic elements underlying the extremophile lifestyle of this species.
The crucifer genus Thellungiella includes a number of species that have recently been recognized as models for identifying processes, pathways, and genes of importance in plant abiotic stress tolerance (Bressan et al., 2001; Xiong and Zhu, 2002; Taji et al., 2004; Amtmann et al., 2005; Griffith et al., 2007). In particular, Thellungiella salsuginea, previously known as Thellungiella halophila (Al-Shehbaz and O'Kane, 1995; Amtmann, 2009), has been studied for its extreme salt, cold, and freezing tolerance and for its efficient mobilization of resources in poor or degraded soils (Kant et al., 2008). Another species, Thellungiella parvula, has been reported to have slightly higher salt and drought tolerance, being otherwise comparable to T. salsuginea in cold and freezing response characteristics (Orsini et al., 2010). The stress tolerance behavior of both species is appropriate to their natural habitats: of the several T. salsuginea ecotypes that have been collected, all are from stress-prone locations, such as the delta of the Yellow River in China, the Yukon Territory of Canada, and the Rocky Mountains of the United States. Similarly, the T. parvula ecotype used in this study originated from a salt flat in central Anatolia, Turkey.
Because of their close phylogenetic relationships to Arabidopsis (Arabidopsis thaliana; O'Kane and Al-Shehbaz, 2003) and because many transcripts have nucleotide sequence identities with the better known model in the 90% range (Wang et al., 2004; Taji et al., 2008), focusing on Thellungiella spp. in order to derive a molecular-level understanding of adaptation to challenging environments appears particularly appropriate. Both Thellungiella species are comparable to Arabidopsis in terms of growth rates and size under similar growth conditions and in terms of fertility, seed number, and ease of transformation by flower dipping (Inan et al., 2004).
A number of tools for comparative molecular characterizations have been developed for T. salsuginea, including tagged mutants, EST collections, and full-length cDNA collections (Inan et al., 2004; Taji et al., 2004, 2008; Wang et al., 2004, 2006; Gong et al., 2005; Wong et al., 2005). To date, results of stress challenge studies have identified transcriptome responses that are broadly similar to those shown by Arabidopsis. Some universally stress-responsive genes, however, appear to be highly expressed in Thellungiella even in the absence of challenges. Others are induced only at much higher levels of stress than their isologs in Arabidopsis. In addition, altered gene expression patterns appear to be more specific to the stress that is experienced. This contrasts with transcriptional responses in Arabidopsis, which are much less discriminating and typically are global (Gong et al., 2005; Kant et al., 2006).
In Arabidopsis, salt tolerance, albeit weak, is based on the action of a complex of two proteins (SALT OVERLY SENSITIVE3 [SOS3], a sensor, and SOS2, a protein kinase) involved in sensing and responding to the influx of sodium ions. This complex activates a number of proteins (Halfter et al., 2000; Cheng et al., 2004; Quan et al., 2007; Lin et al., 2009), including SOS1, a plasma membrane-located sodium/proton antiporter. Activation of SOS1 mediates the export of the Na+ ions, which may also be redistributed throughout the plant (Qiu et al., 2002; Quintero et al., 2002; Shi et al., 2002, 2003). The SOS complex exists in many other species, including T. salsuginea, suggesting that it is a general feature of plants. Recently, the importance of SOS1 in stress tolerance in T. salsuginea has been established using RNA interference to generate lines with reduced SOS1 transcript and protein abundances. These lines showed salt sensitivity nearly as pronounced as that exhibited by Arabidopsis (Oh et al., 2009).
Like Arabidopsis, both T. parvula and T. salsuginea have relatively small genomes. The Thellungiella species have seven pairs of chromosomes (Orsini et al., 2010). For T. salsuginea, the diploid genome size is about twice that of Arabidopsis, approximately 260 Mb (Inan et al., 2004). We now estimate, based on flow cytometry, a diploid genome size of approximately 180 Mb for T. parvula. These values, in combination with the Arabidopsis reference genome structure against which sequences can be compared, make genomic sequence analysis approachable.
Here, we report on our initial analyses of the T. parvula genome structure based on sequencing using the Roche-454 platform. Our objectives were to shed light on differences in the overall gene structure and composition of the T. parvula genome in comparison with those of Arabidopsis and T. salsuginea. We compare regions in the T. parvula genome with two bacterial artificial chromosome (BAC) sequences from orthologous regions in T. salsuginea (Deng et al., 2009; Nah et al., 2009) as well as with orthologous segments in the Arabidopsis genome and, in a three-species comparison, analyze gene and genome structures. We include an analysis of promoter structures, the presence and organization of repeats, and transcript expression for one of the genes in this region, SOS1, in an attempt to distinguish characteristics of the glycophytic and halophytic versions of this important salt tolerance determinant.
T. parvula genome sequences were determined by scaffold generating de novo assemblies constructed with Roche-454 GS-FLX Titanium single and paired-end sequencing. The average read lengths were 360 bp or greater in each paired-end library sequenced, a significant improvement compared with the predecessor version GS-FLX. Further sequencing and a draft genome assembly are in progress. We deduced a minimal genome size of 163.6 Mb, based on the total length of the preliminary assembly contigs. An independent estimate to the genome size of about 180 Mb was obtained through flow cytometric analysis of propidium iodide-stained nuclei (Supplemental Fig. S1).
Overview of T. parvula Genome Assembly and Analyses of a Scaffold
Table I summarizes the statistics of a genome draft assembly. The single end sequence assembly by Newbler and cleanup gave some 21,000 pieces that contain 163.6 Mb of contig sequences, which constitutes the minimal predicted genome size of T. parvula. Addition of paired-end libraries further assembled the contigs into 3,149 scaffolds encompassing 153.7 million bases. The scaffolds contain gaps, and incorporation of the reads from Illumina sequencing with higher coverage is in progress to fill in the gaps. Upon completion, the genome sequence will be released to the public.
One of the larger scaffolds (scaffold 00254) of 10,011,287 bp was subjected to further analysis. Scaffold 00254 had a GC content of 35.79%, comparable to that of the Arabidopsis genome (36%). It had 346 gaps, with a mean predicted gap size of 757 bp, comprising 5.43% of the scaffold sequence. The scaffold contained overall 1.9% of repetitive sequences, including simple repeats, transposons, and retrotransposons, predicted by RepeatMasker. The distribution of repetitive sequences was not homogeneous, with the repeat contents usually very low at less than 0.5% over most of the sequences and dramatically increasing up to 15% toward the end of the scaffold sequence (Supplemental Fig. S2A). An ab initio open reading frame (ORF) prediction with FGENESH (http://linux1.softberry.com/berry.phtml) identified 2,229 ORFs, whose distribution showed a pattern opposite to the distribution of the repetitive sequences (Supplemental Fig. S2B). All 2,229 ORFs showed homology with an Arabidopsis gene locus at least for part of their sequences when subjected to BLASTn search against the Arabidopsis reference mRNA database (Supplemental Fig. S2C). When ORFs with total identity higher than 70% with Arabidopsis were considered, 73%, 15%, and 10% of the total predicted ORFs in scaffold 00254 showed the highest similarity with an Arabidopsis gene in chromosome 5, 3, and 2, respectively. To visualize the synteny between scaffold 00254 and Arabidopsis chromosomes, we provide a Circos plot linking the locations of the homologous ORFs between Arabidopsis and T. parvula. Scaffold 00254 showed extensive synteny with parts of Arabidopsis chromosomes 2, 3, and 5, with several rearrangement and inversion events, especially in the region showing colinearity with Arabidopsis chromosome 5 (Fig. 1).
T. parvula Sequences Compared with Arabidopsis and T. salsuginea
Scaffold 00254 contained regions orthologous to two recently published BAC sequences of T. halophila (now T. salsuginea; Deng et al., 2009; Nah et al., 2009). Here, we focus on these regions for a three-species comparison of genome sequences (Fig. 1, red bands on scaffold 00254). The gaps in the two focus regions were seamlessly filled with genomic PCR and Sanger sequencing with perfect matches in the adjacent sequences. Nah et al. (2009) isolated a T. salsuginea BAC sequence of 193.0 kb (termed here TS-1; National Center for Biotechnology Information [NCBI] GenBank accession no. FJ386403). The orthologous Arabidopsis genomic sequence is a 146.3-kb region (AT-1) located on chromosome 2. The corresponding genomic sequence in the T. parvula genome (TP-1; HM222924) is 134.3 kb in length. The second sequence analyzed here is orthologous to a randomly selected T. salsuginea BAC clone (82.9 kb; termed TS-2 here; DQ226510) presented by Deng et al. (2009). It is compared with a 79.3-kb DNA segment from Arabidopsis chromosome 5 and an orthologous 63.7-kb segment of T. parvula (TP-2; HM222925). Taken together, the overall G+C contents of the two regions in the Thellungiella species are slightly higher than those in Arabidopsis: 35.65% (Arabidopsis), 37.6% (T. salsuginea), and 36.3% (T. parvula).
Both regions for the three species were visualized using dot-plot alignments (Supplemental Fig. S3). The comparison identifies extensive colinearity between the species, with the highest degree between the two Thellungiella species. Several local rearrangements and deletions are obvious, indicated by boxes in Supplemental Figure S3, A and B, for the TP-1 region with respect to the AT-1 and TS-1 regions. In comparison with AT-2, TP-2 showed a large sequence inversion (Supplemental Fig. S3C). In contrast, TP-2 and TS-2 showed colinearity, with several expansions in TS-2 relative to the more compact TP-2 (Supplemental Fig. S3D).
Annotation of T. parvula Genes
The ORFs of the two T. parvula sequences were initially predicted ab initio using FGENESH. Homologous Arabidopsis and T. salsuginea genes were identified using BLASTx to the protein database of both species. Final ORF sequences were determined by FGENESH+ using the homologous Arabidopsis proteins as references. Supplemental Tables S1 and S2 list the predicted ORFs for TP-1 and TP-2, respectively, in comparison with the Arabidopsis and T. salsuginea sequences. ORFs for AT-1 and AT-2 were as annotated in TAIR9, and those for TS-1 and TS-2 as described by Nah et al. (2009) and Deng et al. (2009), respectively. The average gene densities are one per 5.17, 7.15, and 4.58 kb for TP-1, TS-1, and AT-1, respectively, and one per 2.89, 3.45, and 2.78 kb for TP-2, TS-2, and AT-2, respectively.
Ka/Ks (the ratio of the rate of nonsynonymous substitutions [Ka] to the rate of synonymous substitutions [Ks]) between the orthologous genes of three species were calculated, excluding genes of ambiguous orthology (Supplemental Table S3). For the two regions, 11 and 12 sets of genes, respectively, were analyzed. All gene pairs showed Ka/Ks ratios smaller than 1, indicating evolutionary selectivity among the orthologs favoring functional conservation. In all gene pairs, estimated divergences were older when comparing Arabidopsis with the two Thellungiella species than between T. parvula and T. salsuginea, averaging 12.3 million years ago between T. parvula and Arabidopsis and 12.4 million years ago between T. salsuginea and Arabidopsis but only 8.5 million years ago between T. parvula and T. salsuginea (Supplemental Table S3).
Synteny and Similarity
A graphical representation of the three-way comparison of distribution and spacing of genes in the two regions is given in Figure 2. TP-1 showed linear similarity in the arrangement of the genes with both AT-1 and TS-1 (Fig. 2A). One conspicuous difference was the absence of transposons in TP-1 that were present in AT-1 and TS-1; the putative deletion sites are indicated with black arrows in Figure 2A. Both TP-1 and TS-1 contained longer intergenic regions between the genes encoding SOS1 and their associated 5′ gene models compared with AT-1 (Fig. 2A, red arrows). Orthologs of At_c (TET14/AT2G01960) were missing in both TP-1 and TS-1 (Fig. 2A, red box; Supplemental Table S1). In parts of the sequence, however, TP-1 showed higher similarity with AT-1 than with TS-1. For example, orthologous gene pairs Tp05/At_b (AT2G01920) and Tp18/At_d (AT2G02061) are absent in TS-1, while Th_A and Th_B, similar to AT1G14610 and AT2G02030, respectively, were not found in either TP-1 or AT-1 (Supplemental Table S1). TP-2 showed similarity in gene order with TS-2 (Fig. 2B), but both TP-2 and TS-2 showed an inversion of genes relative to AT-2 in part of the sequences. The break point for the inversion was at the position of At15 (AT5G66410; Fig. 2B, red arrow; Supplemental Table S2). In both Thellungiella species, sequence fragments with homologies to AT5G66410 were identified at the break point of the inversion. A gene homologous to AT5G66410, however, could not be identified in the entire T. parvula genome sequence (M. Dassanayake and D.-H. Oh, unpublished data).
Supplemental Figure S4 presents a detailed analysis of exon/intron structures and the overall sequence similarities. In comparing TP-1 and TP-2 with corresponding Arabidopsis and T. salsuginea sequences, the plot identified all exons of the orthologous genes; levels of identities were usually higher than 70%. Short stretches of sequence with significant (greater than 75%) identities were also present in intergenic regions, possibly designating regulatory elements or noncoding RNAs. However, we could not identify any tRNA, microRNA genes, or known small interfering RNA loci in the intergenic regions in any of the three species compared here.
In TP-1, several intergenic regions lacked homology with either Arabidopsis or T. salsuginea [Supplemental Fig. S4A, denoted (a)–(c)]. These discontinuities appear to be based either on a disruption of synteny by the insertion or deletion of genes (a) or transposable element insertion (b). The discontinuity in (c), however, is not explained. The intergenic region upstream of TpSOS1 (TP1-10) was particularly interesting as it revealed homology with T. salsuginea but not with Arabidopsis (Supplemental Fig. S4A, dashed box). In contrast, differences in the intergenic regions between TP-2 and AT-2 involved translocations in TP-2 of genes whose Arabidopsis orthologs were from chromosomes other than chromosome 5: TP2-5, -7, and -16 are orthologs of AT3G53210, AT1G77370, and AT3G47840, respectively [Supplemental Fig. 4B, denoted (a)]. TP2-16 showed similarity with the AT-2 sequence for the latter half of the exons (Supplemental Fig. 4B, dashed box), which had also been observed in the comparison of TS-2 and AT-2 sequences (Deng et al., 2009).
Paucity of Repetitive Sequences in T. parvula
The three species show significant differences in repeat composition and frequency. Repeats in the form of transposable elements, however, are underrepresented (Fig. 2A) to absent (Fig. 2B) in T. parvula compared with the other two species. In both TP-1 and TP-2, analyses using RepeatMasker and searches using other databases failed to identify a complete transposable element, while corresponding T. salsuginea and Arabidopsis sequences contained various retrotransposons and/or DNA transposable elements. In total, these elements span 17.6% (TS-1), 13.5% (TS-2), 8.7% (AT-1), and 3.0% (AT-2) of the sequences (Deng et al., 2009; Nah et al., 2009). In TP-1, small stretches of sequences, less than 100 bp, suggested similarity with Arabidopsis transposable elements (Fig. 2A); such sequences amounted to less than 0.4% of the total length. TP-2 does not present even suggestions of transposable elements (Fig. 2B).
The paucity of repeats in T. parvula is less apparent, however, when tandem repeats are considered (Table II). As observed with transposable elements, sequence 2 has fewer tandem repeats than sequence 1 in all three species. Among the short tandem repeats, the dinucleotide ATn is found at a much higher frequency than other dinucleotide, mononucleotide, trinucleotide, and tetranucleotide repeats in all three species. A survey of short tandem repeats in plants reported ATn as the most abundant short tandem repeat in plant nuclear sequences (Wang et al., 1994).
Comparison of SOS1 Genes
SOS1 was originally identified in Arabidopsis as an important element in the salinity stress tolerance pathway (Shi et al., 2000). Figure 3 presents a comparison of the SOS1 promoters and coding regions in the two Thellungiella species and Arabidopsis. Homology between the promoter regions of AtSOS1, on the one hand, and TpSOS1 or TsSOS1, on the other, was distinctly lacking (Fig. 3A), while PipMaker plots identified conserved motifs between the promoters of TsSOS1 and TpSOS1. Although in the SOS1 gene, TpSOS1 showed higher identity with TsSOS1 than with AtSOS1 in general, different regions exhibited different levels of similarity. For example, TsSOS1 showed higher similarity to TpSOS1 in the N-terminal regions (Fig. 3B, exons 1–15), while AtSOS1 and TsSOS1 showed similar levels of similarity to TpSOS1 in the C-terminal regions (Fig. 3B, exons 17–22).
Comparative Analyses of Transcription
Comparison of SOS1 expression by reverse transcription (RT)-PCR previously revealed higher SOS1 mRNA levels in T. parvula than in Arabidopsis under both normal and high-salt conditions, while housekeeping genes were expressed at similar levels in both species (Oh et al., 2009). Here, we compared the expression patterns of genes surrounding SOS1 in the genome with that of SOS1, using quantitative real-time RT-PCR (Fig. 4; Supplemental Table S4). SOS1 showed 2.6- and 1.5-fold higher mRNA levels in T. parvula shoot and root tissues, respectively, than in Arabidopsis in the absence of stress. Salt stress induced SOS1 expression up to 9.5- and 4.7-fold higher in T. parvula shoots and roots, respectively, while induction was only 1.59- and 2.3-fold in Arabidopsis shoots and roots (Supplemental Table S4). Similar increases in T. parvula were found also after the addition of 350 mm NaCl, a concentration lethal for Arabidopsis ecotype Columbia. The higher SOS1 level in T. parvula compared with Arabidopsis is similar to the previous observation for SOS1 in T. salsuginea (Oh et al., 2009). For other genes tested in the vicinity of SOS1, differences in gene expression between the species were less dramatic (Fig. 4B; Supplemental Table S4).
Also included in the real-time PCR analyses (Fig. 5) were three genes not located on Arabidopsis chromosome 5 but found in TP-2 as genes that disrupted the colinear order of genes: AT3G53210, AT1G77370, and AT3G47840. Analysis was extended to the AT5G66460 ortholog adjacent to AT3G47840 and induced by abscisic acid and salt stress (Zimmermann et al., 2004). The AT3G53210, AT1G77370, and AT3G47840 orthologs were found as single-copy genes in the T. parvula genome sequenced by a combination of Roche-454 and Illumina/Solexa sequencing, which is at present in the final assembly phase (M. Dassanayake and D.-H. Oh, unpublished data). As expected, quantitative real-time RT-PCR amplified single species of amplicons using T. parvula cDNA for all three genes. The most dramatic difference in expression between Arabidopsis and T. parvula was observed with AT3G53210, annotated as encoding a nodulin family protein, in which the mRNA was significantly higher in the shoots of T. parvula than in Arabidopsis, and was further induced by salt stress. AT1G77370 showed generally higher mRNA abundance in T. parvula under all conditions, while the AT5G66460 basal expression was at a lower level, in both shoot and root, than in Arabidopsis. Expression of the AT3G47840 ortholog did not show significant differences between T. parvula and Arabidopsis (Fig. 5; Supplemental Table S4).
Analyses of Sequences Upstream of SOS1
Although functions and domains of SOS1 genes have been extensively studied in a few species, little is known about transcriptional regulation and promoter evolution in this gene family. In the three species, searches for cis-elements in the region up to 2,000 bp upstream from the SOS1 translation start site (predicted for T. parvula) identified several known stress-responsive elements. These included abscisic acid-response elements, anaerobic induction elements, a MYB-binding site involved in drought inducibility, heat stress-responsive elements, low temperature-responsive elements, and defense- and generally stress- and hormone-responsive elements (Supplemental Table S5).
A comparison of SOS1 transcript structures of halophytes and glycophytic species revealed a surprising difference between the two groups. When analyzing the 5′ untranslated regions (UTRs) of SOS1 genes, we found differences in CT content of a stretch extending 100 bp immediately upstream of the translation start sites of all available genes in the NCBI nucleotide databases and the published plant genomes available on Phytozome (http://www.phytozome.net). The highest pyrimidine ratio was found in the two Thellungiella species, and the largest difference between two species in the list separated Salicornia, a halophyte, from Manihot, a glycophyte (Fig. 6). Within the SOS1 5′ UTR adjacent to the translation start site, there are 12 and 18 CTT repeats in T. parvula and T. salsuginea, respectively. The Arabidopsis sequence shows three CTT repeats in the homologous region. In addition, these CT-rich sequences are associated with a region that could potentially form stem-loop structures in proximity to the initiation ATG codon (Supplemental Fig. S5).
Abiotic stress tolerance and sensitivity are unequally distributed among plant species. Although most species are stress sensitive, there are, interestingly, also stress-tolerant species in most families. Moreover, there is significant plasticity in environmental adaptation; even many species that are typically viewed as stress sensitive have salt-tolerant ecotypes. Thus, it seems highly likely that the basic machinery for adaptation to a stressful environment is universally endowed in plant genomes. It is unclear, however, whether the potential for adaptation has failed to develop in some environments or whether it has secondarily been lost in less demanding environments or through human invention. Given the importance of fresh water for agriculture and the increasing salinization of many irrigated lands, especially in dry, subtropical areas, it is important that we find the signposts indicating salinity stress tolerance and that we know about the essential processes and genes (Cheeseman, 1988; Hasegawa et al., 2000; Zhu, 2001; Flowers, 2004; Flowers and Colmer, 2008)
In this paper, we address these goals using high-throughput sequencing. Our strategy was to employ closely related plant species that exhibit very different salt stress tolerance characteristics. Although genome sequencing by Joint Genome Initiative is reported to have been completed, the available genomic resources for T. salsuginea are still limited. We have concentrated on two BAC clones for T. salsuginea (Deng et al., 2009; Nah et al., 2009) and the orthologous regions in Arabidopsis and T. parvula. A three-way comparison revealed similarities in the synteny and gene structures among the two genome segments. Synteny, as expected, is more pronounced among the Thellungiella species. Orthologous genes showed functional conservation, as supported by the Ka/Ks values. However, there were differences in genome features and sequences between the Thellungiella species and Arabidopsis and also within the Thellungiella genus. Some of these may be expected to be related to physiological differences that separate the three species.
Differences between the Thellungiella Species and Arabidopsis
Based on the Ks values of orthologous genes, the divergence of T. parvula and T. salsuginea from Arabidopsis can be placed at approximately 12 million years ago, and the Thellungiella species separated approximately 8 million years ago. The observed genome structures, inversions, and breaks in the colinearity are consistent with the time of divergence.
An obvious difference with respect to Arabidopsis and a specific feature of the Thellungiella species identified genome sequences around the SOS1 gene, one of the well-established salt tolerance determinants (Shi et al., 2000). Irrespective of extensive synteny of the ORFs and the conservation of gene structures for SOS1 between Arabidopsis and T. parvula, colinearity falls apart starting upstream of the first exon in SOS1 (Fig. 3). A hypothesis based on the analysis of SOS1 expression in T. salsuginea (Oh et al., 2009) suggested different expression strength or, possibly, transcript stability. This is further supported in T. parvula by the analysis of SOS1 mRNA levels in comparison with Arabidopsis (Fig. 4). Coupled with the significant sequence similarity in the SOS1 5′ UTR of halophytic species (Fig. 6), a closer examination of this transcript structural feature is appropriate because it may point to a fundamental glycophyte-halophyte distinguishing character that is less or not apparent when promoter cis-elements are compared. The hypothesis of a halophytic transcript structure is particularly attractive because of the very similar pyrimidine tracts, CTn, present in the promoters of Chenopodium quinoa and Salicornia brachiata, which are both recognized as halophytes (Sanchez et al., 2003; Lieth et al., 2008). The possibility of TsSOS1 and TpSOS1 5′ UTR sequences involved in the formation of hairpin structures with free energies of −4.0 and −5.40 kcal mol−1, respectively, close to the protein initiation codon enforces the notion that this structure appears to be important (Supplemental Fig. S5). Very similar stem-loop structures also characterize the 5′ UTR of the two other halophytes for which this sequence information is available.
Analyzing the conservation of (CnTn)n repeats on the 5′ UTR sequences in all stress-related and highly expressed genes is beyond the scope of this study. However, we investigated the emerging apparent pattern in genes with high expression levels or highly induced under abiotic stress in Arabidopsis and the two Thellungiella species. The availability of publicly available 5′ UTR sequences from T. salsuginea full-length transcripts limited candidate genes to a small collection. Nevertheless, a general pattern appears to be preserved in highly expressed genes, and the prevalence of CT-rich regions is more prominent in the two Thellungiella species compared with Arabidopsis. Arabidopsis heat shock protein 70 (AT3G09440) is known to be highly expressed under abiotic stress, including salt stress, both in Arabidopsis and T. salsuginea (Taji et al., 2004; Swindell et al., 2007). There is a pyrimidine-rich region close to the ATG initiation codon in the putative homologs of Thellungiella 5′ UTR sequences (Supplemental Fig. S6). Similar observations were found for cytosolic cyclophilin ROC3 (peptidyl-prolyl cis-trans isomerase; AT2G16600) and a transcription factor B3 (AT4G21550). Cyclophilins such as ROC3 are highly abundant cytosolic proteins known to be induced under abiotic stress (Chou and Gasser, 1997; Romano et al., 2004), and in addition to broad-range stress-responsive expression, the transcription factor AT4G21550 is also induced in specific developmental stages (Suzuki et al., 2007). All nine 5′ UTR sequences can form stem-loop structures close to the initiation codon.
Formation of this hairpin structure could affect transcription efficiency or the stability of the transcript, although this topic, while analyzed in the past in bacterial and viral systems (Simoes and Sarnow, 1991; Emory et al., 1992; Hellendoorn et al., 1997), has not received much attention in plants (Klaff et al., 1996). Pyrimidine-rich elements in the 5′ UTR of plants are known to have positive effects on transcription (Bolle et al., 1994), and they appear to be functionally conserved in diverse gene families across the plant kingdom. A similar pyrimidine-rich element in the 5′ UTR has been shown to confer high transcription levels without the need for other upstream cis-elements except for a TATA box in tomato (Solanum lycopersicum) hydroxy-3-methylglutaryl CoA reductase genes (Daraselia et al., 1996). Also, actin genes from Arabidopsis to Physcomitrella include 5′ UTR pyrimidine-rich stretches, which have been associated with very high levels of transcription (An et al., 1996; An and Meagher, 2010).
Finally, the possibility exists that the CTn sequence domains in halophyte species upstream of the ATG codon might be a target of DNA chromatin modifications that could exert additional expression control. Hypermethylation in Arabidopsis SUPERMAN and AGAMOUS genes is thought to be influenced through DNA secondary structures formed by pyrimidine-rich sequences (Jacobsen et al., 2000).
Differences between T. parvula and T. salsuginea
At approximately 180 Mb, the genome of T. parvula is significantly smaller than that of T. salsuginea, which is estimated as at least 260 Mb (Inan et al., 2004; Nah et al., 2009). The estimated genome based on a preliminary assembly permits us to assume approximately 163 Mb for T. parvula (Table I). Among the largest contributors to angiosperm genome size variations are repetitive DNAs and transposable elements (Ma et al., 2004; Meagher and Vassiliadis, 2005; Gaut and Ross-Ibarra, 2008). The comparison of orthologous genomic regions in this study between the two Thellungiella species showed T. salsuginea to be of consistently larger size, with the difference relating to the insertion of various transposable elements (Fig. 2). In contrast, the T. parvula genomic regions analyzed here showed a more compact gene organization than that seen in Arabidopsis, largely due to the absence of transposable elements and small repeat regions. If the prediction of a more compact structure is maintained across the entire genome, it is possible that T. parvula contains a higher total number of genes as compared with Arabidopsis. The lack of repetitive sequences extended to the bigger genomic region (scaffold 00254), with an overall repeat content as low as 1.9%. However, the T. parvula sequence also revealed regions rich in repetitive sequences; for example, the last 50-kb region of scaffold 00254 contained more than 15% repetitive sequences (Supplemental Fig. S2A), possibly indicating that this region was adjacent to a centromeric region in the corresponding T. parvula chromosome.
The genome size and complexity differences of the Thellungiella species can potentially provide explanations for their different growth habits and physiological trait differences. Differences include rosette formation in T. salsuginea but not in T. parvula and differences in stomatal conductance, stomata density, and water loss (Orsini et al., 2010). Differences also include growth parameters, with T. parvula as a fast-growing species that is adapted to high light that shows etiolation under Arabidopsis-type light conditions. In contrast, T. salsuginea shows a much slower growth rate, possibly a reflection of its adaptation to typically resource-poor habitats (Inan et al., 2004; Gong et al., 2005; Oh et al., 2009).
Toward the T. parvula Genome Sequence
T. salsuginea (salt cress) has been studied as a model halophyte, juxtaposing its physiology and genetic structure to that of Arabidopsis. Suggested mechanisms for tolerance to stress and nutrient-limiting conditions include control over stomatal conductance (Inan et al., 2004), greater discriminatory power of K+- and Na+-transport systems (Volkov et al., 2004; Volkov and Amtmann, 2006), and nitrogen utilization efficiency (Kant et al., 2008). However, a genetic basis of the differences in tolerance could only be deduced by attempts at establishing Arabidopsis-like resources and databases (Inan et al., 2004; Taji et al., 2004, 2008; Wang et al., 2004, 2006; Gong et al., 2005; Wong et al., 2005; Zhang et al., 2008; Oh et al., 2009). Merging the forthcoming T. parvula genome sequence in a three-way comparison that includes Arabidopsis and T. salsuginea (pending; http://www.jgi.doe.gov/sequencing/why/50029.html) will provide a powerful way to probe for the essence of halophytism, in particular if other crucifer species and ecotypes of well-studied species are included (Weigel and Mott, 2009; Orsini et al., 2010).
Halophytism is an intriguing ecophysiological adaptation to saline environments. However, defining its essence in molecular or genetic terms has been elusive, due in a large part to the observation that many different strategies have evolved by which species became tolerant or resistant to the stress. What appears to emerge is the existence of a small number of novel genes (Vinocur and Altman, 2005), while novel uses of kingdom-wide existing genes and proteins seem to provide the bulk of the mechanisms that form the basis of the stress-tolerance phenotype. Increasingly, halophyte transcriptomes are being analyzed and contrasted to glycophytic relatives (Kore-eda et al., 2004; Gong et al., 2005; Zouari et al., 2007; Dassanayake et al., 2009; Guo et al., 2009; Chelaifa et al., 2010). With the advent of numerous ongoing whole genome sequencing projects and comparisons throughout the plant kingdom, a multifaceted approach becomes possible. The result will be the integration of results on genome organization, gene and transcript structures, noncoding regulatory RNAs, regulatory mechanisms, biochemical complexity, and hormone- or metabolite-based signaling networks. From integration will emerge understanding of the processes that enabled individual species in all plant orders to use common genes in the evolution of a halophytic way of life.
Plant Material
The ecotype of Thellungiella parvula used here was collected around a saline lake in the Tuz Golu region occupying a depression on the central plateau of Turkey at an elevation of 905 m above sea level. For most of the year, the shallow (1–2 m) saline lake covers an area of approximately 1,500 km2. Water density is 1.225 g cm−3 (32.4% salt). The lake has no outlet; only a few surface streams feed into it, which may run dry in summer. During the summer months (with temperatures of greater than 40°C), the lake can evaporate completely. Precipitation in the area averages 250 mm per year. An inbred line was used for the genome sequencing and analyses.
Plant Growth and Genome Size
Genomic DNA was extracted from 10-d-old seedlings of T. parvula using the Genomic DNA Preparation Kit (Illumina). For gene expression analyses, total RNAs were isolated from 10-d-old seedlings of T. parvula and Arabidopsis (Arabidopsis thaliana) at the stage when the first true leaves emerge. Salt stress treatment and sample harvest were done as described by Oh et al. (2009). Genome size was determined by flow cytometry using a Becton-Dickinson LSR II (BD Biosciences). Homogenates of Arabidopsis and T. parvula were prepared by chopping leaves, and nuclei were stained with 40 μg mL−1 propidium iodide (Galbraith et al., 1983). Samples were analyzed at an event rate of 200 nuclei s−1. The configuration for propidium iodide detection comprised 488-nm laser excitation and a 630/22-nm band-pass filter. Chromosome numbers were determined in root tips after staining of mitotic cells (M. Dassanayake and D.-H. Oh, unpublished data).
DNA Libraries, DNA Sequencing, and Sequence Assembly
T. parvula DNA was isolated and prepared for Roche-454 GS FLX Titanium series long-read and paired-end sequencing and initial flowgram data processing following standard protocols (Roche-454 Life Sciences). Roche-454 sequences from eight single-ended genomic shotgun libraries and the respective titration plates were first assembled using Newbler (version 2.0.01.14) with a 40-bp minimum overlap and 90% identity. Sequences from one 2.5-kb, three 3-kb, two 8-kb, and one 20-kb paired-end libraries were added to the existing contigs to build scaffolds. A total of 24.89 million reads with a total of 5.9 Gb and an average read length of 360 bp were used for the de novo genome assembly with an inferred read error of 1.25%. A total of 99.2% of the bases used had a quality score of Q40 or greater. The two T. parvula genomic regions corresponding to the two BAC clones of Thellungiella salsuginea were selected using BLASTn. The sequence gaps in these regions were filled by PCR with specific primers targeted to flanking regions of the gaps in the assembly. Sanger sequencing of genomic PCR products was used to fill sequencing gaps and to confirm the sequences adjacent to the gaps.
Sequence Analysis and Annotation
The ORFs in genomic sequences were predicted ab initio with FGENESH (http://www.softberry.com) adopting Arabidopsis parameters. The predicted ORFs were annotated using the NCBI nonredundant databases and the TAIR9 database (http://www.arabidopsis.org/) with BLASTN and BLASTP programs. When analyzing TP-1 and TP-2, the ORF predictions were refined using FGENESH+ with Arabidopsis orthologous proteins as references. Repeat elements were identified by RepeatMasker (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker) and the program Tandem Repeats Finder version 4.04 (Benson, 1999). The homologous sequence comparisons were made with PipMaker (Schwartz et al., 2000) and dot-plot analysis (Sonnhammer and Durbin, 1995).
Quantitative RT-PCR
Primers were designed to have identical sequences and amplifying amplicons of identical size from T. parvula and Arabidopsis cDNAs (Supplemental Table S6). Quantitative real-time RT-PCR was performed as described previously (Poroyko et al., 2007; Oh et al., 2010). For the reference genes, orthologs of EF1α (AT5G60390) and cytochrome c (AT5G40810) from the T. parvula transcriptome (M. Dassanayake and D.-H. Oh, unpublished data) were used and gave similar results. Represented in Figures 4 and 5 are results normalized to EF1α from six repeats (two biological and three analytical repeats).
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers HM222924 and HM222925.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Comparison of relative DNA contents by flow cytometry.
Supplemental Figure S2. Analysis of scaffold 00254.
Supplemental Figure S3. Comparison between T. parvula genomic sequences TP-1 and TP-2 with three orthologous genomic regions in Arabidopsis and T. salsuginea.
Supplemental Figure S4. Syntenic relationships and homologies of sequences represented by percent identity plots.
Supplemental Figure S5. Hairpin structures in the 5′ UTR of the halophytic Thellungiella species.
Supplemental Figure S6. Comparison of CT tracts in the 5′ UTRs of various genes among the three species.
Supplemental Table S1. ORFs identified in TP-1, AT-1, and TS-1.
Supplemental Table S2. ORFs identified in TP-2, AT-2, and TS-2.
Supplemental Table S3. Comparison of Ka/Ks ratios in orthologous genes and the estimated divergence time of three species.
Supplemental Table S4. Comparison of relative gene expression levels by quantitative PCR.
Supplemental Table S5. Known cis-elements in SOS1 promoter regions up to 2,000 bp upstream of the translation start site.
Supplemental Table S6. List of PCR primers.
We thank Keith Frazier and Jyothi Thimmapuram (Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign) for advice and generous help.
↵1 This work was supported by King Abdullah University for Science and Technology of Saudi Arabia, by the World Class University Program, Korea (grant no. R32–10148), by the Biogreen 21 Project of the Rural Development Administration, Korea (grant no. 20070301034030), and by University of Illinois at Urbana-Champaign institutional support.
↵2 These authors contributed equally to the article.
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: Dong-Ha Oh (ohdongha{at}life.illinois.edu).
↵[W] The online version of this article contains Web-only data.
- Received August 3, 2010.
- Accepted September 8, 2010.
- Published September 10, 2010.


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Royal Love! Learn 5 Fascinating Facts About Princess Beatrice's Handsome New Beau Edoardo Mapelli Mozzi
It's getting serious! Princess Beatrice and her new beau, Edoardo Mapelli Mozzi, made their relationship red-carpet official when they recently attended the 2019 Portrait Gala in London on March 12. The new couple showed their support at the glam event and helped raise funds for the National Portrait Gallery's "Inspiring People" project.
Nearly a week later, Bea, 30, and her man — who goes by "Edo" — were seen celebrating their newfound love across the pond in New York City. Queen Elizabeth's granddaughter and Edo looked so happy as they walked hand-in-hand down the Manhattan streets.
Royal fans and admirers first began speculating that Prince William and Prince Harry's cousin was dating Edo in November. "Edoardo is a friend of Beatrice's family," a source previously dished to People. Although many outlets reported that the couple just met last fall, Bea and the handsome hunk have apparently known each other for years.
In honor of them recently stepping out in public together for the first time, we're bringing you five things you need to know about Bea's new beau — scroll through the gallery below to check out 5 fun facts about Edo!
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Sixth grader Leah Musson holds on to her Colton Wildcats flag Friday as the wind whips across the football field at Colfax. Kay Meyer keeps a grip on her flag in the background.
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The Carrot Crossbow is a Sniper weapon introduced in the 17.5.0 update. It is obtained after completing all 6 Easter Challenges from the battle pass.
It is a crossbow with a carrot acting as it's projectile, with a scope mounted on it's back. It has high damage, low fire rate, low capacity, and slow mobility. It is currently an 1 shot to the body at maximum efficiency.
It appears as a green crossbow-shaped stalk that has a carrot attached on it. On top of the crossbow there is green scope with a little white detail. The gun has a gray butt and some orange details on it.
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When reloading, the user inserts a carrot into the front socket of the bow. It outputs delay to input delay weapons, and also receives delay from output delay weapons.
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Recommended Maps
Equipment Setups
Bring a strong shotgun/melee/flamethrower/electrothrower to deal with enemies at close range. Furthermore, bring a fast-firing backup or special in case an enemy survives a shot from this weapon. Additionally, a high-mobility weapon is recommended for traversing around the map.
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- This is the second slowest firing sniper in the game, it just fires a bit faster than the One Shot despite the 25 fire rate.
- This is the second weapon that shoots a kind of vegetable,the first being Potato Implant.


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Etowah County
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Koch Foods Investing $50M In New Alabama Feed-mill
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Motus Integrated Technologies Creating 90 New Jobs In Alabama
The interior automotive parts supplier will invest over $15 million and create 90 new jobs at its new manufacturing facility in Gadsden, AL.


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Chu Shu Inc. Declares 'Silver Linings' To Be NY Fashion Week's One And Only Fashionable Shoe Liners
September 15, 2010
New York start-up Chu Shu Inc. declares Silver Linings an important fashion accessory for any 'fashionista' during 'Fashion Week' and the only fashionable antimicrobial shoe liners infused with Agion® silver that extend the life of expensive shoes by keeping feet fresh and dry when wearing them without socks with bacteria-banishing and moisture-wicking features and a non-adhesive grip backing.
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September 15, 2010
- Today, while New York city is abuzz with 'Fashion Week,' New York start-up Chu Shu Inc., proclaims that its fashionable antimicrobial shoe liners, Silver Linings, a must for any 'fashionista' who doesn't want to let her 'look' down with sticky feet and shoes when treading down her own personal runway. These slim and pretty half-soles protect her expensive and favorite shoes, from perspiring feet, when wearing them without socks or stockings. Infused with Agion® silver, these innovative inserts keep feet odor-free and dry, with bacteria-banishing and moisture-wicking features and a non-adhesive grip backing.
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Childhood Developmental Disorders come in many different forms. Many cause an inability to pay attention, exhibit appropriate communication skills, focusing on tasks at hand or even properly taking care of oneself into adulthood.
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Mack Rhoades' gives gut feeling on 2020 season, expounds on Baylor's COVID-19 testing
On Monday Baylor Athletics released their weekly report of both positive and negative COVID-19 tests that have been conducted since student athletes began their return to campus last week. After reporting that three of 59 tests were positive on June 16th, a week later the total had grown to eight with a total of 109 tests conducted.
Four of the athletes who tested positive for the virus were showing mild symptoms while the other four were asymptomatic.
In his weekly segment with SicEm365 Radio, Baylor Vice President and Director of Athletics Mack Rhoades said his staff feels blessed that the number has remained relatively low.
"I think overall we felt fortunate, felt blessed, to test 109 total so far, right, and to only have eight positives—four symptomatic, four asymptomatic," Rhoades said. "Again, felt fortunate. We are extremely grateful that the four symptomatic are mild.
A large concern for any athletes who test positive for the virus is the number of people who they have made contact with, including other players who are returning to campus at similar times. Some programs around the country including Big 12 program Kansas State, have shut down voluntary workouts because of the high exposure after double-digit positive tests to athletes.
Baylor isn't in danger of needing to end voluntary workouts currently, but Rhoades acknowledged they must continue to take proper steps to prevent that from happening.
"When you start doing the contract tracing, the primary contacts, even thought we don't release those numbers, those are relatively low," Rhoades said. "We're not in a position or a place where we're having to shut down anything or adjust anything to date.
"As we all know, this could change very, very quickly, but so far we've been really, really blessed."
With 109 test results back in hand as of Monday, Baylor is now focused on the next moves as they look to bring back the remaining student athletes who participate in fall sports at the beginning of July.
"Now next steps for us is for our infectious disease response team, right?" Rhoades said. "They're meeting today just reviewing and making sure everybody felt comfortable with how we onboarded the previous groups of student athletes and then do we move forward with the fall sports."
For now, dates for return are tentatively set.
"As mentioned before we have targeted right after for Fourth of July, July 6th, for our soccer and volleyball teams, and then July 7th for cross country, men's basketball and then July 13th for women's basketball."
According to Rhoades, the plan being executed has gone well so far, but it takes only a small break in a single link of the chain for it to all come crashing down.
"The first time somebody doesn't wear a mask or the first time somebody doesn't wash their hands, or the first time somebody says hey, you know what, I'm not going to go quarantined for 48 hours, I'm going to go play cards over at this house with 20 people, that's where we get into trouble and we falter," Rhoades said.
"It has, it's been a dose of reality. It's probably made us less optimistic about, you know, completing an entire season without any interruptions because now it's right here in front of us and you're dealing with it, right?
"But, you know, I still—maybe I'm just not very smart, but I'm still optimistic that we'll play and that we'll do it in a really safe manner and a very, very healthy manner, and where our student athletes feel comfortable about it."
Get your Baylor mask today through SicEm365!


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Cheekh Episode 25 ARY Digital – Review
Cheekh comes on board with a thrilling episode which manages to grab our eyeballs through and through. Cheekh episode 25 elucidates some central developments which will turn the tables soon.
Cheekh episode 25 begins with Mannat (Saba Qamar) who undergoes a panic attack when Wajih (Bilal Abbas) comes to visit her. Therefore, she is shifted to the mental ward where, as per Aqil Zada (Shabbir Jan)'s instructions, she is given a heavy dose of medicines so that she loses it. Afterward, Haya (Azikeh Daniel) comes to know about the reality about Wajih's crime and she is dumbfounded. Moreover, Shayaan (Emad Irfani) after witnessing Mannat's bad condition; comes to blows with Wajih and tries to kill himself with a gun.
Cheekh Episode 25 is an absolute entertainer which manages to give us goosebumps at various levels. The episode has a lot to offer, and after a long time, Cheekh has engrossed us in terms of good content. I believe there is something huge going to happen in the next episode, which will open up new avenues of the plot. Haya and Wajih's conversation is classic and summarizes the sibling challenges of our society.
Sacred Games Season 2 Trailer: The Worst is Yet to Come!
The episode entails a lot of good performances: Shabbir Jan's wickedness is to die for. He very masterly delivers a genuine performance whereas Azikeh Daniel outshines in her role categorically. Saba Qamar has less to do in the episode but still performs impactfully. Bilal Abbas is the king of expressions, as he flawlessly displays the damaging sentiments. Emad Irfani looks different when angry, in a good way. On the whole, Cheekh has closed with a cliffhanger and I reckon that a plethora of plot-twisting advancements is on their way.
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Let's talk about the elephant in the room. No, not the on-going and successful 3-day state visit of Donald Trump to the United Kingdom, where post-Brexit negotiations are already happening. No, but now let's take a bit of time to go over what BIG TECH are going to be facing very soon, for their partisan lies over their platforms and search engines, in a war on information, filled with incredible conservative bias.
Yes, Google and Amazon are going to get closer regulatory scrutiny over concerns that they're actually two monopolies, that no-less, only favour one side of political sphere, with a streamlined, biased service provided, which favours the left, leftist views and leftist websites, whilst censoring and limiting the reach of content that's not abiding by their beliefs.
President Donald Trump's DOJ have now launched an official Anti-Trust investigation into Google's parent company, Alphabet Inc. and now the Democrat controlled House Judiciary Committee plans to investigate giant tech companies, promising a "bipartisan look at competition in the digital markets," according to House Judiciary ranking member Doug Collins (R-Ga.)
Shares of both Google and Amazon fell on Monday, over the reports that the two companies would be facing US regulatory scrutiny, after many years of objections from many rivals and plenty of politicians.
Alphabet Inc. stocks fell 6% on Monday, to $1,036.23 and has now surrendered all gains in 2019. Whilst, Amazon stock gave up 5% to finish at $1,692.69, it's lowest stock value since March 2019.
The Federal Trade Commission and Department of Justice are looking closer at Amazon and Google. The DOJ will focus on Google. Whilst, the FTC will focus on Amazon and insider knowledge tells us that both companies are very nervous about what is about to unfold.
Politicians on both sides, Democrat and Republican, have made breaking up big tech companies high up on their agenda, with both Elizabeth Warren and Bernie Sanders backing these moves, as well.
Whilst, President Donald Trump maintains that Google's algorithmic search results are biased against right-leaning content. Many people know this very well, and it's nice that this is now being addressed via the DOJ Anti-Trust probe into Google and Amazon.
This also casts a shadow over Google's online advertising, with critics pondering its ability to combine ad revenue to search results, which offers them an anti-competitive edge.
Facebook also took another hit in the stock market on Monday, with a 7.5% decline down to $164.15 a share on Monday. As we all know, Facebook have faced numerous scandals over its data practices in recent times, and Mark Zuckerberg has recently expressed a will to be regulated, in the light of recent data usage scandals.
However, Google and Amazon do not share the same willingness, to be regulated, but soon comes the probe, launched by the DOJ.
Well, in an ideal world, wouldn't it be nice if Google provided an unbiased search engine, an unbiased advertising service and an unbiased and open service for all people to use equally?
The same can be said about Amazon's bias against conservative voices and websites, in stifling sales revenue for websites that do not adhere to their globalist and socialist view point. Plus, the same can also be said of Instagram and Facebook, for their incredible bias, occurring prior, and right after the US 2016 election, which has been on-going, ever since.
Whatever happened to an equal playing field? So, are we now witnessing a digital war on information and the stifling of different view points in big tech, which aren't aligned with the leftist and socialist mentality?
We believe so, and perhaps this is why the Department of Justice are serving Google with an Anti-Trust probe at this time? Many people cannot wait to see what will happen next regarding this phenomenon of censorship and bias.
Hopefully, this will culminate in a more open and broad minded service provided by the big tech companies, such as with services provided from Google, Amazon, Facebook and the apps that they own, like Instagram, being a level playing field for all.
Since the bias of big tech companies is very well known, and finally now, its going into the investigative stages, to potentially break up, unearth and further regulate them for their biased ways.
Moreover, what Google and other big tech companies are doing in censoring and filtering out opposing voices to their globalist agenda, is in fact, illegal under Section 230 of the Communication Decency Act (CDA), making it a criminal act to not service users with the same right to share information.
The recent outage of Google, Instagram, Snapchat and other apps worldwide, just a few days ago, has been rumoured to be a direct causation of the upcoming investigations into the big tech companies, in that they were trying to hide their censorship tracks. But, you won't ever hear that from them…
Just imagine if refunds and compensation would even be provided to those that have used their biased services, yet had their website reach, advertising revenue and social media stats limited by the big tech companies algorithms' of censorship. Yes, due to their conservative bias, many have not been provided a fair and just service. Well, wouldn't that be nice and fair, if things were finally to change to a level plain.
The DOJ Anti-Trust probe into Google, would probably find out lots of information that the public would wish to know about their biased practices within the world's online sphere. Just maybe? Here's to hoping and have a great day!
Story by The Narrator
Featured Photo Credit: Google


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31-Jul-2020 | Zion Market Research
As per analysts from Zion Market Research, the global beverage cartoners market was capitalized at US$ 619.93 million during 2019 and is estimated to surge at a CAGR of 5%, during 2020–2026. The beverage cartoners are the incorporation of different machines in a single unit. The beverage cartoners are well-designed machines that fabricate cartons from flat blanks, make safe them by pre-cut tabs & slots, and lastly, seal by utilizing heat or adhesives. The global beverage cartoners market is mostly expanded in regions like North America and Europe. The global beverage cartoners market is projected to be mainly swayed by the high demand from applications like dairy products and fruit juices in the beverages industry.
Some of the key manufacturers in the market are SIG Combibloc Group, Inc., Tetra Pak, Econocorp, Inc., Krones AG, Elopak AS, RA Jones & Co. Inc., Visy Industries Holdings Pty Ltd., Gerhard Schubert, and Shanghai Joylong Industry Co. Ltd. Many regional and unrecognized contenders are anticipated to contribute to the worldwide beverage cartoners market during 2020–2026.
Inclination Toward Ready-To-Drink Beverages To Accelerate The Global Beverage Cartoners Market
The beverage cartoners market is projected to be deeply benefitted by the surging influence toward ready-to-drink beverages and the augment in intake of aseptic packaging products. The beverage cartoners form packaging in such a manner that they are helpful for foodservice suppliers who do not have adequate storage space for putting refrigerators. Also, the ergonomics contemplation in the beverage cartoners is proven to be a driving aspect of the market. The key players in the beverage cartoners market are aiming to surge the manufacturing capacity by making it more ergonomic. The augmenting consumption of on-the-go food has resulted in the surge in demand for packaging solutions that augment the shelf-life of the product that drives the market of beverage cartoners.
The global beverage cartoners market is categorized on the basis of the product type, end-use application, output capacity, and geographical classification. Based on product type, the market is segmented into brick carton machines and gable top machines. Based on the output capacity, the market is divided into 9,000-12,000 packages/hr, below 9,000 packages/hr, 12,000-24,000 packages/hr and above 24,000 packages/hr. Based on the end-use application, the market is classified into fruit juices, dairy products, carbonated soda, RTD tea & coffee, water, and alcoholic beverages.
Europe Projected To Report The Highest Growth In The Global Beverage Cartoners Market
Based on the geographical analysis, the global beverage cartoners market is divided into North America, Europe, Asia Pacific, Latin America, and Middle East & Africa. Germany has reported an impressive development in the manufacturing of beverage cartoners. Concerning value, it towers over other nations in Europe and beyond. Germany will contribute to over 10% of the worldwide beverage cartoners manufacturing in the coming years.
Browse the full "Beverage Cartoners Market - By Product Type (Brick Carton Machines and Gable Top Machines), By End-Use Application (Fruit Juices, Dairy Products, Carbonated Soda, RTD Tea & Coffee, Water, and Alcoholic Beverages), By Output Capacity (9,000–12,000 Packages/hr, Below 9,000 Packages/hr, 12,000–24,000 Packages/hr, and Above 24,000 Packages/hr), And By Region - Global Industry Perspective, Comprehensive Analysis, and Forecast, 2020 – 2026." Report at https://www.zionmarketresearch.com/report/beverage-cartoners-market
The global beverage cartoners market is segmented as follows:
By product type:
By output capacity:
By end-use application:
By region:
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Zion Market Research is an obligated company. We create futuristic, cutting-edge, informative reports ranging from industry reports, the company reports to country reports. We provide our clients not only with market statistics unveiled by avowed private publishers and public organizations but also with vogue and newest industry reports along with pre-eminent and niche company profiles. Our database of market research reports comprises a wide variety of reports from cardinal industries. Our database is been updated constantly in order to fulfill our clients with prompt and direct online access to our database. Keeping in mind the client's needs, we have included expert insights on global industries, products, and market trends in this database. Last but not the least, we make it our duty to ensure the success of clients connected to us—after all—if you do well, a little of the light shines on us.
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When Donald Trump ran for office in 2016, immigration reform served as the hallmark of his presidential campaign. Since the president's time in office, he has stood by his vows to reform the American immigration system. This has subsequently attracted a considerable amount of backlash from Democrats and other Trump critics.
Over the course of Trump's presidency, he has repeatedly affirmed his support for legal and merit-based immigration. New developments recently confirmed that immigrants who are likely to rely upon welfare may not receive access to green cards.
Reviewing the Latest Change Regarding Immigrants' Access to Green Cards
The ruling from the Trump administration declares that government officials may employ the "public charge" rule. This rule states that stricter green card access is designed to promote economic independence amongst immigrants while preventing U.S. taxpayers from having to pay for immigrants to reside in America.
A statement from U.S. Citizenship and Immigration Services (USCIS) Acting Director Ken Cuccinelli reads as follows:
"The principle driving it is an old American value, and that's self-sufficiency. It's a core principle, the American Dream itself, and it's one of the things that distinguishes us; it's central to the legal history in the U.S. back into the 1800s."
Cuccinelli moreover noted that immigrants should manage to "stand on their own two feet" thus generating personal success along with America's success.
Green card restrictions are most likely to impact immigrants who may rely upon food stamps, Temporary Assistance for Needy Families (TANF), and Supplemental Security Income (SSI). This stipulations come in addition to the more traditional factors which include education levels, economic standing, age, and physical health.
Criticism of the Trump Administration's Immigration Policies
The Trump administration has faced considerable backlash for their immigration policies. Limiting access to green cards is another measure which many Democrats will likely brand as racist.
When the left censures Trump-era immigration policies, they claim that these policies are rooted in disdain for immigrants or "the other." Those who favor Trump's immigration reform regularly maintain that the left's perception is inaccurate. Instead, conservatives state that merit-based and legal immigration is the ultimate endgame, not the collective prohibition of immigrants altogether.


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Question about Krups Coffee Makers & Espresso Machines
Posted by Joseph Danick on
SOURCE: My Krups won't brew
The culprit is probably the thermal fuse. It's under the bottom cover, and you have to have Torx security bits (the ones with the holes in the middle) to remove the three screws holding it in place. Sadly, the thermal fuse is not resettable. Once it's tripped, it's dead. If you have this problem, forget about getting Krups or their lame service agent, smallappliance.com, to repair it. Both contacts I had with Krups were unsatisfactory. The factory/customer service folks say that there are no parts available and to buy a new coffee pot. Great solution. You can damn well believe it won't be a Krups. Or is it ****? An otherwise perfectly functioning unit gets sent to the landfill because the folks at Krups can't be troubled to find the part number. Idiots. For your info, there are thermal interrupters made by several manufacturers. The trip temperature on the breaker is 318 degrees, C. Something in the neighborhood of 200 degrees C would probably work, but would have to be soldered in. If you are comfortable with doing that, it's a solution.
Posted on Apr 17, 2008
SOURCE: stops brewing before all water empties from reservoir
I went to Hamilton Beach site and read the troubleshooting. I followed there cleaning instructions and reduced grind time on my coffee and the machine is working perfectly again. I stressed over this problem for a long time before following their operating instructions. Have had the machine for over two years now and it's back to working perfectly. i.e. Run a pint of white vinegar through it. Wait 30 minutes and run another full vat of water....wait again and run another full vat of water. If you're grinding your own coffee, you may be grinding it too fine. Otherwise Hamilton Beach recommends the kind of coffee to use. Also the problem maybe too dense of a filter. This is all info I got on the H. Beach troubleshooting site. Good Luck !!! Mine is back to working great !
Posted on Dec 13, 2008
SOURCE: black and decker spacesaver coffee maker
There are two heat stages in the Spacesaver. One is the carafe heater you can see while the other is an assembly within the main housing. This assembly contains a heater, thermostat, and a section of metal tubing with high temperature silicon connectors at each end. when the water storage tank is inserted it fills the heated section of the tube and when activated the water boils as it passes through the one way valve and forces the water into the coffee container. This steam driven surge repeats until all water has been moved to carafe. There are several failure modes associated with this assembly an I have two. 1. is the heater opening which I have and 2. is the thermostat degrading causing incomplete transfer and ultimately no transfer because it brings the temp down prematurely. Another is crud on the check valve so many problems - no solutions
Posted on Jun 01, 2009
SOURCE: cleanded brew unit as instructed - brew unit miss error
check small switch behind brew unit in machine maybe your brew unit dont touch switch.
Posted on Aug 30, 2009
SOURCE: Coffee maker won't start brewing process
brew light is off
Posted on Feb 08, 2010
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