text:
Plant parasitism represents an extraordinary interaction among flowering plants: parasitic plants use a specialized organ, the haustorium, to invade the host vascular system to deprive host plants of water and nutrients. Various compounds present in exudates of host plants trigger haustorium development. The two most effective haustorium inducing factors (HIFs) known for the parasitic plant Triphysaria versicolor (T. versicolor) are peonidin, an antioxidant flavonoid, and 2,6-dimethoxybenzoquinone (DMBQ), an oxidative stress agent. To date, two genes involved in haustorium initiation in T. versicolor have been identified: TvQR1, a quinone oxidoreductase that generates the active HIF from DMBQ, and TvPirin, a transcription co-factor that regulates several other DMBQ- responsive and –non-responsive genes. While the expression of these genes in response to DMBQ is well characterized, their expression in response to peonidin is not. In addition, the pattern of polymorphisms in these genes is unknown, even though nucleotide changes in TvQR1 and TvPirin may have contributed to the ability of T. versicolor to develop haustoria. To gain insights into these aspects, we investigated their transcriptional responses to HIFs and non-HIF and their natural nucleotide diversity.
Here we show that TvQR1 and TvPirin are transcriptionally upregulated by both DMBQ and peonidin in T. versicolor roots. Yet, while TvQR1 also responded to juglone, a non-HIF quinone with toxicity comparable to that of DMBQ, TvPirin did not. We further demonstrate that TvPirin encodes a protein shorter than the one previously reported. In the T. versicolor natural population of Northern California, TvQR1 exhibited remarkably higher molecular diversity and more recombination events than TvPirin, with the highest non-synonymous substitution rate in the substrate recognition and catalytic domain of the TvQR1 protein.
Our results suggest that TvQR1 and TvPirin have most likely evolved highly distinct roles for haustorium formation. Unlike TvPirin, TvQR1 might have been under diversifying selection to maintain a diverse collection of polymorphisms, which might be related to the recognition of an assortment of HIF and non-HIF quinones as substrates for successful haustorial establishment in a wide range of host plants.
Keywords:Parasitic plants; Haustorium; Nucleotide diversity; Allelic polymorphism; Population genetics; Transcriptional responses
The plant kingdom is usually associated with autotrophy as most plants produce their own nutrients via photosynthesis. Plant parasitism presents a divergence from this generalization as parasitic plants derive all or part of their nutrients and water from their host plants. About 4,000 parasitic plant species are widely distributed among various taxa and over diverse environments, ranging from arctic to tropical climates. Some of the best known parasitic plants include the Christmas ornament mistletoe, the world largest blooming flower Rafflesia, the fragant oil producing sandalwood, and the debilitating agricultural weeds dodder, witchweed (Striga) and broomrape (Orobanche). Although diverse in morphology, reproductive aspects, and life habitats, all parasitic plants share a specialized organ - the haustorium - which they evolved to assure a successful life cycle [1]– [4]. Parasitic plants develop haustoria either near their root tips or along their stems in response to selective chemical factors released by their hosts [5,6]. The haustorium initiates via localized cell expansion and division that is coupled with haustorial hair growth in many species. Following attachment and penetration of the host's roots or stems, the haustorium establishes a vascular continuity between the parasite and its host through which water and nutrients are channeled to the parasite to benefit its own growth and development. By so doing, parasitic plants disturb many developmental and physiological aspects of host plants [1,2,5,6]. Consequently, parasitic plants have a tremendous impact on community ecology among plants, animals, microbes, and even the surrounding physical environment [7]. Therefore, studies on parasitic plants encompass a range of interests, including anatomy and development, cellular physiology, gene regulation, population genetics, phylogeny, ecology and evolution. Here, we focus on a variety of aspects of host perception and recognition by parasitic plants.
The perception and recognition by parasitic plants of haustorium inducing factors (HIFs) released by host plants is best documented in root parasites of the Orobanchaceae family (many previously classified with Scrophulariaceae). The first three HIFs identified, xenognosin (i.e., host recognition factor) A, xenognosin B, and 2,6-dimethoxybenzoquinone (DMBQ), are phenolic compounds [8]– [10]. Additionally, various quinone and flavonoid HIFs were subsequently identified and characterized [11,12]. Further studies on HIF biogenesis, activity, and recognition in the two root hemiparasites Striga spp. and Triphysaria spp. have shed light onto the chemical signaling by DMBQ, which is involved in the first steps of the haustorium developmental pathway [13]– [16]. The genus Striga contains prolific weeds causing substantial damage to cereal crops mostly in Africa [17,18], whereas Triphysaria is a wild native genus in the grassland communities of Northern American coastlands [19]. The current model for haustorium initiation proposes that certain unidentified peroxidases or laccases released by parasitic roots catalyze the production of DMBQ at the host contact site, which diffuses towards the parasitic plant [14,15]. Next, a quinone oxidoreductase from the parasite converts DMBQ from its inactive form into the transiently active single-electron free radical form with the suitable redox potential for haustorium induction. This process is hypothesized to be the first step of the haustorium organogenesis pathway [13,16].
To date, only two parasitic plant genes involved in haustorium development have been identified, TvQR1 and TvPirin, both from the root parasite Triphysaria versicolor (T. versicolor) [13,20]. The TvPirin protein is a general transcription co-factor that up-regulates several DMBQ-responsive and –non-responsive genes [20]. TvQR1 encodes the aforementioned quinone oxidoreductase, which catalyses the reduction of quinones [13]. In comparison to the HIF DMBQ, this enzyme displays even higher catalytic activities for three other non-HIF quinones, one of which is juglone [13]. Consistent with its biochemical role, TvQR1 gene expression is rapidly up-regulated in T. versicolor root tips exposed to either maize or Arabidopsis root exudates containing active HIFs, DMBQ or the non-HIF juglone [11,21,22]. By contrast, up-regulation of TvPirin in root tips only occurs in response to DMBQ and Arabidopsis root exudates [21].
Most identified HIFs are quinones like DMBQ, which are known to be abundant in plant exudates and have also been associated with allelopathic effects (i.e., inhibition of neighboring plant growth and development) and defense against pathogens [23]. At concentrations below 10-4 M, quinone HIFs induce haustorium development in parasitic plants [8,11,24,25]. However, at concentrations between10-4 M and 10-3 M, they impart the negative effects of oxidative stress to parasitic plants that result in root necrosis and a reduction in the frequency of haustorium formation [8,11,24,25]. Further underscoring this quinone toxicity is the enrichment of transcripts involved in oxidative stress and detoxification pathways in T. versicolor root tips following exposure to DMBQ or host root exudates [21]. Like DMBQ, juglone is a well-known allelopathic compound present copiously in root exudates of the black walnut tree [26], but unlike DMBQ, juglone lacks the haustorium-inducing capability [21,24]. The fact that juglone is a better substrate for TvQR1 than DMBQ [13] correlates with the faster and stronger up-regulation of TvQR1 expression in T. versicolor root tips upon juglone exposure [21,22]. The only non-quinone HIF known to date is the flavonoid peonidin [11]. Peonidin belongs to the anthocyanin class of compounds present in red colored flowers and fruits that act as potent antioxidants by scavenging free radicals, and play various roles in plant development and stress protection [27]– [29]. Although peonidin is a HIF as potent as DMBQ for T. versicolor[11], its role in the regulation of TvQR1 and TvPirin gene expression during haustorium development has not yet been investigated. Furthermore, the availability of peonidin, a non-toxic HIF, DMBQ, a toxic HIF, and juglone, a toxic non-HIF, offers the opportunity to utilize these three chemicals to de-couple the two seemingly intertwined pathways triggered by the quinone HIFs - namely the oxidative stress response and haustorium organogenesis.
To understand how TvPirin and TvQR1 contribute to the perception of various phytochemicals present in the rhizosphere and, thus, to the initiation of haustorium development, it is essential to survey their patterns of polymorphism in natural populations of T. versicolor. Since so far only a single allele of each of these two genes has been reported [13,20]– [22], we examined the pattern of nucleotide polymorphism of TvQR1 and TvPirin by using 20 T. versicolor individual plants from natural populations of the Northern Californian grasslands. We found strikingly higher allelic diversity in TvQR1 compared to TvPirin, with the highest non-synonymous substitution rate in the catalytic domain of the TvQR1 protein. In addition, we provide evidence that TvPirin encodes a protein shorter than the one previously reported [20], and show that TvQR1 and TvPirin are differentially regulated by HIFs and non-HIF. Our results suggest that TvQR1, unlike TvPirin, has maintained a high level of nucleotide diversity, which might reflect its important role in the perception of various phytochemicals from host plants.
Results and discussion
TvQR1 and TvPirin are differentially regulated by haustorium inducers and non-inducers
To compare the regulation of TvQR1 and TvPirin, we selected two HIFs and a non-HIF displaying overlapping and differential biological properties on parasitic plants: DMBQ with the dual role of an oxidative stress agent and haustorium inducer, peonidin as a non-toxic HIF, and juglone as a non-HIF quinone and strong oxidative stress agent. In T. versicolor in vitro haustoria formation assays, both DMBQ and juglone reduced haustorium formation frequency and caused root necrosis at concentrations of ≥ 50 μM (Table 1 and Figure 1A), in agreement with previous observations [11,24]. Although either 10 μM DMBQ or 30 μM juglone alone did not result in root necrosis or a reduced frequency of haustorium formation, the combination of both treatments did (Table 1), further substantiating quinone toxicity to the parasite. On the other hand, peonidin had no toxic effects on T. versicolor, even at 1 mM concentration (Table 1 and Figure 1A). Using Northern blot hybridizations, we examined the spatial and temporal pattern of transcript levels of TvQR1 and TvPirin in response to DMBQ, peonidin, and juglone in three areas of T. versicolor seedlings - root tips, the remainder of the roots, and shoots - at 1, 3, and 5 hours (h) after treatment. TvQR1 was more strongly up-regulated by the non-HIF juglone than by the HIF DMBQ (Figure 1B), corroborating its higher substrate affinity for juglone [13]. On the other hand, TvPirin was similarly up-regulated by both the HIFs DMBQ and peonidin, but not by the non-HIF juglone (Figure 1B). These up-regulation patterns were observed only in roots but not in shoots (Figure 1B), consistent with a role for TvQR1 and TvPirin in haustorium development only in the root. We further confirmed the differential expression of TvQR1 and TvPirin in response to these HIFs and non-HIFs in T. versicolor root tips at 2–3 h post-treatment by reverse transcription quantitative polymerase chain reaction (RT-qPCR) (Figure 1C). Our results suggest that TvQR1 is associated with both the oxidative stress response and haustorium initiation, while TvPirin appears to be mainly involved in haustorium development. Determining whether TvPirin is exclusively regulated by HIFs and only involved in haustorium initiation would require the analysis of additional non-toxic HIFs, which so far have not been identified.
Table 1. Effects of different HIFs and non-HIFs on haustorium formation and root necrosis in T. versicolor
Figure 1. Expression of TvQR1 and TvPirin under haustorium-inducing and -non-inducing conditions. (A) Toxicity effects of HIFs and non-HIFs on T. versicolor roots and haustorium formation. D20: 20 μM DMBQ, D100: 100 μM DMBQ, J50: 50 μM juglone, P1000: 1 mM peonidin. (B) Northern blot analysis of the spatial and temporal expression of TvQR1 and TvPirin following treatment with HIFs and non-HIFs. 0: no treatment, H: water, E: 0.1% ethanol (solvent of peonidin), D: 20 μM DMBQ, J: 10 μM juglone, P: 30 μM peonidin. TvRuvB DNA helicase is a constitutively expressed gene. (C) Differential expression of TvQR1 and TvPirin under different HIFs and non-HIFs measured by qRT-PCR. H: water, J: 10 μM juglone, D: 20 μM DMBQ, P: 30 μM peonidin.
TvPirin encodes a 288 amino acid long protein
The TvPirin gene was initially identified as an incomplete EST (Accession BE574904.1) from a suppressive subtractive hybridization (SSH) library enriched for T. versicolor root tip transcripts that are up-regulated by DMBQ [22,30]. To determine the sequence of the full length TvPirin transcript, we performed 5′ and 3′ rapid amplification of cDNA ends (RACE) reactions and RT-PCR with primers complementary to both ends of the RACE clones using a pooled RNA population isolated from T. versicolor root tips with haustoria induced by 20 μm DMBQ. Alignment of the 5, 6, and 4 independently recovered 5′ RACE, 3′ RACE, and full-length cDNA clones, respectively, revealed a TvPirin open reading frame (ORF) encoding a 288 amino acid (aa) long protein with a 5′ untranslated region (UTR) of 64–68 base pairs (bp) (Additional file 1). However, the previously published TvPirin cDNA sequence (Accession JN606867 in [20]; clone TrVeBC2_244713 in [31] and in http://ppgp.huck.psu.edu/search.php webcite) has a coding capacity for a 322 aa long protein [20]. This cDNA sequence was created by contig assemblies following 454 and Illumina sequencing of the T. versicolor transcriptome [31]. In comparison to the sequence of the TvPirin full length cDNA reported here (Additional file 2), that of the TrVeBC2_244713 clone contains an additional 80 bp in the 5′UTR and an extra 100 bp in the 3′UTR ( http://ppgp.huck.psu.edu/search.php webcite; [31]). The extended 5′UTR in clone TrVeBC2_244713 contains an ATG codon that was assigned as the translation start codon and explains the longer coding sequence of the previously published TvPirin ORF (Accession JN606867 in [20]). In order to resolve these discrepancies regarding the TvPirin transcript 5′UTR and ORF, we performed in silico experiments and quantitative transcriptional assays.
Additional file 1. Identification of TvPirin full-length cDNA and open reading frame.
Format: PDF Size: 263KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 2. TvPirin open reading frame encodes a 288-amino acid long protein.
Format: PDF Size: 306KB Download file
This file can be viewed with: Adobe Acrobat Reader
First, BLAST-searches for the presence of other TvPirin contig assemblies from the T. versicolor transcriptome ( http://ppgp.huck.psu.edu/search.php webcite, [31]) uncovered seven clones with homology to the TvPirin cDNA clone TrVeBC2_244713 (Additional file 2A). Two of these clones (TrVe62GuB1_70666 and TrVe3GB1_60414) had the same orientation as the TvPirin cDNA and lacked the extended 5′UTR. The remaining 5 clones had the reverse orientation and 5′ UTRs of variable lengths. One of these clones (TrVeGnuB1_1027) had the same length and shared 100% identity in reverse complementation order to the TrVeBC2_244713 TvPirin cDNA. Another reverse-complemented clone (TrVe61FuB1_8246) shared discontinuous identity with the TvPirin cDNA sequences in both orientations (Additional files 2A and 3). These observations suggest that these seven clones represent either transcripts from genes other than TvPirin or artifacts of the contig assembly. Similar BLAST-searches in the T. versicolor SSH root tip contig libraries enriched for transcripts up-regulated by DMBQ ( http://www.plantsciences.ucdavis.edu/yoder/lab/Sequence_index.html webcite) identified two clones, Contig5118 and Contig492, with homologies to the 5′ and 3′ half of our TvPirin cDNA sequence, respectively, and without the extended 5′UTR of the TrVeBC2_244713 clone (Additional file 2A).
Additional file 3. Discontinuous identity between Accession JN606867 and clone TrVe61FuB1_8246.
Format: PDF Size: 539KB Download file
This file can be viewed with: Adobe Acrobat Reader
Second, we performed RT-qPCR on total RNA isolated from T. versicolor root tips following exposure to two HIFs (DMBQ and peonidin) and a non-HIF (juglone), using primers chosen for amplification of either the extended 5′UTR or the shorter 5′UTR of the TvPirin transcripts. The shorter 5′UTR amplicon displayed the same up-regulated expression pattern as the TvPirin ORF amplicon, whereas the prolonged 5′UTR amplicon did not show any significant up-regulation in response to either DMBQ or peonidin (Figure 1C), indicating that the extended 5′UTR does not authentically belong to the TvPirin transcript.
Collectively, our in silico and RT-qPCR results support that the shorter 5′UTR is the bona-fide 5′UTR of the full-length TvPirin cDNA. Consequently, the TvPirin protein contains 288 aa (Additional file 1B) and not 322 aa as previously reported [20,31] (Additional file 2B). The formerly published TvPirin cDNA sequence likely represents an artifact of 454 and Illumina sequence assembly. The artifactual nature of the TvPirin TrVeBC2_244713 clone is further supported by the fact that BLAST searches for TvQR1 sequences in the T. versicolor transcriptome ( http://ppgp.huck.psu.edu/search.php webcite, [31]) did not yield any TvQR1 complete ORF, suggesting that caution should be applied when using the T. versicolor transcriptome assembled by 454 and Illumina sequencing methods to assign gene structures. Our findings reinforce that for T. versicolor, gene annotation for individual gene studies needs a careful and thorough molecular cloning approach coupled with verification by gene expression analysis until a completely and properly annotated genome is available.
TvQR1 and TvPirin exhibit distinctive natural allelic diversity
The TvQR1 cDNA (Accession AF304461), originally isolated from the same SSH library used to clone the full-length TvPirin cDNA [20,22,30], is a partial cDNA clone containing the complete ORF but lacking the 5′ and 3′UTRs. We cloned the full-length TvQR1 cDNA using the same procedures carried out for TvPirin as described above, and recovered 9 independent full-length TvQR1 cDNA clones representing 9 different TvQR1 cDNA alleles (Additional file 4). The aforementioned 4 independently recovered full-length TvPirin cDNA clones also corresponded to 4 different TvPirin cDNA alleles (Additional file 1). In order to understand the patterns of nucleotide polymorphisms of these genes, we conducted a molecular population genetic analysis by using natural strains collected from a population in Northern Californian grasslands (Figure 2). We randomly selected 20 plants from the in vitro haustorium formation assays, of which 6 were responsive to DMBQ (designated as R1-R6), 8 responsive to peonidin (r1-r8), 3 non-responsive to DMBQ (N1-N3), and 3 non-responsive to peonidin (n1-n3) (see Methods). Genomic sequences from the start codon to the stop codon were obtained for each gene from the same plant by the Sanger sequencing method.
Figure 2. Map showing the T. versicolor seed collection sites.
Additional file 4. Multiple alignment of full-length TvQR1 cDNA clones.
Format: PDF Size: 141KB Download file
This file can be viewed with: Adobe Acrobat Reader
The complete alignments of all 40 alleles from these 20 plants showed sequences of 1962 and 2011 nucleotides long for TvQR1 and TvPirin, respectively (Additional files 5 and 6). We found striking differences in nucleotide diversity between the two genes. While there were only 9 single nucleotide polymorphisms (SNPs) in TvPirin (Additional file 6, Table 2), TvQR1 contained 350 segregating sites in addition to 31 insertions/deletions (indels) throughout the entire gene body, with indels present only in introns, and with the highest diversity in intron 3 (Additional file 5, Table 2, Figure 3A). This confers TvQR1 nucleotide diversity two orders of magnitude higher than that of TvPirin. Likewise, the numbers of synonymous substitutions and non-synonymous changes in TvQR1 are 55 and 27 times higher, respectively, than those of TvPirin (Table 2). Furthermore, four-gamete tests detected a higher level of recombination in TvQR1 (Rm = 32) compared to TvPirin (Rm = 2) (Table 2, Figure 3B), which might have contributed to the generation of the extremely high number of haplotypes in TvQR1. In TvQR1, the highest number of recombination events was estimated in the last exon (Figure 3B). As all these individual plants came from one single population of a restricted geographical location (Figure 2), this high level of intra-populational variations of TvQR1 is even more extraordinary.
Table 2. Population genetic summary statistics of TvQR1 and TvPirin
Figure 3. Distinctive natural allelic polymorphisms of TvQR1 and TvPirin. (A) Sliding window analysis of nucleotide diversity of TvQR1 and TvPirin. Solid and dashed lines are silent (synonymous and intron) and non-synonymous nucleotide diversity, respectively. Shaded regions indicate exons. (B) Minimum estimates of recombination across TvQR1 and TvPirin. Within each line, at least one recombination event was detected by the four-gamete test. Shaded regions indicate exons.
Additional file 5. Multiple sequence alignment of all genomic TvQR1 alleles.
Format: PDF Size: 547KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 6. Multiple sequence alignment of all genomic TvPirin alleles.
Format: PDF Size: 381KB Download file
This file can be viewed with: Adobe Acrobat Reader
Next, we examined the diversity of TvQR1 and TvPirin genes at the protein level by aligning the aa sequences deduced from the ORFs of their genomic and full-length cDNA clones. InterProScan runs from Swissprot for these two proteins identified the alcohol dehydrogenase (ADH) GrosE-like and the ADH_N_2 Rossmann domains in TvQR1 (Additional file 7), and the Pirin N- and C-terminal domains in TvPirin (Additional file 8). Although the crystallized structure of the human Pirin homolog revealed the β-barrel structure of the N- and C-terminal domains [32], no biochemical functions for either domain have been identified. By contrast, in redox enzymes such as ADH or quinone oxidoreductases, the ADH GrosE-like domain is the catalytic site with the oxidoreductase activity and substrate specificity [33,34], while the ADH_N_2 Rossmann domain is the NAD(P)-binding site where the one-electron addition/reduction reaction takes place [35]– [37].
Additional file 7. TvQR1 protein domains and amino acid diversity.
Format: DOC Size: 79KB Download file
This file can be viewed with: Microsoft Word Viewer
Additional file 8. TvPirin protein domains and amino acid diversity.
Format: PDF Size: 199KB Download file
This file can be viewed with: Adobe Acrobat Reader
As predicted from the higher number of non-synonymous substitutions described above, the TvQR1 protein exhibited more aa variations than the TvPirin protein (Additional files 7 and 8). A total of 13 aa changes were present in the 288-aa TvPirin protein with no preferential domain of high polymorphic level, and 12 of these variations belonged to rare alleles where the polymorphism occurred only once as singletons in the 34 deduced proteins (Additional file 8). On the other hand, only 13 of the 33 aa variation sites in the 329-aa TvQR1 protein sequence were singletons among the 29 deduced proteins (Additional file 7). The ADH GrosE-like domain of TvQR1 presented the largest cluster of aa polymorphisms. Its 14 changes within the 64-aa stretch (22%) are 3-fold higher than the 17 changes distributed over the remaining 266 aa residues of the protein (6.4%). Notably, the other domain of TvQR1, ADH_N_2 Rossmann, contained only 9 changes in the 130-aa stretch (7%), with 2 residues at positions 289 and 305 having 4 and 3 variants, respectively (Additional file 7). We further looked for aa changes in these two proteins that occurred exclusively in the non-responsive plants in order to identify potential mutations associated with the loss of haustorium formation phenotype. However, such changes were not found (Additional files 7 and 8), suggesting that loss-of-function mutations could reside in the promoter and/or other regulatory elements of the genes.
Collectively, these population genetic analyses revealed a remarkably higher level of molecular diversity, at both nucleotide and aa levels, of TvQR1 compared to TvPirin in the T. versicolor natural populations from Northern California. It is noteworthy that the highest aa polymorphism in TvQR1 appears in the ADH GrosE-like domain, which determines substrate specificity. Thus, by analogy to the extremely divergent LRR domain of the plant resistance R genes involved in specific pathogen recognition [38]– [43], the ADH GrosE-like domain of TvQR1 may have been under diversifying selection that sustains its molecular labile state in order to enable rapid changes aimed at recognizing a large variety of quinone substrates from host root exudates. At least 19 species belonging to 14 different families in both monocot and dicot clades are natural hosts of T. versicolor, and multiple hosts are often parasitized simultaneously by T. versicolor in the fields [19]. Therefore, having the ability to recognize a multitude of host factors from such a wide range of hosts would confer this parasitic species an evolutionary advantage. We hypothesize that parasitic plants might have evolved a mechanism to exploit the abundant allelopathic chemicals in their environment and turn the phytotoxicity they initially encountered into a haustorium-inducing cue for their benefit. Maintenance of TvQR1 molecular diversity could be a mechanism enabling this evolutionary process. High level of nucleotide diversity in synonymous sites as well as in non-synonymous sites of TvQR1 may suggest that the long-term selection has been acting on this gene, as it is expected to elevate nucleotide diversity of closely linked sites while non-synonymous sites are more likely to be the direct target of selection. Those patterns are indeed observed in the plant R genes that are suggested to be under long-term balancing selection (e.g. [41,42]). TvPirin, on the other hand, as a transcription co-factor, probably interacts with other proteins with specific conformations for DNA binding, and thus would be subject to more evolutionary constraints in order to maintain specific protein-protein interactions, which might have led to the much lower molecular diversity observed here.
Our molecular analysis has shown that, in the natural T. versicolor populations from Northern California, the only two genes known to date to be required for haustorium initiation, TvQR1 and TvPirin, display highly contrasting patterns of molecular polymorphisms. Especially, the nucleotide diversity of TvQR1 is 97-times higher than that of TvPirin, suggesting that these two genes would have been subjected to different selective pressures, possibly reflecting their distinct roles in phytochemical perception during host recognition. This difference is further manifest in their differential regulation by HIFs and non-HIFs. Since TvQR1 responds to both HIFs and non-HIFs, it might not only function in haustorium development but also in the oxidative stress response. The non-toxic HIF peonidin provides a useful tool to dissect these two pathways in future search for additional parasitic plant genes involved in haustorium development. Differential responses of parasitic genes under peonidin, other toxic HIFs, and toxic non-HIFs bear the potential to separate genes only involved in the detoxification pathway from genes specific to the haustorium signaling pathway. Furthermore, although peonidin is a very effective HIF in T. versicolor, its ability to induce haustoria in other parasitic plant species has not yet been investigated. How peonidin functions as a HIF, e.g., with regard to its active form and receptor on the parasite, also awaits further studies. Both TvQR1 and TvPirin genes have orthologs in non-parasitic plants. Thus, further phylogenic investigations of these genes in different plant taxa might elucidate the mechanism(s) by which parasitic plants have, during the evolutionary course from complete autotrophy to heterotrophy, co-opted a quinone oxidoreductase and a transcription co-factor from their existing genetic reservoir and adapted them to fulfill the new function of developing the organ that embodies plant parasitism: the haustorium.
Plant and chemical materials
T. versicolor seeds were collected and pooled from wild populations growing in the grasslands of Northern California, USA. For T. versicolor cDNA clones and Northern blot analysis, seeds were collected in Napa (Napa county) (Figure 2). For in vitro haustorium and root necrosis assays, qRT-PCR experiments, and population genetics study, seeds were collected in Marysville (Yuba county) (Figure 2). DMBQ and juglone were obtained from Pflatz & Bauer (Waterbury, CT, USA) and Alfa Aesar (Karlsruhe, Germany). Peonidin was obtained from Indofine Chemicals (Belle Mead, NJ, USA) and Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).
Haustorium induction and toxicity assays
T. versicolor seeds were surface-sterilized, germinated, grown on agar plates, and seedlings treated with various haustorial inducers (10 or 100 μM DMBQ, 30 μM or 1 mM peonidin) and non-inducers (50 μM juglone), treatment combinations (10 μM DMBQ + 30 μM juglone), or mock-treated with water or 0.1% EtOH (which is the peonidin solvent) as described previously [11,21]. Each plate contained 10–20 seedlings. For each treatment, haustorium formation and root necrosis were scored 24 h after treatments in 60–66 seedlings.
Nucleic acid extraction
For Northern blot analysis, total RNA was isolated from root tips, remaining roots, and shoots of 100–200 T. versicolor plants treated with water, 0.1% ethanol, 20 μM DMBQ, 10 μM juglone, or 30 μM peonidin at 1, 3, and 5 h after treatment. For RT-qPCR, 20 μM DMBQ or 30 μM peonidin was applied twice to the same seedling plates at 2-day intervals and haustorium formation was scored 24 h after each time to identify responsive and non-responsive plants. Then the third treatment was applied, and 2 h later, root tips were collected from three batches of 20 each inducer-responsive plants representing three biological replicates. One application was performed for mock (water and 0.1% ethanol) or 10 μM juglone treatments to collect root tips for three biological replicates. Total RNA was extracted from root tips by the Trizol method (Invitrogen, Carlsbad, CA, USA). From the haustorium formation assays performed for the RT-qPCR experiments, 20 individual plants responsive or non-responsive to DMBQ or peonidin were randomly selected for genomic DNA extraction using CTAB and chloroform, followed by isopropanol precipitation and ethanol washes [44].
Transcription assays
Northern blot hybridization was performed with 10 μg of total RNA from each sample as described elsewhere [22]. cDNA probes for the RNA blot were synthesized by PCR-amplifying the TvQR1, TvPirin, and TvRuvB DNA helicase partial cDNA clones from the SSH library [21], randomly labeled by nick-translation with CTP 32 as per the manufacturer's protocol (Ambion, now Invitrogen, Carlsbad, CA, USA), and used at 107 cpm/ml of hybridization buffer. The radioactive signals on the filters were captured on the Phosphor screen and images were scanned by the Storm PhosphorImager (GMI, Ramsey, MN, USA). The same filters were sequentially hybridized to different probes following the stripping of each previous probe and exposure to the Phosphor screen to check for residual signals. For RT-qPCR, two technical replicates were carried out with 1 μl of 1:20 diluted cDNAs reverse-transcribed from 2 μg each of three total RNA biological replicates in a 20 μl volume using SYBR Green I dye in an Applied Biosystem 7500 real-time PCR machine (Applied Biosystem). PCR parameters were as followed: 50°C/20 sec, 95°C/10 min, 40× (95°C/15 sec, 60°C/1 min). Melting curve analysis was done at the end of the PCR reactions to confirm single PCR product amplifications at 95°C/1 min, 60°C/30 sec, 95°C/1 min, 60°C/15 sec. Relative quantification of target transcripts were normalized to the reference transcript TvRuvB DNA helicase, which is constitutively expressed, and the relative expression levels of each transcript species under inducing and non-inducing conditions were compared to the control (water) treatment by the Δ ΔCt method [45].
Gene cloning
5′ and 3′ RACE reactions of TvQR1 and TvPirin were performed with total RNA isolated from DMBQ-responsive T. versicolor root tips using the Invitrogen's RLM RACE kit per the company protocol. The amplified cDNAs were cloned into the pCR2.1TOPO vector (Invitrogen) and sequenced; the transcriptional start and stop sites of these sequences were identified and used to design new primers complementary to both ends of each cDNA clone for subsequent PCR amplification of the full-length cDNA clones from the same cDNA source used for RACE and from T. versicolor genomic DNA with high-fidelity polymerases Pfu or Phusion Taq (NEB). PCR parameters were typically as follows: 94°C/2 min, 30× (94°C/15 sec, 60°C/25 sec, 72°C/2.5 min), 72°C/5 min, with the 94°C steps being replaced with 98°C when Phusion Taq was used. The full-length cDNA PCR products were cloned into pCRII vector (Invitrogen) and sequenced. The genomic DNA PCR product from each individual plant was directly sequenced by the Sanger sequencing method and the sequence chromatograms visually inspected for sites where two nucleotide peaks of similar height were present which indicated heterozygosity. Sequences of both strands of heterozygotes were visually inspected to confirm the same two nucleotide peaks. The genomic DNA of the identified heterozygotes was subcloned into the pDrive vector, and the corresponding inserts isolated from 2–4 independent clones were subsequently sequenced to identify the SNPs in each allele.
Sequence analyses
Contig assembly was done manually or by using the CLC Main Workbench 6.6.1 software (CLC bio) coupled with visual inspection for base call error and heterozygosity detection. BLASTN 2.2.13 and BLASTN 2.2.26+ [46] were used to search for TvPirin sequences in the T. versicolor genome sequence databases at http://ppgp.huck.psu.edu/search.php webcite and http://www.plantsciences.ucdavis.edu/yoder/lab/Sequence_index.html webcite. Multiple sequence alignments were performed with MultAlin ( http://multalin.toulouse.inra.fr/multalin/ webcite[47] or ClustalW2 ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ webcite, [48,49]), and the multiple alignments presented in the BOXSHADE format ( http://www.ch.embnet.org/software/BOX_form.html webcite, Kay Hofmann and Michael D. Baron). The protein PFAM domains were identified from Swissprot database using InterProScan ( http://www.ebi.ac.uk/Tools/pfa/iprscan/ webcite).
Population genetic analysis
Sequence alignments were performed using ClustalX installed in MEGA5 [50]. Minor adjustments to optimize the alignments were made by eye. Various population genetic analyses were performed using DnaSP v5 [51]. Nucleotide diversity was corrected using the Jukes-Cantor method [52]. In the sliding window analysis, window length was 50 bp and step size was 10 bp.
Primers (5′ to 3′)
TvQR1 full-length cDNA: forward CTGGAATTCATTTTCAAGCTTCTCA, reverse CTTATGGCTCGACAACGATTTT.
TvPirin full-length cDNA: forward ATTCCTCATAAACATCAAATCCCCA, reverse TAGGTTAACAAAAGATCTCATATCAAAACA.
TvQR1 RT-qPCR (164 nt amplicon): forward TGATAAAGTTGTTGCTATGCTTGGC, reverse GTTGGGTGAGGGCCATGTG.
TvPirin RT-qPCR (90 nt amplicon): forward TCCAAGGACAAAATGATTGAACCT, reverse CACTTTTACTTCAACCCCATCTTTTT.
Bona-fide TvPirin 5′UTR RT-qPCR (60 nt amplicon): forward ATTCCTCATAAACATCAAATCCCCAAT, reverse ATGATATAACGTCGAGAAGACGGGTTT.
TrVeBC2_244713 5′UTR RT-qPCR (102 nt amplicon): forward GAAATGCCACTATTACTCAGCTGGT, reverse TGGGGATTTGATGTTTATGAGGA.
TvRuvB DNA helicase RT-qPCR (133 nt amplicon): forward TAAACCGAGCTCTGGAGAACGAC, reverse ATGAGCAGACGGTCGAGAAAATC.
Accession numbers
Sequence data from this article can be found in the GenBank data libraries under accession numbers XX000000.
T. versicolor: Triphysaria versicolor; HIF: Haustorium inducing factor; DMBQ: 2,6-dimethoxybenzoquinone; SNP: Single nucleotide polymorphism; Indel: Insertion/deletion; RT-qPCR: Reverse transcription - quantitative polymerase chain reaction.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
QAN conceptualized and designed the study. QAN, HA, and TT carried out experiments, analyzed the data, and wrote the manuscript. UG provided the infrastructure, research materials and reagents, and analysis tools. All authors critically read and commented on the manuscript and approved the final version for submission.
Authors' information
QAN studied Triphysaria versicolor and Triphysaria pussilla during her PhD program at the Genetics Graduate Group of the University of California - Davis during the years of 2000–2003, focusing on cloning and functional characterization of Triphysaria candidate genes involved in haustorium development, and on the development of Triphysaria transformation systems for gene functional test by RNAi-mediated gene silencing. Since 2007, QAN is a postdoctoral fellow at the University of Zürich. HA established root exudates as haustorium inducers in T. versicolor and identified peonidin as a HIF in T. versicolor during her postdoctoral work from 1993 to 1997 at the University of California -Davis. TT studies the genetic basis of adaptation from 2010–2011 as a postdoctoral fellow at the University of Zürich and, since 2012, at the Gregor Mendel Institute in Vienna. In this study, he contributed to the analysis of the nucleotide polymorphism in T. versicolor. UG is heading the Department of Plant Developmental Genetics at the Institute of Plant Biology of the University of Zürich and studies developmental, ecological, and evolutionary aspects of plant reproduction.
We thank Dr. John Yoder (University of California – Davis) for providing laboratory space for the Northern blot experiments and gene cloning, Dr. Denneal Jamison, Dr. Jean-Michel Petit, Dr. Russel Wrobel (all at University of California – Davis), and Dr. Philipp Schlüter (University of Zürich) for helpful discussions, the Division of Biological Sciences Sequencing Center (University of California - Davis) and Daniela Guthörl and Dr. Edith Schlagenhauf (University of Zürich) for sequencing assistance, Arturo Bolanos and Peter Kopf (University of Zürich) for general laboratory support. This work was supported in part by a Jastro-Shields graduate grant from the University of California – Davis to QAN. QAN was further supported through teaching assistantships from the Genetics Graduate Group and the Molecular and Cell Biology Department of the University of California – Davis, and the University of Zürich. TT was supported by an EMBO long-term postdoctoral fellowship.
Heide-Jorgensen HS: Parasitic Flowering Plants. Leiden, The Netherlands: Brill Academic Publishers; 2008.
Kuijt J: The Biology of Parasitic Flowering Plants. Berkeley: University of California Press; 1969.
Musselman DL, Press MC: Introduction to parasitic plants. In Parasitic Plants. Edited by Press MC, Graves JD. London: Capman and Hall; 1995:1-13.
Nickrent DL, Musselman DL: Parasitic flowering plants. American Phytopathological Society APSnet Education Center, the Plant Health Instructor web publication The Plant Health Instructor; 2004. Publisher Full Text
Musselman LJ, Dickison WC: The structure and development of the haustorium in parasitic Scrophulariaceae.
Bot J Linnean Soc 1975, 70:183-212. Publisher Full Text
Riopel JL, Timko MP: Haustorial initiation and differentiation. In Parasitic Plants. Edited by Press MC, Graves JD. London: Chapman and Hall; 1995:39-79.
Press MC, Phoenix GK: Impacts of parasitic plants on natural communities.
New Phytol 2005, 166(3):737-751. PubMed Abstract | Publisher Full Text
Chang M, Lynn DG: The haustorium and the chemistry of host recognition in parasitic angiosperms.
J Chem Ecol 1986, 12:561-579. Publisher Full Text
Lynn DG, Steffens JC, Kamut VS, Graden DW, Shabanowitz J, Riopel JL: Isolation and characterization of the first host recognition substance for parasitic angiosperms.
J Am Chem Soc 1981, 103:1868-1870. Publisher Full Text
Steffens JC, Lynn DG, Kamat VS, Riopel JL: Molecular specificity of haustorial induction in Agalinis purpurea (L.) Raf. (Scrophulariaceae).
Albrecht H, Yoder JI, Phillips DA: Flavonoids promote haustoria formation in the root parasite Triphysaria versicolor.
Plant Physiol 1999, 119(2):585-592. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Riopel JL, Timko MP: Signals and regulation in the development of Striga and other parasitic angiosperms. Boca Raton: CRC Press; 1992.
Bandaranayake PC, Filappova T, Tomilov A, Tomilova NB, Jamison-McClung D, Ngo Q, Inoue K, Yoder JI: A single-electron reducing quinone oxidoreductase is necessary to induce haustorium development in the root parasitic plant Triphysaria.
Plant Cell 2010, 22(4):1404-1419. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Keyes WJ, O'Malley RC, Kim D, Lynn DG: Signaling organogenesis in parasitic angiosperms: xenognosin generation, perception, and response.
J Plant Growth Regul 2000, 19(2):217-231. PubMed Abstract
Kim D, Kocz R, Boone L, Keyes WJ, Lynn DG: On becoming a parasite: evaluating the role of wall oxidases in parasitic plant development.
Chem Biol 1998, 5(2):103-117. PubMed Abstract | Publisher Full Text
Smith CE, Ruttledge T, Zeng Z, O'Malley RC, Lynn DG: A mechanism for inducing plant development - the genesis of a specific inhibitor.
PNAS 1996, 93:6986-6991. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Scholes JD, Press MC: Striga infestation of cereal crops - an unsolved problem in resource limited agriculture.
Curr Opin Plant Biol 2008, 11(2):180-186. PubMed Abstract | Publisher Full Text
Timko MP, Gowda BS, Ouedraogo J, Ousmane B: Integrating New Technologies for Striga Control: Towards Ending the Witch-Hunt. Edited by Ejeta G, Gressel J. Singapore: World Scientific; 2007:115-128.
Thurmann LD: PhD thesis: Genecological studies in Orthocarpus subgenus Triphysaria (Scrophulariaceae). Berkeley: University of California; 1966.
Bandaranayake PC, Tomilov A, Tomilova NB, Ngo QA, Wickett N, de Pamphilis CW, Yoder JI: The TvPirin gene is necessary for haustorium development in the parasitic plant Triphysaria versicolor.
Plant Physiol 2012, 158(2):1046-1053. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Matvienko M, Torres MJ, Yoder JI: Transcriptional responses in the hemiparasitic plant Triphysaria versicolor to host plant signals.
Plant Physiol 2001, 127(1):272-282. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Matvienko M, Wojtowicz A, Wrobel R, Jamison D, Goldwasser Y, Yoder JI: Quinone oxidoreductase message levels are differentially regulated in parasitic and non-parasitic plants exposed to allelopathic quinones.
Plant J 2001, 25(4):375-387. PubMed Abstract | Publisher Full Text
Li ZH, Wang Q, Ruan X, Pan CD, Jiang DA: Phenolics and plant allelopathy.
Molecules 2010, 15(12):8933-8952. PubMed Abstract | Publisher Full Text
Jamison DS, Yoder JI: Heritable variation in quinone-induced haustorium development in the parasitic plant Triphysaria.
Plant Physiol 2001, 125(4):1870-1879. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Palmer AG, Chen MC, Kinger NP, Lynn DG: Parasitic angiosperms, semagenesis and general strategies for plant-plant signaling in the rhizosphere.
Pest Manag Sci 2009, 65(5):512-519. PubMed Abstract | Publisher Full Text
Rietveld WJ: Allelopathic effects of juglone on germination and growth of several herbaceous and woody species.
J Chem Ecol 1983, 9:295-308. Publisher Full Text
Cao G, Sofic E, Prior RL: Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships.
Free Radic Biol Med 1997, 22(5):749-760. PubMed Abstract | Publisher Full Text
Gould KS, Lister C: Flavonoid function in plants. In Flavonoids: Chemistry, biochemistry and applications. Edited by Andersen M, Markham KR. Boca Raton, FL: CRC Press; 2006:397-441.
Wang H, Cao G, Prior RP: Oxygen radical absorbing capacity of anthocyanins.
J Agric Food Chem 1997, 45:304-309. Publisher Full Text
Torres MJ, Tomilov AA, Tomilova N, Reagan RL, Yoder JI: Pscroph, a parasitic plant EST database enriched for parasite associated transcripts.
BMC Plant Biol 2005, 5:24. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Wickett NJ, Honaas LA, Wafula EK, Das M, Huang K, Wu B, Landherr L, Timko MP, Yoder J, Westwood JH: Transcriptomes of the parasitic plant family Orobanchaceae reveal surprising conservation of chlorophyll synthesis.
Current Biology: CB 2011, 21(24):2098-2104. PubMed Abstract | Publisher Full Text
Pang H, Bartlam M, Zeng Q, Miyatake H, Hisano T, Miki K, Wong LL, Gao GF, Rao Z: Crystal structure of human pirin: an iron-binding nuclear protein and transcription cofactor.
J Biol Chem 2004, 279(2):1491-1498. PubMed Abstract | Publisher Full Text
Murzin AG: Structural classification of proteins: new superfamilies.
Curr Opin Struct Biol 1996, 6(3):386-394. PubMed Abstract | Publisher Full Text
Taneja B, Mande SC: Conserved structural features and sequence patterns in the GroES fold family.
Protein Eng 1999, 12(10):815-818. PubMed Abstract | Publisher Full Text
Bashton M, Chothia C: The geometry of domain combination in proteins.
J Mol Biol 2002, 315(4):927-939. PubMed Abstract | Publisher Full Text
Rubach JK, Plapp BV: Amino acid residues in the nicotinamide binding site contribute to catalysis by horse liver alcohol dehydrogenase.
Biochemistry 2003, 42(10):2907-2915. PubMed Abstract | Publisher Full Text
Thorn JM, Barton JD, Dixon NE, Ollis DL, Edwards KJ: Crystal structure of Escherichia coli QOR quinone oxidoreductase complexed with NADPH.
J Mol Biol 1995, 249(4):785-799. PubMed Abstract | Publisher Full Text
Dunning FM, Sun W, Jansen KL, Helft L, Bent AF: Identification and mutational analysis of Arabidopsis FLS2 leucine-rich repeat domain residues that contribute to flagellin perception.
Plant Cell 2007, 19(10):3297-3313. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ellis JG, Lawrence GJ, Luck JE, Dodds PN: Identification of regions in alleles of the flax rust resistance gene L that determine differences in gene-for-gene specificity.
Plant Cell 1999, 11(3):495-506. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
McDowell JM, Dhandaydham M, Long TA, Aarts MG, Goff S, Holub EB, Dangl JL: Intragenic recombination and diversifying selection contribute to the evolution of downy mildew resistance at the RPP8 locus of Arabidopsis.
Plant Cell 1998, 10(11):1861-1874. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Rose LE, Bittner-Eddy PD, Langley CH, Holub EB, Michelmore RW, Beynon JL: The maintenance of extreme amino acid diversity at the disease resistance gene, RPP13, in Arabidopsis thaliana.
Genetics 2004, 166(3):1517-1527. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Seeholzer S, Tsuchimatsu T, Jordan T, Bieri S, Pajonk S, Yang W, Jahoor A, Shimizu KK, Keller B, Schulze-Lefert P: Diversity at the Mla powdery mildew resistance locus from cultivated barley reveals sites of positive selection.
Mol Plant Microbe Interact 2010, 23(4):497-509. PubMed Abstract | Publisher Full Text
Yahiaoui N, Brunner S, Keller B: Rapid generation of new powdery mildew resistance genes after wheat domestication.
Plant J 2006, 47(1):85-98. PubMed Abstract | Publisher Full Text
Porebski S, Grant Bailey L, Baum BR: Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components.
Plant Mol Biol Report 1997, 15(1):8-15. Publisher Full Text
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method.
Methods 2001, 25(4):402-408. PubMed Abstract | Publisher Full Text
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res 1997, 25(17):3389-3402. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Corpet F: Multiple sequence alignment with hierarchical clustering.
Nucleic Acids Res 1988, 16(22):10881-10890. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R: Clustal W and Clustal X version 2.0.
Bioinformatics 2007, 23(21):2947-2948. PubMed Abstract | Publisher Full Text
Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res 1994, 22(22):4673-4680. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.
Mol Biol Evol 2011, 28:2731-2739. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Librado P, Rozas J: DnaSP v5: a software for comprehensive analysis of DNA polymorphism data.
Bioinformatics 2009, 25:1451-1452. PubMed Abstract | Publisher Full Text
Jukes TH, Cantor CR: Evolution of protein molecules. In Mammalian Protein Metabolism. Edited by Munro HN. New York: Academic; 1969:32-41.


text:
Madonna's Chopard Bling: Love It or Hate It?
Madonna's Chopard Bling: Love It or Hate It?
by Fashion
Like us on Facebook
Hard Candy
Love It or Hate It
Celebrity Designers
POPSUGAR, the #1 independent media and technology company for women. Where more than 75 million women go for original, inspirational content that feeds their passions and interests.
keiraz 7 years
madonna can do no wrong :-D
keiraz 7 years
madonna can do no wrong :-D
gaelgirl 7 years
what is wrong w/ this woman & what has she done to her face???
kbyersobrien 7 years
I swear about 90% of the time Madonna looks like a tranny. Honey, you're almost 50, tone it down.
marie-lee 7 years
Yes vintage is in. Should she be recycling stuff from wardrobe that she wore back in the 90's - no.
JaimeLeah526 7 years
Tacky. Spend your money on beautiful things not crap that you won't want in 2 years.
linday_love 7 years
madge i love you but the backdrop has got to go!
ElectroPopTart 7 years
It works for her cuz she's HER! =D
fsofield 7 years
don't be mad at me cuz you're pushing 50 and your old tricks no longer work
shini 7 years
she should be becoming more classy with age, and not tarty...
DCStar 7 years
WHAT is up with the prize-fighter belt? That's all I want to know...
lizzylu49 7 years
If I have to see her in one more spread-legged pose....
Imani33 7 years
HaHa she looks like she just won a WWF tourney!
Lovely_1 7 years
I couldn't pull it off, but it looks GREAT on her!
Lovely_1 7 years
I couldn't pull it off, but it looks GREAT on her!
emalove 7 years
Yuck. I hate it.
Dana18 7 years
not for me!
Dana18 7 years
not for me!
berrymix 7 years
Not liking it.
berrymix 7 years
Not liking it.
Lala77 7 years
Now why am I loving this ? :D
Lala77 7 years
Now why am I loving this ? :D
Nicwolf 7 years
She always does crazy and over-the-top - that's what makes her Madonna!
aimeeb 7 years
aimeeb 7 years

subset: commoncrawl


text:
Sherlock Holmes is coming to The Simpsons.
Benedict Cumberbatch, the man behind the world's most famous detective on BBC America's sleek remake, has signed on to voice two roles on an episode of the Fox animated smash this fall.
Cumberbatch will portray the British Prime Minister and a snake-like character in an episode titled "Love Is A Many-Splintered Thing." No air date has been announced yet.
As we await news installments, visit our section of The Simpsons quotes now - and just try not to laugh.


text:
Search Results
Refine SearchDate Source
- - Blogs (692)
- - Print Articles (655)
- - Partners Articles (23)
- - Online Articles (5)
- All Authors
- - Philip Morrison
- - F. D. McHugh
- - Alexander Klemin
- - Albert A. Hopkins
- - James R. Newman
The Oldest Eggs in the World
Steamed dinosaur eggsNeosauropods sought out geothermal vents to keep their eggs warm.
Device for Testing Eggs
Desiccated and Frozen Eggs
Eggs of Curious Forms
Some Observations on EggsScientific Principles That Enter Where Least Expected and Are Carried Out Unrecognized
Testing Eggs by PhotographySome of the Things Which the Eyes of the Camera Sees When it Looks Through the Hen's Creations
To Make Hens' Eggs Hard, "Flu" Experiments on Monkeys
How Large is the Human Egg?Some of the Findings of Recent Research Upon the Eggs of the Human Species, and Those of Other Mammals
The hunt for a perfect fishy fatherFemale scissortail sergeants assess potential partners with 'test' eggs.
The Coevolution of a Butterfly and a VineHeliconius butterflies lay their eggs only on Passiflora Vines. In defense the vines seem to have evolved fake eggs that make it look to the butterflies as if eggs have already been laid on them
Does Plate Glass Fade? How do Rats Carry Eggs?
Incubator BirdsThese extraordinary fowl hatch their eggs not by sitting on them but by putting them in a hot place. Some species even rake together dead leaves, the decay of which heats the eggs
Marsupial FrogsSome tropical frogs incubate eggs on the mother's back, often in a special pouch. Certain features of the adaptation recall pregnancy in mammals and the eggs and embryos of birds; others are unique
Beyond the Piano: Musicians Explore the Acoustics of Kelp and Clay and Ostrich Eggs
Shape-Shifting Science: Molding Hard-Boiled EggsA crafty food science project from Science Buddies
Mickey Has Two MomsNo sperm needed: mice are born from two eggs
Good news after the Gulf spill: Turtle rescue plan succeedsSafe hatching for eggs that were relocated to avoid oil spill.
Good news after the Gulf spill: Turtle rescue plan succeedsSafe hatching for eggs that were relocated to avoid oil spill.
Unorthodox Methods of Sperm TransferThe eggs of some animals are not fertilized by the usual methods, which raises a number of interesting questions. Consider the sponge, the bedbug, the spider and the leech


text:
More about this preview.

Change DPI Base size is 150
What we do and how we do it.
See last week's winners
Explore Our fabrics
Optional Filters
I'm going to use this fabric to make...
I'm looking for a fabric that is...
Receive our weekly email with news about contests, giveaways and prodution releases.
Email Address
No Thanks
© 2008-2015 Spoonflower Inc


text:
ABATE 38 Toy Run
( Los Ninos Toy Run)….It is being put on by ABATE Local 38.
( Los Ninos Toy Run)….It is being put on by ABATE Local 38.
(The 26th Annual Holiday Concert)….The Annual Christmas gift to the Imperial Valley from Jimmie Cannon's Valley Jazz.
(Early morning air pollution)….Unhealthy air was reported at 2:00 am Monday.
(Scammer loses his cool)…Tax Season is still a couple months away.
(Grinch strikes in Calexico)…Somebody stole Christmas gifts from a vehicle in El Centro recently.
(Another partnership with Mexicali)…A ceremonial signing will be next week.
(Man found dead)…He was found in an apartment on Park Avenue in El Centro
(Tabarez submits letter of resignation)….Lisa Tabarez submitted the letter Thursday.
Page 201 of 622


text:
- Age / Gender:
- 19, Male
- Location:
- Why should I tell you!?
- Joined:
- 4/21/11
- All Stats >
User Statshot
- Community Stats
Level 4 Blank Slate
Garbage Whistle
Ranked as Civilian
Contact Info / Websites
Favorite Movies
Animator vs. Animation IV Rated 4.5358 Stars The Second Coming of The Chosen One. Comedy - Original Animator vs. Animation II Rated 4.4126 Stars The stick figure is back, and now he's got laser eyes! Comedy - Original Animator vs. Animation Rated 4.3677 Stars It's a struggle between a stick figure and his creator, using items on the Flash toolbar! Comedy - Original J Jonah Jameson Is Mean Rated 4.0083 Stars J Jonah Jameson laughs Packer's ass off about random stuff. Comedy - Parody
Favorite Games
Road of the Dead 2 Rated 4.5132 Stars Survive the massive outbreak in Evans City as two soldiers fighting to live. Action - Other Space Flash Arena Rated 4.0807 Stars Space combat sim in full 3D! Action - Shooter - First Person SCGMD4 Rated 4.4579 Stars The super crazy guitar maniac is back with more songs than ever! And he's not alone... Rhythm SC Guitar Maniac Dx 2 Rated 4.3584 Stars Rhythm game packed with 14 Newgrounds songs, 6 guitars and a crazy stickman. Rhythm
Favorite Audio
|~NK~ "Blacklight" FULL
|Dubstep Song
|Defy the Faith
|Heavy Metal Song
|Heavy Metal Song
|-= xKore - Fire (RMX) =-
|Dubstep Song
Favorite Art
Recent Game Medals
Reloader II 50 Points Reload a weapon 25 times. Medal Stats.
Item Collector 25 Points View all equipment items. Medal Stats.
Stealth 10 Points Fire a silenced weapon 25 times. Medal Stats.
Aggressor II 50 Points Fire a weapon 100 times. Medal Stats.
Reloader I 10 Points Reload a weapon 5 times. Medal Stats.
Aggressor I 10 Points Fire a weapon 10 times. Medal Stats.
Goron Mania is Rolling Wild 10 Points Click on Darunia when he Mentions Andre the Biggoron, Brother! Medal Stats.
Operation Moonfall 5 Points click on the "where is the next one coming out?" button Medal Stats.
SECRET MEDAL 10 Points Unlock this medal to see it's details. Medal Stats.
Total Medals Earned: 95 (From 12 different games.)
Latest Shared Creations
Imagine Greater (alternate endin Added to songs for Punk-o-matic 2 Mar 3, 2012. Load Level
Live Free or Die Added to songs for Punk-o-matic 2 Jun 6, 2011. Load Level
Imagine Greater Added to songs for Punk-o-matic 2 Jun 6, 2011. Load Level
Heavy Metalloid Added to songs for Punk-o-matic 2 Jun 5, 2011. Load Level


text:
Cummed XD
Thanks for helping us associate the correct Pornstars to this video!
Help Us: Do these Pornstars appear in this video?
Pornstar Name:
Thanks for helping us sort this video!
All Comments (15)
Cummed XD
few hours after looking this vid i was peeing in a public toilet,my cock started to stick up..it become longer and longer, a man beside me was looking my hard cock ,i felt an unbeleavable pleasure...12 cockshots 2 feet up the wall..
cs at :11 made ME cum! (i didn't get very far)
Bryan, or better yet, imagine you were the other guy, and he shot his cum all over your cock, lubing your hand and cock with his hot cum!
whats that music at 6:00?
Who is the guy at 2:41? I wouldn't mind getting impaled by him.
:11 if Peter didn't point his beatyful cock downwards cumshots would have arrived to the ceiling
:11 what a cockshot!!!! my wife gasps while she watches...
the first guys to cum of her tits.they both have nice big hard cocks and nice creamy hot cum.i want to suck off both those guys and eat their creamy cum.
biguy- Nice thought.
:11- i can't take my eyes off his cock and SHE didn't even look?!
@eddy i wish i'd been there looking at you showing off your rock hard, long, cock,i'm sure you love that men put their eyes on your cock when it's sticking up like a pole.....


text: Many posts are showing invalid TeX formulae, even though they are valid. For example, https://www.physicsforums.com/showthread.php?t=265656 When I click on the invalid TeX block, it shows [ tex ]...[ tex ] (or [ itex ]...[ itex ]), while I definitely typed [ tex ]...[ /tex ] (or [ itex ]...[ /itex ]). (Extra spaces added so we don't go into TeX mode.) I'm using a Mac, if that makes any difference.


text:
|Boxing Champions | Boxing Schedule | Boxing Video | Boxing History | Pound For Pound | Lounge | The Wire | Audio | Arcade
Big Bad Booty Daddy
Join Date: Sep 2003
Location: New York
Posts: 20,781
Quoted: 388 Post(s)
Rep Power: 200000
Points: 2,502,114,854,178.98
Bank: 0.00
Total Points: 2,502,114,854,178.98
Comments Thread For: Random Acts of Ring Rambling: Ironfist, Punisher, More
By Thomas Gerbasi - Lets not mince words here. The odds of Dereck Chisora capturing lightning in a bottle and pulling off a Buster Douglas-esque upset of Vitali Klitschko in their WBC heavyweight championship fight this Saturday in Germany are perhaps even slimmer than slim and none.
But Ill be watching. And odds are that you will too. Because for all the rightful criticism the heavyweight division gets these days (and for the last few years), in the back of my mind, this is still the sports glamour division, the home for guys like Jack Dempsey, Joe Louis, Rocky Marciano, Muhammad Ali, George Foreman, and Mike Tyson.
Sure, Vitali and his brother Wladimir are not the charismatic figures the aforementioned group was, but they are the best big men in a sport that has always celebrated those over 200 pounds, and theyve dominated for longer than most. Vitali, in particular, may be the most underrated heavyweight champion of the last 30 years simply because he hasnt had the dance partners some of his colleagues have had. [Click Here To Read More]
Undisputed Champion
Join Date: Mar 2010
Posts: 5,729
Quoted: 63 Post(s)
Rep Power: 21
Points: 5,000,010,000,071,086.00
Bank: 2,016,758,459,029.69
Total Points: 5,002,026,758,530,116.00
Gerbasi is an excellent writer:
I remember well when the heavyweight division reigned !!
Sonny Liston I feel is so overlooked, for his short run he was a devastating monster !
It took the " GREATEST " to whup him and the other ATG's, Frazier, Foreman and etc.....
Hats off to V. and W. for their reign as KINGS !
Pac does have the toughest fight. He chose the toughest fight, but that is not uncommon for him to do.
All this crying about weight draining is so silly. Fighters agree to the weights made, like with Cotto and ODH. Cotto was fast and sharp, his legs stayed with him until he was nailed by that wicked left and right of Pac, and ODH never made the excuse about his weight, if he came in light whose fault is that ,,, PAC's ??? silly
Mayweather does his usual thang mang. He fights the EEEEEEEEEEEAsy fights mang, he waits as usual as he has in the past , even Corralles sp "UNTIL", his man is:
Beat half to death
wee little
before he walks in to whup up on they heads mang and claim the nice throne, LOL !!!!!!!
good ole JR
The reason NO ONE WILL EVER whup him is because he will never be found taking Risks ! The reason he smiles is because he has duped the public once again into paying to watch him fight.
Take the fight with Cotto. He should have fought and whupped Cotto when they both were in their primes back in 2007 or 8,,,,but no he had to retire and his back up excuse was the dirty word,,,,,,,
LOL,,,, if he had wanted that fight then he would have worked around ARUM.
He is very happy to fight a beat up man that has QUIT TWICE,
Jr will be smiling as he goes to his ropes and does the rope a dope , make his doe and hopes Bradley stomps a mudhole in Pac so he can wipe his brow of the past three years of ducking.
Happy ? LOL
Undisputed Champion
Join Date: Mar 2011
Posts: 5,459
Quoted: 17 Post(s)
Rep Power: 16
Points: 55,818.90
Bank: 0.00
Total Points: 55,818.90
if it happens Canelo will smoke Williams...Liston was great & see his fights of the late 50's ve everyone...awesome/unbeatable, never hurt, especially his 2 vs Cleveland Williams, makes 1 wonder was the Clay fights on the up&up...
Up and Comer
Join Date: Sep 2011
Location: Phoenix, AZ
Age: 29
Posts: 81
Quoted: 1 Post(s)
Rep Power: 5
Points: 2,736.97
Bank: 961.12
Total Points: 3,698.09
There's More
Seth Mitchell is not the only American heavyweight that has the potential of super-stardom. I never hear anyone talk about David Rodriguez. This guy is 6'5 and a solid 250lbs. He also boasts an amazing record of 36-0 with 34 KOs! This guy really needs to get some more attention.
Ph.D in the Sweet Science
Join Date: Oct 2009
Posts: 17,361
Quoted: 402 Post(s)
Rep Power: 56
Points: 1,050,000,000,000,090,240.00
Bank: 572,720,073,079,479,424.00
Total Points: 1,622,720,073,079,569,664.00
David Rodriguez has potential but he doesn't fight anybody good.
It's hard to know how talented he really is until we seem him against someone in the top 15.
|Share This With Friends
|Thread Tools
Cookie Policy -
Privacy Policy - Submit
News - Feedback
- Site Map - Advertise
with Us
Copyright © 2003-2015 BoxingScene LLC All rights reserved.
Copyright ©2000 - 2015, Jelsoft Enterprises Ltd.
Copyright © 2003-2015 BoxingScene LLC All rights reserved.


text:
From the article: "Culdcept was one of the greatest underappreciated games the PlayStation 2 had to offer. It was a bizarre mashup of Monopoly's property buying and Magic: The Gathering's monster battling with cards. It's really a game who have to experience for yourself to truly get a feel for.
Because of that, Xbox 360 owners are in luck today as a demo of the sequel, Culdcept Saga, is now up on the Xbox Live Marketplace. "


text:
I bought second-hand a Gossen Multisix (hand-held reflected light and incident light exposure meter) and a Minolta Spotmeter F (hand-held spot reflected light e.m.). They are OK but, frankly, an integrated solution like the Sekonic L-758 would be much more practical.
So if money is no object and if one wants to buy new, and has slides in his photographic habits, I would go for a "do-it-all" so that the expense is made only once, it takes less space in the bag etc.
Overall I think that a spot lightmeter is useful only when using slides, or when using the zone system. So for most photographers it is not necessary until the day when they regret not to have bought an integrated one


text:
If you can't cook, soak. That's what I've started doing when I want to prepare grains without raising the temperature -- the stove's, the kitchen's or mine.
It's not a new idea. The technique goes back to traditional preparations for that classic Middle Eastern parsley-and-bulgur salad, tabbouleh. Rather than boil water and pour it over the bulgur (parcooked cracked wheat), such traditionalists as the eminent Claudia Roden call for the grain to be soaked for a longer time in cold water, then squeezed dry so it can absorb the salad's other flavors.
I didn't have to make tabbouleh -- as much as I love it -- to realize the benefits of such a technique when concocting other warm-weather, grain-based dishes.
For instance, couldn't I apply the same idea to another quick-to-cook "grain" (really a pasta), instant couscous? I used to start preparing it the same way as bulgur, by putting a kettle on to boil, but no more. Couscous soaks up cool water in less than an hour; bulgur -- the coarse variety, anyway -- soaks in about 90 minutes.
The best thing about this way of doing things, besides the no-heat benefit, is that you can combine grains with water (or vegetable broth, for more flavor), cover them and refrigerate them overnight -- or all day.
When I use a 1:1 ratio of grain to water, the grains soak up what they need and then stay put, not becoming soggy as they sit longer.
I've been using my soaked bulgur and couscous interchangeably, putting them to good use in two dishes: beets and their greens dolloped with garlicky yogurt, and a cold vegetable-and-chickpea salad doused with a sesame-miso dressing.
In case you're wondering, this soak-instead-of-cook idea isn't something you can do with all grains. Many of the bigger/tougher ones -- barley, rice, farro and the like -- will start to ferment and/or sprout long before they are tender enough to eat raw, so those require cooking.
Here, the couscous is soaked in cold water and the greens are simply massaged. Store-bought roasted and peeled beets are used (to keep the oven turned off), and the dish is served at room temperature. But you can cook the couscous (or any other grain) by more traditional methods, saute the greens and cook raw beets; serve that version warm. Recipe adapted from Martha Rose Shulman in "The Simple Art of Vegetarian Cooking" (Rodale, 2014).
Makes 4 servings.
1 cup whole-wheat couscous (or substitute coarse bulgur)
1 cup no-salt-added or homemade vegetable broth or water
1 tablespoon plus 1 teaspoon extra-virgin olive oil
2 bunches Swiss chard, beet greens or combination (12 ounces)
2 tablespoons chopped fresh mint leaves, dill or parsley leaves or combination
1/2 teaspoon fine sea salt
1/2 teaspoon black pepper
1 tablespoon fresh lemon juice, plus more to taste
1 cup whole-milk Greek-style yogurt (or low-fat or nonfat)
2 cloves garlic, finely chopped
1 pound cooked and peeled beets, cut into 1/2-inch cubes
3 tablespoons chopped walnuts
To soak couscous: In medium bowl, combine couscous, broth or water and 1 teaspoon oil. Cover. Let rest for at least 45 minutes at room temperature or refrigerate overnight. (Note: If using coarse bulgur, it will be ready after about 90 minutes and can also be refrigerated overnight.) When ready to serve, fluff with fork.
To prepare greens: Strip greens from stems. Discard stems or reserve for another use. Rinse and dry leaves thoroughly. Slice thinly. Transfer to mixing bowl. Using hands, pick up greens and squeeze/massage. Repeat, working through bowlful. Continue squeezing/massaging until greens turn darker green and silky. Toss greens with 1 tablespoon chopped herbs, remaining 1 tablespoon oil, 1/4 teaspoon salt, 1/4 teaspoon pepper and lemon juice. Taste. Add lemon juice if needed.
To prepare yogurt: In medium bowl, whisk together yogurt, garlic and remaining 1/4 teaspoon salt and 1/4 teaspoon pepper.
To prepare beets: Toss beets with remaining 1 tablespoon chopped herbs.
To assemble: Divide couscous among individual bowls or plates. Top each portion with -- in following order -- greens, beets, dollops of yogurt mixture and sprinkling of walnuts. Serve immediately.
Nutrition information: One serving provides 310 calories, 14 grams protein, 53 grams carbohydrates, 8 grams fat, 2 grams saturated fat, 5 milligrams cholesterol, 510 milligrams sodium, 7 grams dietary fiber, 14 grams sugar.
This cool, refreshing grain-based salad, enlivened with a sesame-miso dressing, comes together quickly -- and without heating up THE kitchen. Recipe adapted from Washington Post food editor Joe Yonan, author of "Eat Your Vegetables: Bold Recipes for the Single Cook" (Ten Speed Press, 2013).
Makes 6 servings.
1 cup coarse bulgur (or substitute whole-wheat couscous)
1 cup no-salt-added or home-made vegetable broth or water
3 cups no-salt-added, cooked chickpeas, drained and rinsed
1 medium cucumber, peeled and cut into small dice
1/2 pint cherry or grape tomatoes, each cut in half
1 scallion, trimmed and thinly sliced
1 tablespoon white miso paste
1 tablespoon toasted sesame oil
1/4 cup unseasoned rice vinegar
3 tablespoons vegetable oil
1 tablespoon honey
2 cups fresh watercress leaves
1 avocado, peeled and cut into cubes
To soak bulgur: In medium bowl, combine bulgur and broth or water. Cover. Let rest for at least 90 minutes at room temperature or refrigerate overnight. (Note: If using whole-wheat couscous, it will be ready after 45 minutes but can also refrigerate overnight.) When ready to serve, fluff with fork.
To assemble salad: Add chickpeas, cucumber, tomatoes and scallion to bulgur. Toss to incorporate.
To make dressing: In medium bowl, whisk together miso, sesame oil, vinegar, vegetable oil and honey. Reserve 2 tablespoons. Pour remaining dressing over bulgur mixture. Toss to incorporate.
To serve: Divide watercress leaves among salad plates or bowls. Drizzle with remaining dressing. Mound some bulgur mixture on top of each portion. Add avocado. Serve.
Nutrition information: One serving provides 380 calories, 12 grams protein, 50 grams carbohydrates, 17 grams fat, 2 grams saturated fat, 0 cholesterol, 150 milligrams sodium, 13 grams dietary fiber, 9 grams sugar.


text:
Audio Visual
Sound, Lighting, Presentation Display Systems for Large Venue, Campus, and Commercial Space. Continue reading
Sound, Lighting, Presentation Display Systems for Large Venue, Campus, and Commercial Space. Continue reading
Complete Design, Installation, CAD, Inspection, Service, Upgrade, Maintenance, and Monitoring.
Continue reading
UPS and Generator Backup, IT, AV, CCTV, and Low Voltage related Power Outlet installation. Continue reading
Card Access, CCTV, Intrusion Detection, Intercom, Perimeter Protection, Visitor Management.
Continue reading
Data Centers, Campus, Office, Education, Government, Financial, and Health Care facilities. Continue reading
Traditional and Voice Over IP Business Telephone Systems and Service for both Large and Small needs. Continue reading
Hats off to Guy Raymond, Eddie Keith, Jordan Phillips, Tracy Byrd, Will Hall, and Sam Jackson…literally! This Strategic Connections team participated…
Strategic Connections has joined the ranks of Triangle Business Journal's 2014 Fast 50 winners. In this 22nd year of celebrating…
Posted on February 14, 2015
Lenovo Buildings 7 and 8 Working long shifts with multiple crews performing prefab assembly in our warehouse and simultaneous installation on…
Posted on September 8, 2014
Strategic Connections recently completed the massive cabling infrastructure and campus backbone project for RTP giant's 5th lab building, over 155,000…


text:
We thought you might enjoy a few flyers from Citizen Dick's shows at the Vogue and RKCNDY and a Cliff Poncier T-shirt special…
We thought you might enjoy a few flyers from Citizen Dick's shows at the Vogue and RKCNDY and a Cliff Poncier T-shirt special…
Here's the front cover of the upcoming 7″ for Citizen Dick's "Touch Me, I'm Sick". This Record Store Day exclusive is a single-sided 7" with an etching on the b-side. All the RSD releases on tap for April 18th can be found at the official Record Store Day site.
Happy Saturday. Thought it might be fun to look back on Cameron's Pearl Jam 1993 cover story for Rolling Stone. It was the band's first real in-depth interview just as Vs. was making its debut. There's an even longer version that we might share one day, but for now, check out the published version.
Five Against the World
Pearl Jam emerge from the strange daze of superstardom with a new album full of rage and warrior soul.
There are two Eddie Vedders. One is quiet, shy, barely audible when he speaks. Loving and loved in return. The other is tortured, a bitter realist, a man capable of pointing out injustice and waging that war on the home front, inside himself. On a warm and windy late-spring day in San Rafael, California, it's easy to see which Eddie Vedder is shooting baskets outside the Site, the recording studio where Pearl Jam are finishing their second album. It is tortured Eddie, the one with the deep crease between his eyebrows.
"Your shot," calls Jeff Ament, the group's bassist. He bounces the ball to Vedder, who takes a long outside jumper. It rattles into the basket and rolls away. By the time Ament retrieves the ball, Vedder has already disappeared into the studio. His mind is on a new song, "Rearviewmirror." This is the last day of recording at the Site, and the track's fate hangs in the balance. It's a song about suicide… but it's too "catchy."
Greg here. Since I live in Seattle and with today marking Singles 20th anniversary, I thought it would be fun to take a look at some of the filming locations and how they look now. I've included stills from the movie along with recent pictures that I've taken. It's crazy how many locations are gone or completely changed, while others have stood the test of time. We hope you enjoy Part 1 of this trip down memory lane.
Tidbit: The fountain was created out of styrofoam for the film and the building is only two stories, so there's not actually an elevator. In reality, Cliff's apartment was in the laundry room and storage area (located in the basement of the apartment complex).
Cameron shares some of his favorite concert set lists in the December 2011 issue of Filter magazine. He discusses the reasons for each inclusion along with the actual scan of the set list. Selections include Pearl Jam, Joni Mitchell, U2, Eddie Vedder, Simon and Garfunkel and My Morning Jacket. You can check out the full article in the Journalism section.
Cameron and the members of Pearl Jam shared their favorite rock documentaries with Reuters as part of the Pearl Jam Twenty press at the Toronto International Film Festival back in September. Here's their picks:
Cameron Crowe
Can I do two? Okay, Gimme Shelter and Don't Look Back. Gimme Shelter, because it's just amazing, on-the-fly film-making and Don't Look Back because it just captures the white-hot heat of somebody like Bob Dylan, who's exploding and there are cameras and microphones everywhere.
Eddie Vedder
The Kids are Alright and Last Waltz. It reminded of the time I got caught smoking pot and got grounded for a week in the summer. And it wasn't that bad, because all I did was listen to Last Waltz for 18 hours a day.
Matt Cameron
I saw Jimi plays Berkley and Jimi Hendrix (film). Those two films kind of blew my mind as a teen. And I also remember watching Freddie and his friends, about Freddie Mercury's home life. It had nothing to do with the band Queen, just how Mercury was as a person. It was mind-blowingly cool.
Jeff Ament
There was a documentary that came out three or four years ago called American Hardcore. That was sort of the music that I learned to play and to see the footage of some of the Detroit bands and the Boston bands was pretty amazing.
Stone Gossard
Spinal Tap has got to be in there too. It really is something that affected me hugely.
Mike McCready
I would say one life changing film was Woodstock. My first concept of a rock singer ever was probably Roger Daltrey at Woodstock doing his thing. And Hendrix, of course his version of The Star Spangled Banner was the one that went into my soul and I grabbed it, grabbed it and I ran with it.
Please chime in! What are your favorite rock documentaries?
Now that most of you have seen Pearl Jam Twenty, I thought it was a good time to share the Toronto International Film Festival press conference with you. This footage is courtesy of Now magazine. My videos didn't turn out nearly this good. I've also transcribed the press conference, if you'd like to read it, you can here.


text:
Advisory Commission on Intergovernmental Relations. 1980. The Federal Role in the Federal System:The Dynamics of Growth. Hearings on the Federal Role. Washington, DC: Government Printing Office.
Ayres, B. Drummond Jr. 1981 b. "Georgia Governor Calls on U.S. to Assist States." The New York Times, 9 August.
Bickel, Alexander M. 1970. The Supreme Court and the Idea of Progress. New York: Harper & Row.
Broder, David. 1981. "The Governors Have Their Fingers Crossed." The Washington Post, 16 August.
-----, and Naughtie, James M. 1981. "Governors Suggest Federal-State Responsibility Swap." The Hartford Courant, 10 August.
Chayes, Abram. 1976. "The Role of the Judge in Public Law Litigation." 89 Harvard Law Review1281.
Choper, Jesse. 1977. "The Scope of National Power vis-à-vis the States: ."The Dispensability of Judicial Review 86 Yale Law Journal1552.
Cohen, Richard E. 1981. "For Spending Cuts, Only the Beginning." National Journal, 8 August.
Cox, Archibald. 1978. "Federalism and Individual Rights under the Burger Court." 73 Northwestern University Law Review1, 18.
Davis, Albert J. 1981. "Stage Two: Revenue Return." Intergovernmental Perspective 7, 2 (Spring).
-----, and Shannon, John. 1981. "Stage Two: Revenue Turnbacks." Intergovernmental Perspective 7, 2 (Spring).
Diamond, Martin. 1969. "On the Relationship of Federalism and Decentralization." In Cooperation and Conflict: Readings in American Federalism, ed. Daniel J. Elazar. Itasca, IL: F. E. Peacock.
Elazar, Daniel J. 1962. The American Partnership. Chicago, IL: University of Chicago Press.
-----. 1971. "Community Self Government and the Crisis of American Politics." Ethics, January.
-----, ed. 1969. Cooperation and Conflict: Readings in American Federalism. Itasca, IL: F. E. Peacock.
-----. 1981. "The Rebirth of Federalism: The Future Role of the States as Polities in the Federal System." Commonsense 4, 1.