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- American Society of Plant Biologists
Tocopherols (vitamin E) are lipophilic antioxidants that are synthesized by all plants and are particularly abundant in seeds. Two tocopherol-deficient mutant loci in Arabidopsis thaliana were used to examine the functions of tocopherols in seedlings: vitamin e1 (vte1), which accumulates the pathway intermediate 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ); and vte2, which lacks all tocopherols and pathway intermediates. Only vte2 displayed severe seedling growth defects, which corresponded with massively increased levels of the major classes of nonenzymatic lipid peroxidation products: hydroxy fatty acids, malondialdehyde, and phytoprostanes. In the absence of pathogens, the phytoalexin camalexin accumulated in vte2 seedlings to levels 100-fold higher than in wild-type or vte1 seedlings. Similarly, gene expression profiling in wild-type, vte1, and vte2 seedlings indicated that increased levels of nonenzymatic lipid peroxidation in vte2 corresponded to increased expression of many defense-related genes, which were not induced in vte1. Both biochemical and transcriptional analyses of vte2 seedlings indicate that nonenzymatic lipid peroxidation plays a significant role in modulating plant defense responses. Together, these results establish that tocopherols in wild-type plants or DMPBQ in vte1 plants limit nonenzymatic lipid peroxidation during germination and early seedling development, thereby preventing the inappropriate activation of transcriptional and biochemical defense responses.
Tocopherols, which collectively constitute the essential dietary micronutrient vitamin E, are lipophilic antioxidants synthesized exclusively by plants, algae, and some cyanobacteria. There are four naturally occurring forms of tocopherols, α, β, γ, and δ, which differ in the presence and position of methyl groups on the chromanol ring. Because tocopherols are essential for human nutrition, tocopherol functions and chemistry have been studied most extensively in animal systems and in vitro. Although the biological activities of tocopherols may differ between plants and animals, the chemical properties of tocopherols are presumed to be the same in both kingdoms. Tocopherols scavenge a variety of reactive oxygen species (ROS) and free radicals and can quench singlet oxygen (Fukuzawa et al., 1982; Brigelius-Flohe and Traber, 1999; Wang and Quinn, 2000). The chromanol ring of tocopherol allows the donation of a hydrogen atom while maintaining a resonance-stabilized configuration (Liebler, 1993; KamalEldin and Appelqvist, 1996); other aromatic antioxidants must donate two hydrogen atoms to attain a stable state (Liebler and Burr, 2000). Donation of a hydrogen atom generates a tocopherol radical, which can be recycled back to the original tocopherol by direct interaction with ascorbate or another reductant (Liebler, 1993).
Although water-soluble antioxidants such as ascorbate and glutathione also scavenge ROS, in vitro and animal studies have established that tocopherols have the unique ability to protect polyunsaturated fatty acid (PUFA) acyl chains from oxidative damage because they are localized within membranes (reviewed in Schneider, 2005). ROS-generated radicals readily attack the double bonds of PUFAs, remove an electron, and create a lipid radical, which reacts with oxygen to form a lipid peroxyl radical (Figure 1A ). In the densely packed environment of lipid bilayers or other lipid aggregates, lipid peroxyl radicals will in turn attack neighboring PUFAs, propagating a chain reaction of lipid peroxidation through the membrane (Figure 1A). Tocopherols scavenge lipid peroxyl radicals by donating a hydrogen atom to these radicals, converting them to lipid peroxides and thereby blocking the propagation of lipid peroxidation in membranes (Figure 1A). The lipid peroxides produced are either enzymatically or spontaneously converted to relatively inert hydroxy fatty acids or other products in vivo.
Although a good deal is known about the chemistry and functions of tocopherols in animal systems and in vitro, much less is known about their functions in photosynthetic organisms (Fryer, 1992; Munne-Bosch and Alegre, 2002). Some important insights have been made in recent years from the analysis of tocopherol biosynthetic mutants in the cyanobacterium Synechocystis sp PCC6803 (Maeda et al., 2005; Sakuragi et al., 2006) and from the treatment of Chlamydomonas reinhardtii with an herbicide that disrupts both tocopherol and plastoquinone synthesis (Trebst et al., 2002, 2004). However, neither organism is multicellular, and understanding tocopherol functions in plants has only been possible with the isolation of mutants disrupting tocopherol synthesis in Arabidopsis thaliana, including vitamin e1 (vte1; tocopherol cyclase) and vte2 (homogentisate phytyl transferase) (Porfirova et al., 2002; Sattler et al., 2003, 2004; DellaPenna and Pogson, 2006). vte1 and vte2 mutants both lack tocopherols, but vte1 mutants accumulate the tocopherol pathway intermediate 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ), which lacks the characteristic chromanol ring of tocopherols but is still redox-active (Sattler et al., 2003, 2004). DMPBQ levels in vte1 mutant tissues are comparable to tocopherol levels in wild-type tissues, and DMPBQ is below the limit of detection in all wild-type and vte2 tissues (Sattler et al., 2003, 2004).
The vte mutants have allowed several groups to initiate studies of tocopherol functions in plants. Because tocopherols are only synthesized by photosynthetic organisms, are localized in chloroplasts, and accumulate during both abiotic and biotic stresses, it has been assumed that tocopherols play a central role in protecting chloroplast membranes from ROS generated by excessive light energy (Fryer, 1992; Munne-Bosch and Alegre, 2002). Surprisingly, mature vte1 and vte2 plants were indistinguishable from wild-type plants both under normal growth conditions (Porfirova et al., 2002; Sattler et al., 2003, 2004; Maeda et al., 2006) and during high light stress (Porfirova et al., 2002; Havaux et al., 2005; Maeda et al., 2006). It was only during extreme abiotic stress, when low temperature was combined with high light, that measurable physiological differences were observed between vte1 and wild-type plants (Havaux et al., 2005). Tocopherols have since been shown to play essential roles in low-temperature adaptation (Maeda et al., 2006), a response that is independent of light level. Thus, many of the functions deduced for tocopherols by comparing the responses of vte1 and wild-type plants with combined high-light and low-temperature treatment (Havaux et al., 2005) were likely attributable to the loss of low-temperature tocopherol functions (Maeda et al., 2006). These combined data suggest that the role of tocopherols in the response to high light is more limited than has been assumed in mature leaves.
In addition to temperature adaptation, tocopherols have also been show to play critical roles in seed and seedling physiology; both vte1 and vte2 have significantly reduced seed longevity compared with wild-type plants (Sattler et al., 2004). During germination, vte2 seedlings also displayed a range of defects, including impaired root growth, cotyledon expansion defects, and slowed storage lipid catabolism, all of which were correlated with dramatic increases in nonenzymatic lipid peroxides and the corresponding hydroxy fatty acids (Sattler et al., 2004). The vte2 mutant clearly demonstrates that an essential function of tocopherols is to control lipid peroxidation during germination and early seedling development. The observation that germinating vte1 seedlings were nearly identical to wild-type seedlings suggested that DMPBQ, which accumulates in vte1, can functionally replace tocopherol in this regard (Sattler et al., 2004).
In living organisms, nonenzymatic lipid peroxidation is a natural, continually occurring process, but under oxidative stress conditions, which enhance ROS and radical production, the levels of nonenzymatic lipid peroxidation can increase dramatically. In plants, nonenzymatic lipid peroxidation is known to increase during many abiotic stresses (Baryla et al., 2000) and in response to both bacterial and fungal pathogen attack (Muckenschnabel et al., 2001; Thoma et al., 2003). Linoleic (18:2) and linolenic (18:3) acids are highly abundant in plant membrane lipids and are the main reactants in nonenzymatic lipid peroxidation (Berger et al., 2001; Mueller, 2004; Weber et al., 2004). Peroxyl radicals, which are unstable and highly reactive, are formed on PUFA acyl chains and undergo a series of reactions resulting in at least three distinct classes of products (Figure 1) (Porter et al., 1995). The lipid peroxyl radical can be reduced to the corresponding hydroxy fatty acid, fragmented into a variety of reactive aldehydes including malondialdehyde (MDA), nonenal, hexanal, and hexenal, or cyclized to form a large class of compounds known as isoprostanes (Figure 1; phytoprostanes in plants) (Mueller, 1998). The initial phytoprostane formed in the membrane is PPG1, but it is highly unstable and either spontaneously rearranges to form other phytoprostanes or fragments, releasing MDA (Figure 1A). 18:2 peroxyl radicals primarily form hydroxy fatty acids and fatty acid fragments, whereas 18:3 peroxyl radicals form all three classes of compounds (Mueller, 2004; Weber et al., 2004).
Nonenzymatic lipid peroxidation products can also be separated into two groups based on chemical functionality: electrophiles that contain highly reactive α/β unsaturated carbonyl groups (Vollenweider et al., 2000; Stintzi et al., 2001; Almeras et al., 2003; Weber et al., 2004), and less reactive classes lacking α/β unsaturated carbonyl groups. Electrophiles, which include MDA and some of the phytoprostanes, can have deleterious effects on plant cells by covalently modifying thiol groups of peptides as well as reacting with other cellular components. Moreover, most nonenzymatic lipid peroxidation products initially accumulate within membranes and can disrupt the selective permeability of the lipid bilayer (Mueller, 2004).
The ability of enzyme-derived lipid peroxidation products, the oxylipins (JA and OPDA) in plants (Figure 1B) and the eicosanoids (prostaglandins, leukotrienes, and thromboxanes) in animals, to act as signals and to regulate gene expression is well established (reviewed in Funk, 2001; Creelman and Mulpuri, 2002; Howe and Schilmiller, 2002). There is a growing body of evidence that certain nonenzymatic lipid peroxidation products can similarly act as signals and regulate gene expression in both the plant and animal kingdoms. In animals, nonenzymatic lipid peroxidation products, including isoprostanes, have been shown to act at low levels as signaling molecules that modulate the function of the immune and vascular systems (Bochkov et al., 2002; Hazen and Chisolm, 2002; Traidl-Hoffmann et al., 2005), whereas high levels are clearly detrimental (Leitinger, 2003). In plants, externally applied MDA and specific phytoprostanes (PPB1, PPA1, dPPJ1, and PPE1) have been shown to induce the expression of several stress-related genes (Vollenweider et al., 2000; Almeras et al., 2003; Thoma et al., 2003; Weber et al., 2004; Iqbal et al., 2005; Loeffler et al., 2005). To better understand the roles of tocopherols in plants, we examined nonenzymatic lipid peroxidation products in vte1 and vte2 and the effects of these mutations on global gene expression. These two mutants allow us to delineate the direct functions of tocopherols from those as a lipophilic antioxidant and to address how the absence of the plant's main lipophilic antioxidant affects gene expression and stress responses in Arabidopsis seedlings.
MDA Analysis
In a prior study, lipid-soluble peroxides and hydroxy octadecadienoic fatty acids (9- and 13-HODEs derived from 18:2) were found to be increased up to 5- and 100-fold, respectively, in germinating vte2 seedlings compared with wild-type or vte1-1 seedlings (Sattler et al., 2004). The stereoisomeric and enantiomeric compositions of the HODEs detected in vte2 seedlings were indicative of high levels of nonenzymatic lipid peroxidation (Sattler et al., 2004). However, hydroxy octadecatrienoic acids (HOTEs; 18:3-derived hydroxy fatty acids) were ∼20-fold less abundant than HODEs in vte2 seedlings (Sattler et al., 2004), even though 18:2 and 18:3 fatty acid levels in Arabidopsis seed are nearly equal in abundance and 18:3 autooxidizes far more readily than 18:2 in vitro and in vivo. Thus, the question remained, is 18:3 not being oxidized in vte2 seedlings, or are products other than HOTEs being formed?
To address this issue, we measured a broader spectrum of nonenzymatic lipid peroxidation products in germinating seedlings. PUFA peroxyl radicals formed in the membrane during lipid peroxidation can subsequently decompose, resulting in an instantaneous release of MDA and other volatile alkane and alkene compounds. In Arabidopsis leaves, ∼75% of the MDA is formed from 18:3 (Weber et al., 2004). Total MDA levels in 0-, 1-, 3-, and 6-d-old seedlings were measured using gas chromatography–mass spectrometry (GC-MS) and found to be significantly increased in vte2 seedlings at all time points relative to wild-type or vte1 seedlings (Figure 2 ). In vte2 seedlings, MDA levels increased dramatically at 1 d to levels 14-fold higher than in either wild-type or vte1 seedlings. MDA levels peaked at 3 d in vte2 at levels fourfold higher than in either wild-type or vte1 seedlings. These data underscore the importance of tocopherols (or DMPBQ in vte1) in preventing the formation MDA during germination, an oxidatively intense period of the plant life cycle.
Phytoprostane Analysis
Another major group of nonenzymatic lipid peroxidation products, the phytoprostanes, are formed from 18:3 when linolenic peroxyl radicals cyclize to form PPG1. Spontaneous rearrangements result in the numerous species of phytoprostanes found in plant tissues (Figure 1B) (Mueller, 2004). We analyzed the formation of total F1-phytoprostanes (free and esterified PPF1) in 0-, 1-, 3-, and 6-d-old seedlings, because PPF1 are a class of nonreactive, chemically stable phytoprostane end products that are excellent markers for nonenzymatic lipid peroxidation (Imbusch and Mueller, 2000a). Similar to the results for MDA (Figure 2), lipid peroxides, and HODEs (Sattler et al., 2004), total PPF1 levels in wild-type and vte1 seedlings (Figure 3 ) were low and comparable to levels observed in mature leaves (Imbusch and Mueller, 2000a). By contrast, even in dry vte2 seeds collected immediately from plants, PPF1 levels were already increased (4.78 nmol/g seed) compared with those in the wild type, which were below the limit of detection (0.03 nmol/g seed). PPF1 levels in vte2 seeds increased almost ninefold after 5 d of imbibition at 6°C (42.5 nmol/g dry weight at 0 d of growth). At 1, 3, and 6 d of growth, PPF1 levels were 100- to 200-fold higher in vte2 relative to wild-type or vte1 seedlings, reaching a maximum level at 3 d before beginning to decline. At later time points, 14 and 35 d, the PPF1 levels in mature vte2 plants were similar to those in wild-type plants (Figure 3B), suggesting that PPF1 is turned over or that its production is limited by tocopherol-independent mechanisms in mature leaves. These data are consistent with a previous report showing that lipid peroxide levels in vte2 approached wild-type and vte1 levels by 18 d of growth (Sattler et al., 2004).
The extremely high phytoprostane levels in 3- and 6-d-old vte2 seedlings explain the relative differences in abundance between HODEs (derived from 18:2) and HOTEs (derived from 18:3), because 18:3 and HOTEs are prone to undergo double oxygenation as well as cyclization reactions after free radical attack. Therefore, phytoprostanes, MDA, and other oxidized lipids are formed instead of HOTEs in the absence of tocopherols (or DMPBQ in vte1) in vte2 seeds and seedlings.
JA, OPDA, and Oxylipin Analysis
Enzymatically driven oxidation of 18:3 can produce a number of oxylipins, including the phytohormone JA and its precursor OPDA. To determine the levels of JA or OPDA in vte seedlings, quantitative analysis of jasmonates was performed by GC-MS on 0-, 1-, 3-, and 6-d-old seedlings. Free OPDA levels were similar in vte2 and wild-type seedlings at all time points but were twofold to fourfold lower in vte1 than in the wild type at 3 and 6 d (Figure 4 ). In the wild type, JA levels were near or below the detection limit (2 to 4 pmol/g dry weight) for all time points (Figure 4), and they were increased in both vte1 and vte2 relative to the wild type at 3 and 6 d. Although these results suggest that tocopherol deficiency modestly induces JA accumulation, the maximal JA level in vte2 of 0.49 nmol/g dry weight (corresponding to ∼38 pmol/g fresh weight) is still severalfold lower than the 171 pmol/g fresh weight in unwounded Arabidopsis leaf tissue and 200 times lower than the 8570 pmol/g fresh weight induced by wounding Arabidopsis leaf tissue (Weber et al., 1997).
An oxylipin profiling method (Weber et al., 1997) was also applied to vte2-1 seedlings to assess the level of other known enzymatically generated oxylipins and to survey for the presence of potentially novel oxylipins in addition to JA and OPDA. This method is able to detect >20 different oxylipins as well as other stress-related compounds (Mueller et al., 2006). Only four compounds, none of which is an oxylipin, were highly increased in vte2 relative to the wild type and vte1. These compounds were two free fatty acids, oleic (18:1) and eicosenoic (20:1) acids, the phytoalexin camalexin, and its putative biosynthetic precursor indole 3-carboxylic acid. The levels of both 18:1 and 20:1 were ∼20-fold higher in 3- and 6-d-old vte2 seedlings relative to wild-type or vte1 seedlings.
Camalexin in vte2
Camalexin in seedlings was quantified using an HPLC-based method. Camalexin was below the detection limit (0.5 nmol/g dry weight) in wild-type and vte1 seedlings at all time points and at 0 and 1 d in vte2. Camalexin levels in vte2 increased to ∼50 nmol/g dry weight at 3 and 6 d, a >100-fold increase relative to the wild type or vte1 (Figure 5A ). Generally, camalexin is produced in response to plant pathogen attack, but its synthesis can also be induced by treating plants with redox-active heavy metals such as silver, copper, or iron (Mert-Turk et al., 2003; Glawischnig et al., 2004; Loeffler et al., 2005; Schuhegger et al., 2006). However, all seedlings were grown on sterile Murashige and Skoog medium, and pathogens or exogenous heavy metals were not present.
To further examine the basis of camalexin production in vte2 seedlings, double mutants between vte2 and two camalexin-deficient mutants, phytoalexin-deficient3 (pad3) and pad4, were isolated. PAD3 is a cytochrome P450 protein in the camalexin biosynthetic pathway (Zhou et al., 1999; Schuhegger et al., 2006), whereas PAD4 has similarity to triacylglycerol lipases and is involved in defense signaling leading to camalexin biosynthesis (Jirage et al., 1999). As expected, 3- and 6-d-old pad3, pad4, and pad3 vte2 seedlings lacked camalexin (Figure 5B). However, pad4 vte2 seedlings contained camalexin at levels threefold higher than vte2 seedlings at 3 d and at levels comparable to vte2 at 6 d (Figure 5B), indicating that the induction of camalexin synthesis is PAD4-independent in vte2 seedlings. The presence of camalexin in vte2 seedlings in the absence of pathogen attack suggests that the high level of nonenzymatic lipid peroxidation in vte2 seedlings triggers a PAD4-independent signaling pathway leading to camalexin synthesis. This result suggests that tocopherols play a role in regulating biotic stress responses, such as camalexin synthesis, by modulating the level of nonenzymatic lipid peroxidation.
Expression Profiling in the vte Mutants
To gain further insight into how seedlings respond to the absence of tocopherols and to increased levels of lipid peroxidation products, we examined global gene expression profiles in 1- and 3-d-old vte1, vte2, and wild-type seedlings using an Affymetrix ATH1 GeneChip. The chip contained 22,810 probe sets, representing nearly the entire Arabidopsis genome, and 15,293 probe sets were called present for at least one of the conditions. As a whole, the expression of relatively few genes was either significantly increased or decreased (≥2.5-fold) relative to the wild type in either vte1 or vte2 at 1 or 3 d after imbibition. Our criteria for the expression of a gene to be considered increased (or decreased) relative to the wild type required that the probe set was called present for all three chips of the mutant (or called present in the wild type for decreased) and the signal log ratio was ≥1.3 (or ≤−1.3 for decreased) for all three comparisons of the mutant versus wild-type chips. In vte2 seedlings, the transcript levels for 21 and 160 probe sets were increased relative to the wild type at 1 and 3 d, respectively (see Supplemental Table 1 online). In vte1 seedlings, the transcript levels for 24 and 12 probe sets were increased relative to the wild type at 1 and 3 d, respectively (see Supplemental Table 1 online). In vte2, the transcript levels for 5 and 3 probe sets were decreased relative to the wild type at 1 and 3 d, respectively (see Supplemental Table 2 online). In vte1, the transcript levels for 0 and 6 probe sets were decreased relative to the wild type at 1 and 3 d, respectively (see Supplemental Table 2 online).
Interestingly, there was only a single probe set that was upregulated at 1 d in both vte1 and vte2 relative to the wild type, At5g39410, an expressed gene. There was only a single probe set that was downregulated in both vte1 and vte2 at 3 d, At3g62950, a glutaredoxin-like protein. At 3 d after imbibition, 6 of the 12 probe sets that were upregulated in vte1 were also upregulated in vte2. Genes for all six of these probe sets encode heat shock proteins (HSPs) and correspond to four classes of cytosolic HSPs (17, 70, 81, and 101). This set of six HSPs appears to represent the only genes whose regulation is similarly affected in vte1 and vte2 (i.e., tocopherol deficiency–specific) during early seedling growth and development (see Supplemental Table 1 online).
Curiously, at 1 d, the vte1 global expression pattern was consistent with a transient delay in germination and subsequent development, as had been reported previously in germinating vte1 seedlings (Sattler et al., 2004). Eight of the 24 probe sets upregulated at 1 d in vte1 are annotated as late embryogenesis abundant proteins. This expression profile may indicate a transient delay in vte1 development at 1 d but does not suggest an apparent cause for this delay. Evidence of a developmental delay is no longer apparent by 3 d in vte1.
The most dramatic changes observed in vte2 seedlings were at 3 d after the imbibition treatment. The majority of the 160 upregulated probe sets at day 3 were annotated as responsive to either abiotic or biotic stress. These transcripts encode gene products that are localized in nearly all subcellular compartments, including the cytosol, chloroplast, mitochondria, cell wall, and secretory pathway. Probe sets for 17 transcription factors were upregulated in the 3-d-old vte2 seedlings, including many that are stress-associated, such as NAMs, ZAT12, WRKYs, and DREB2-like (Dong et al., 2003; Rizhsky et al., 2004; Davletova et al., 2005; Vogel et al., 2005). Probe sets for nine HSPs were upregulated (including the six heat shock probe sets also upregulated in vte1), along with four probe sets related to proteolysis, which suggests increased protein damage in vte2. Probe sets for eight glutathione S-transferase (GST) genes, five cytochrome P450 genes, and eight oxidoreductase genes were also upregulated.
Probe sets for three Trp biosynthetic genes were upregulated in vte2 as well as the probe set for PAD3 (CYP71B15), which catalyzes the final step in camalexin synthesis (Zhou et al., 1999; Schuhegger et al., 2006) (Figure 6 ; see Supplemental Table 1 online). The probe sets for two additional cytochrome P450s, CYP79B2 and CYP79B3, which convert Trp to indole-3-acetaldoxime, an intermediate in the synthesis of camalexin, auxin, and indole glucosinolates (Glawischnig et al., 2004), were called increased for all three comparisons with the wild type, and the P values were highly significant (P = 2.5 × 10−6 and 1.2 × 10−8, respectively), but their signal:log ratios for at least one comparison were slightly below our cutoff of 1.3. The Trp metabolic pathway is upregulated specifically in vte2, evidently in response to the absence of tocopherols or DMPBQ during germination.
The 20 most highly upregulated transcripts in 3-d vte2 seedlings relative to the wild type are indicative of responses to both abiotic and biotic stresses (Table 1 ). These transcripts include the pathogen-related genes AIG1 and Defensin1.4 (Reuber and Ausubel, 1996) and a DREB2-like transcription factor. Members of the DREB class of transcription factors have been shown to activate transcriptional responses to abiotic stresses, including drought, salinity, and temperature (Liu et al., 1998; Shinozaki and Yamaguchi-Shinozaki, 2000). Two pEARLI genes, which encode proteins with a putative lipid-transfer motif, were highly upregulated in vte2 (Wilkosz and Schlappi, 2000). The pEARLI genes were originally isolated for their cold-responsiveness, but they have since been shown to be induced by a number of stresses (Bubier and Schlappi, 2004; Vogel et al., 2005). SAG13, a senescence-associated transcript (Lohman et al., 1994; Miller et al., 1999), was also highly upregulated in vte2, as was an iron deficiency–inducible metal transporter, IRT1 (Varotto et al., 2002). Transcripts of stress-protective enzymes, GST5, a peroxidase, and Glu decarboxylase were also induced in vte2 (Watahiki et al., 1995; Turano and Fang, 1998; Bouche et al., 2004).
To confirm the Affymetrix GeneChip results indicating that defense/pathogen-responsive genes were induced in vte2 seedlings, RNA gel blot analysis was performed on total RNA extracted from 3-d-old seedlings with two defense- or pathogen-related genes, OPR1 (At1g76680) and PAD3 (Figure 7A ). These results show that both transcripts were specifically induced in vte2 relative to the wild type or vte1. Expression of the jasmonate biosynthetic gene AOS was not detectable in the wild type, vte1, or vte2 (Figure 7B) in the absence of exogenously administered methyl jasmonate (MeJA). However, the jasmonate biosynthetic gene OPR3 was detectable in all three genotypes and marginally induced in vte2 seedlings relative to wild-type or vte1 seedlings in the absence of exogenous MeJA (Figure 7B), which is consistent with slightly increased levels of JA in vte2 (Figure 4B). The expression of the JA-dependent gene VSP1 was also not detectable in wild-type, vte1, and vte2 seedlings in the absence of exogenous MeJA treatment. The JA-inducible gene CORI1 was also present in all three genotypes in the absence of exogenous MeJA (Figure 7B). Upon exogenous MeJA treatment, the expression of all four genes (AOS, CORI, OPR3, and VSP1) was strongly induced (Figure 7B). Together, these RNA gel blot results suggest that the levels of jasmonates in both vte1 and vte2 seedlings are not sufficient to induce JA-responsive gene expression in the seedlings, but that 3-d-old wild-type, vte1, and vte2 seedlings are capable of mounting a transcriptional response when JA levels are adequately increased.
Tocopherols and Germination
The phenotype conferred by vte2 clearly demonstrates that tocopherols are irreplaceable antioxidants during germination and early seedling growth that cannot be compensated for by other mechanisms. Indeed, in this regard, it was quite surprising that the expression of most genes involved in the biosynthesis and recycling of ascorbate and glutathione was similar in the wild type and vte2, expect for the induction in vte2 of 1 of 3 extracellular ascorbate oxidase genes (At4g39830) and 8 of the 52 GST genes in the genome. Likewise, genes encoding ascorbate peroxidases (8 genes) and the ROS-scavenging enzymes catalase (3 genes) and superoxide dismutase (11 genes) are also not induced in vte2 seedlings relative to wild-type seedlings. These results suggest either that the expression of genes encoding these ROS-scavenging enzymes is already at nearly maximal levels during germination and early seedling development or that young seedlings are unable to increase the expression of these genes in response to the compromised tocopherol antioxidant system of vte2. Alternatively, the seedling response to tocopherol deficiency may simply not include the induction of water-soluble antioxidant or ROS-scavenging systems, because the oxidative damage in vte2 is most likely confined to the lipid-soluble cellular compartments.
In comparing the global transcriptional response of vte1 and vte2 seedlings at 1 and 3 d after imbibition, the increased or decreased expression of only eight genes could be attributed specifically to the absence of tocopherol in both genotypes. Six genes are HSPs, some of which have been shown to act as chaperones, and are upregulated in response to a variety of abiotic stress conditions. These results suggest that as a whole, the Arabidopsis transcriptome has relatively few genes that respond directly to the presence of tocopherol at this stage of development. These observations are in contrast with studies in animal systems, in which direct, antioxidant-independent roles for tocopherols in modulating signaling pathways by inhibiting phospholipase A2 and protein kinase C activity and activating gene transcription have been reported (Tran et al., 1996; Yamauchi et al., 2001; Chandra et al., 2002; Clement et al., 2002). It is still possible that tocopherols have analogous antioxidant-independent functions at other stages of plant development, but this is clearly not a major function for tocopherols in seedlings. However, the induction of six HSP genes in both vte1 and vte2 indicates that there are biological consequences of tocopherol deficiency and that there may be a group of proteins that are specifically protected by tocopherols in germinating seedlings. Increased nonenzymatic lipid peroxidation was not required to trigger this HSP response, because vte1 and the wild type had nearly identical levels of lipid peroxides and lipid peroxidation products: HODEs (Sattler et al., 2004), MDA (Figure 2), and PPF1 (Figure 3). Based on these data, we can also conclude that the DMPBQ accumulated in vte1 can fully compensate for the absence of tocopherols in limiting the formation of nonenzymatic lipid peroxidation during germination and early seedling development. Whether DMPBQ plays a similar compensatory role in mature vte1 leaf tissue remains to be determined.
Tocopherols, Nonenzymatic Lipid Peroxidation, and the Control of Gene Expression
Although we find little evidence supporting a direct role for tocopherols in the regulation of gene expression, our analysis clearly indicates that, similar to in vitro and animal studies, tocopherols play a critical role in controlling nonenzymatic lipid peroxidation. The products generated by this pathway most likely play a key role in the global response observed in vte2 seedlings and likely in selected biological responses of wild-type plants. We observed high levels of lipid peroxidation products in vte2 seedlings, hydroxy fatty acids from 18:2 (Sattler et al., 2004) and 18:3 (MDA and PPF1), the predominant PUFAs in Arabidopsis seedlings (Figures 2 and 3). Hydroxy fatty acids and most phytoprostanes are relatively stable molecules, initially remaining esterified in the membrane, and are indicative of the oxidative history of a tissue. However, during membrane turnover and repair, phytoprostanes are released from membranes and rapidly metabolized (Imbusch and Mueller, 2000a; Mueller, 2004). By contrast, MDA is produced and released instantaneously by fragmentation of oxidized membrane lipids and is a member of a group of short-lived, lipid-derived reactive electrophile species (RES) produced in response to stress. For these reasons, MDA and PPF1 do not reach similar levels or follow the same temporal patterns of accumulation in vte2 seedlings.
In vte2 seedlings, MDA levels peaked at 3 d and declined rapidly to near wild-type and vte1 levels by 6 d. This rapid decline is consistent with prior studies, suggesting that plants have mechanisms to control the levels of MDA (Weber et al., 2004) and indicating that these mechanisms are tocopherol-independent in seedlings. RES, such as MDA, have strong biological activities in plants and stimulate the expression of genes involved in cell protection/detoxification (e.g., OPR1 [Almeras et al., 2003] and GST1 [Vollenweider et al., 2000; Loeffler et al., 2005]) as well as genes implicated in resistance to stresses such as heat shock and dehydration (Weber et al., 2004). GST1 has been shown to be stress- and MDA-inducible and is strongly upregulated in vte2. Likewise, OPR1, which is absent from the Affymetrix ATH1 GeneChip, is strongly upregulated in vte2 based on RNA gel blot analysis (Figure 7) and in response to a number of abiotic and biotic stress conditions (Biesgen and Weiler, 1999; Almeras et al., 2003; Weber et al., 2004; Mezzari et al., 2005).
In addition to MDA, the phytoprostanes, a second major class of nonenzymatic lipid peroxidation products derived from 18:3, were increased in vte2 seedlings to some of the highest levels ever observed in plants (Imbusch and Mueller, 2000a). The phytoprostane class measured, PPF1, has not been shown to have biological activity when administered exogenously, possibly because this class is polar and not readily taken up by cells, as is the case for the analogous F-type of mammalian prostaglandins in cells lacking prostaglandin uptake transporters (Schuster, 2002). However, the PPF1 class is an excellent marker for nonenzymatic lipid peroxidation and indicative that other classes of more labile and reactive phytoprostanes (PPA1, PPB1, PPD1, PPE1, PPG1, and dPPJ1) are likely to have been generated also (Imbusch and Mueller, 2000a). Exogenously supplied PPA1 and PPB1 have also been shown to trigger biological responses in plants, including activating mitogen-activated protein kinase kinase, inducing phytoalexin synthesis, and activating gene transcription (Thoma et al., 2003; Loeffler et al., 2005), including 17 GST genes. Previous work has suggested that some of the GSTs and P450s induced by phytoprostanes may function in detoxifying potentially damaging products of nonenzymatic lipid peroxidation and electrophilic xenobiotics (Baerson et al., 2005; Loeffler et al., 2005; Mezzari et al., 2005). In vte2 seedlings, 4 of these 17 GST genes were also upregulated relative to the wild type.
Within the phytoprostane family of compounds as well as in other lipid peroxidation products, there are several species that contain α/β unsaturated carbonyl groups and, like MDA, are electrophilic and reactive. There is substantial evidence that such electrophiles, which include PPA1, dPPJ1, β-hydroxyacrolein (a tautomeric form of MDA), and the JA biosynthetic intermediate OPDA, have potent effects on gene expression, possibly attributable to their electrophilic properties (Vollenweider et al., 2000; Stintzi et al., 2001; Almeras et al., 2003; Mueller, 2004; Weber et al., 2004). PPA1, dPPJ1, and OPDA are all cyclopentenones containing an unsaturated α/β carbonyl group within a five-carbon ring. Expression profiling of Arabidopsis suspension cells treated with either PPA1 or PPE1 affected the expression of ∼800 genes (S. Berger and M.J. Mueller, unpublished data). The expression of 59 of the 160 genes upregulated in vte2 seedlings at 3 d is also induced by either PPA1 or PPE1 treatment of Arabidopsis suspension cells. Together, these data indicate that nonenzymatic lipid peroxidation products generated in vivo play an important role in the transcriptional response observed in vte2 seedlings and provide further evidence that electrophilic lipid peroxidation products play a significant role in modulating gene expression and cellular responses in plants.
The 160 transcripts induced in 3-d-old vte2 seedlings relative to wild-type seedlings (see Supplemental Table 1 online) were examined for their responses to both abiotic and biotic conditions using GENEVESTIGATOR (Zimmermann et al., 2004, 2005) (Figure 8 ). Most of these genes were unchanged in expression in 3-d-old vte1 seedlings, which further highlights how dissimilar the tocopherol-deficient vte1 and vte2 transcription profiles are to each other (Figure 8). Generally, many of the vte2-1–induced genes were also induced in response to biotic stress (Pseudomonas, Alternaria, and Botrytis infection), but surprisingly few of these genes were induced in response to abiotic stress treatments, including drought, wounding, heat, or cold stress. In addition, many of the 160 vte2-1–induced genes were also induced by ozone, salt, and silver nitrate but not by MeJA treatment. Ozone is itself a ROS and readily penetrates membranes, causing nonenzymatic lipid peroxidation of PUFAs (Pauls and Thompson, 1980; Pryor and Church, 1991), whereas exposure of plants to heavy metals such as silver nitrate is also known to induce nonenzymatic lipid peroxidation (Baryla et al., 2000; Imbusch and Mueller, 2000b; Thoma et al., 2004).
The meta-analysis in Figure 8 suggests that from a biological perspective, the transcriptome of vte2 seedlings responds as if under pathogen attack and that the nonenzymatic lipid peroxidation occurring in vte2 seedlings plays a role in triggering aspects of plant responses to pathogens. The fact that the majority of the genes induced in both vte2 and during pathogen attack are also not induced by MeJA treatment suggests that nonenzymatic lipid peroxidation is a component of previously described JA-independent responses of plants to pathogens (Kloek et al., 2001; Roetschi et al., 2001; Almeras et al., 2003; Bohman et al., 2004). Most surprisingly, this analysis also suggests that abiotic stresses such as drought, heat, wounding, or cold stress do not have a strongly regulated nonenzymatic lipid peroxidation component. Although tocopherols show increased accumulation under drought and heat (Munne-Bosch and Alegre, 2002), tocopherols may not play as crucial a role in protecting plants from these abiotic stresses as has long been assumed. A plausible alternative explanation is that increased tocopherol levels in response to these abiotic stresses may limit nonenzymatic lipid peroxidation and thereby prevent the inappropriate induction of biotic stress responses. Further analysis of the response of tocopherol-deficient plants to abiotic stresses is needed to test this intriguing hypothesis.
Induction of Trp Metabolism
Trp provides the indole ring for the biosynthesis of a variety of important plant compounds, including auxin, and the indole glucosinolates and phytoalexins (Glawischnig et al., 2004), which are important for defending plants against insect herbivory and pathogens, respectively. In plants, Trp biosynthetic genes are upregulated in response to a variety of biotic and xenobiotic stresses as well as to amino acid starvation (Zhao and Last, 1996; Zhao et al., 1998) and in response to electrophilic vinyl ketones (Almeras et al., 2003). The expression of PAD3 (CYP71B15), a P450 involved in camalexin biosynthesis, is upregulated in vte2 seedlings (Figures 6 and 7) and in response to PPB1 treatment (Loeffler et al., 2005). PAD3 catalyzes the final step in camalexin synthesis, acting downstream of CYP79B2 and CYP79B3 (Glawischnig et al., 2004; Schuhegger et al., 2006), whose expression levels were likewise increased in vte2 relative to the wild type, although slightly below our 1.3 signal:log ratio criterion. Because several Trp biosynthetic genes are upregulated in vte2 seedlings (Figure 6), our data suggest that nonenzymatic lipid peroxidation may be a key signal for the increase of Trp metabolism in response to biotic stress in the wild type. Interestingly, the spontaneous lesion mutant acd2 also accumulates camalexin in the absence of pathogens (Zhao and Last, 1996), although any possible connection between nonenzymatic lipid peroxidation and the acd2 mutant remains to be determined.
PAD4 has similarity to triacylglycerol lipases (Jirage et al., 1999) and has been shown to be a member of a defense signaling complex together with EDS1 and SAG101 (Feys et al., 2001, 2005). pad4 mutants are unable to fully induce several defense responses, including camalexin synthesis in response to specific pathogens (Glazebrook et al., 1997). Interestingly, the camalexin level in the pad4 vte2 double mutant was significantly higher than that in vte2 at 3 d and nearly equal to that in vte2 at 6 d, indicating that camalexin synthesis in the vte2 mutant is either independent of or repressed by PAD4. These data support the model that both PAD4-dependent and -independent pathways lead to camalexin synthesis (Glazebrook et al., 1997). Our data further define an important role for nonenzymatic lipid peroxidation in inducing the PAD4-independent pathway, but they do not exclude the possibility that nonenzymatic lipid peroxidation might also be involved in the PAD4-dependent pathway. The oxidative burst associated with incompatible host–pathogen interactions would create the biochemical environment necessary to form a variety of nonenzymatic lipid peroxidation products and trigger downstream defense responses such as camalexin synthesis. Our data suggest that tocopherols play a role in modulating such defense signals in seedlings under normal conditions or in locations removed from the site of pathogen attack. Whether such biotic interactions are also affected in mature vte2 leaves remains to be determined.
Nonenzymatic versus Enzymatic Lipid Peroxidation Pathways
Both plants and animals have evolved enzymatic pathways for the production of lipid peroxidation products (octadecanoids in plants and eicosanoids in animals), and the molecular, genetic, and biochemical roles of these compounds in mediating/modulating biological responses, including defense responses, have been well studied. In plants, the best-described oxylipin is JA, which affects a wide range of signal transduction pathways, including reproductive development, pathogen defense, and insect defense (Howe and Schilmiller, 2002). However, there has been comparably little analysis of the products of nonenzymatic lipid peroxidation as signaling compounds, probably because the production of these compounds occurs independently of enzymatic control and leads to a wider range of products. Undoubtedly, nonenzymatic lipid peroxidation occurs ubiquitously in organisms because it requires only PUFAs, oxygen, and free radicals. In plants, these nonenzymatic and enzymatic pathways appear to have diverged, and vte2 seedlings are strong genetic evidence that the two pathways have separate roles.
The potential of RES to act as regulators of gene expression in plants has, until now, lacked genetic validation (Farmer et al., 2003). The data presented here provide a strong genetic demonstration of RES activity in vivo and underscore the potential of nonenzymatic lipid peroxidation products to act as signals. In addition, these data provide clear genetic evidence separating enzymatically derived oxylipins and nonenzymatic lipid peroxidation products in signaling pathways. The levels of JA were increased slightly in both vte1 and vte2, and the levels of OPDA were similar in vte2 and wild-type seedlings, which suggests that JA and OPDA are not major factors contributing to the vte2 seedling phenotype. The observation that well-characterized JA-inducible genes (AOS, CORI1, and VSP1) were not altered in expression in vte2, vte1, and wild-type seedlings but were similarly induced upon exogenous MeJA treatment in 3-d-old seedlings of all genotypes supports this conclusion. These genes were clearly responsive to JA at this stage of development, but the endogenous JA levels in all three genotypes were insufficient for their induction (Figure 7B). vte2 was crossed to the JA signaling mutant coi1, and the progeny of vte2 coi1/+ (heterozygous for coi1) were not altered in their development compared with vte2 seedlings alone, indicating that JA signaling is not a key factor in the vte2 seedling phenotype (S.E. Sattler, unpublished data). Finally, analysis of public GeneChip data for MeJA and ozone-induced transcripts (≥2.5-fold relative to their respective controls; see Methods) showed substantial overlap (61%) between vte2 and ozone-induced genes, but only a small number (7.5%) of vte2-induced genes were in common with MeJA-induced genes (Figure 9 ). It should also be stressed that the MeJA-treated tissue (7-d-old seedlings) were developmentally more similar to 3-d-old vte2 seedlings than were the 2-week-old plants used for ozone treatment. Together, these results suggest that enzymatic oxylipins, including JA and ODPA, play at best a minor role in the vte2 seedling phenotype and that the major responses of vte2 seedlings are mediated by various products of nonenzymatic lipid peroxidation.
Plants and Growth Conditions
vte1-1 and vte2-1 were isolated and described previously (Sattler et al., 2003, 2004). pad3-1 and pad4-1 were obtained from the ABRC. Arabidopsis thaliana plants were grown at 22°C under a 16-h photoperiod (120 μmol photons·m−2·s−1 or μE) in a vermiculite and potting soil mixture and fertilized with 1× Hoagland solution. Alternatively, seedlings were grown on Petri plates containing 0.5× Murashige and Skoog basal medium (Sigma-Aldrich) and 1.0% phytoagar under a 12-h photoperiod (120 μmol photons·m−2·s−1 ) after 5 d of imbibition at 6°C to break dormancy, as described previously (Sattler et al., 2004). For the MeJA treatment, 3-d-old seedlings were sprayed with 10 μM MeJA (Sigma-Aldrich) in 0.05% Tween 20 and incubated for 3 h. For the negative control, 3-d-old seedlings were sprayed with 0.05% Tween 20 and incubated for 3 h.
Oxylipin Profiling
Seedlings were harvested from plates, weighed, and immediately frozen in liquid nitrogen. A total of 500 mg of frozen tissue was ground in a mortar to a fine powder and added to 3.5 mL of ice-cold methanol containing 100 ng of tetrahydro-12-OPDA and 100 ng of dihydrojasmonic acid as internal standards and 12.5 μL of trimethyl phosphite (Fluka). The solution was immediately homogenized with a Polytron (Kinematica) for 1 min on ice. Extraction of fatty acids was continued by rotating the samples in tubes for 2 h at 4°C. Ice-cold water (1.5 mL) was added. After centrifugation, 80 μL of 1 M NH4OH was added, and the supernatant was passed through an 8-mL C18-SPE column (JT Baker; Bakerbond 8ml 500 mg spe Glass Octadecyl), which had been prewashed with methanol followed by methanol:water (70:30, v/v). The column was washed with an additional 7 mL of methanol:water (75:25, v/v), both eluates were combined and acidified with 120 μL of 10% formic acid, and the volume was adjusted to 50 mL using water. The column was reconditioned with 6 mL of methanol containing 40 μL of 10% formic acid, followed by 5 mL of methanol, 6 mL of diethyl ether, 5 mL of methanol, and finally twice with 7 mL of water. Elute was loaded on the column and washed with ethanol:water (15:85, v/v). The oxylipins were eluted with 10 mL of diethyl ether, dried over anhydrous MgSO4, and evaporated under a stream of N2 at 35°C. Oxylipins were resusupended in 60 μL of methanol and methylated with 450 μL of diazomethane in ether, dried under N2, and redissolved in 120 μL of acetonitrile. Samples were derivatized with 60 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (Pierce) for 30 min at 60°C, dried under N2, and redissolved in 500 μL of hexane. The samples were loaded onto an 8-mL Florisil-SPE column (JT Baker; Bakerbond 8ml 500 mg spe Glass Florisil), which had been prewashed first with diethyl ether (3 mL) and then with hexane (10 mL). The loaded column was washed with 4 mL of hexane, and the oxylipins were eluted with 7 mL of ethyl acetate:diethyl ether (1:1, v/v). After evaporation of the solvent under N2, the samples were taken up in 10 μL of hexane and stored at −80°C. Oxylipins were analyzed by GC-MS (Hewlett-Packard 5890 gas chromatograph linked to a Hewlett-Packard 5927 mass spectrometer, electron ionization mode, 70-eV electron potential, 11-p.s.i. column head pressure, HP-5MS column 30 m × 0.25 mm). The temperature gradient was 100°C for 1 min, 100 to 160°C at 20°C/min, 160 to 238°C at 3°C/min, and 238 to 300°C at 30°C/min, as described previously (Weber et al., 1997). Camalexin was identified in oxylipin profiles by its characteristic retention time (23.70 min) and mass spectrum: m/z (%) = 200 (M+, 100), 173 (4), 142 (30), 115 (17), 83 (25), 58 (50). Similarly, for indole 3-carboxylic acid, retention time = 14.41 min, m/z (%) = 175 (M+, 53), 144 (100), 116 (22), 89 (21), 63 (9).
Camalexin Analysis
For quantitative camalexin analysis, seedlings were harvested and immediately frozen in liquid nitrogen. A total of 500 mg of frozen seedlings was ground in a mortar to a fine powder, added to 3.5 mL of 80% methanol, and incubated at 80°C for 20 min. After centrifugation, the supernatant was removed, the volume was reduced to 1 mL by drying with N2 gas, and 0.5 mL of water was added. The solution was extracted twice with 1.5 mL of chloroform, and samples were vacuum-dried and resuspended in 100 μL of hexane:isopropanol (93:7, v/v). HPLC analysis was performed using an Agilent 1100 series HPLC apparatus on a ReliaSil Silica 5-μm, 250 × 4.6-mm normal phase column (Column Engineering) at 30°C with a flow rate of 1 mL/min and hexane:isopropanol (93:7, v/v). Camalexin was detected with an excitation wavelength of 318 nm and an emission wavelength of 385 nm using a fluorescence detector. A camalexin standard (a gift from Ray Hammerschmidt, Michigan State University) was used to identify the compound based on HPLC retention time and to construct standard curves for quantification.
MDA Analysis
Approximately 200 mg of seedlings was harvested and stored in liquid nitrogen until MDA quantification. Total MDA levels were determined by GC-MS according to a protocol modified from Weber et al. (2004). Tissues were ground in liquid nitrogen and resuspended in 0.4 mL of PBS containing 1 mM deferoxamine mesylate (Sigma-Aldrich), 0.08 mM butylated hydroxytoluene (Sigma-Aldrich), and 0.5 nmol of the internal standard [2H2]MDA. After rapid vortexing, 15 μL of 6.6 N sulfuric acid was immediately added and the sample was incubated for 10 min on ice. After hydrolysis of the bound MDA pool, proteins were precipitated with 112 μL of 0.3 M sodium tungstate (Fluka) and the samples were centrifuged. The supernatant was combined with 450 μL of buffer containing 0.58 M sodium phosphate, 0.21 M sodium citrate, and 75 μL of pentafluorophenylhydrazine (5 mg/mL; Fluka) in a new Eppendorf tube and incubated for 30 min at room temperature while shaking. The reaction was stopped by adding 15 μL of 9 N sulfuric acid, and the MDA–pentafluorophenylhydrazine products were extracted with 125 μL of isooctane (Fluka). Preparation of the internal standard [2H2]MDA from [2H2]1,1,3,3-tetraethoxypropane (CDN Isotopes) and GC-MS analyses were performed as described previously (Weber et al., 2004).
PPF1 and Jasmonate Analysis
Total levels of PPF1 (free and esterified) were determined after alkaline hydrolysis of plant extracts containing [18O3]PPF1 as an internal standard. Extraction, saponification, derivatization, and quantitation of PPF1 by GC-MS in negative ion chemical ionization mode were performed as described (Imbusch and Mueller, 2000a, 2000b). Levels of free OPDA and JA were determined as described (Parchmann et al., 1997).
RNA Extraction and Affymetrix GeneChip
Approximately 100 mg of seedlings was frozen in liquid nitrogen, and total RNA was isolated using the RNAqueous RNA extraction kit and the Plant RNA Isolation Aid (Ambion) according to the manufacturer's protocol. Biotinylated target RNA (copy RNA) was prepared from 15 μg of total RNA using the Affymetrix GeneChip One-Cycle Target Labeling kit (Affymetrix) according to the manufacturer's protocols. The three biological replicates for each time point and genotype were hybridized to the Affymetrix Arabidopsis ATH1 GeneChip (Affymetrix) and analyzed as described previously (Fowler and Thomashow, 2002).
Affymetrix GeneChip Data Analysis
The Affymetrix Microarray Suite 5.0 was used to analyze the microarray data. Output from all GeneChip hybridizations was scaled globally such that its average intensity was equal to an arbitrary target intensity of 500, allowing comparisons between GeneChips. Signal (gene expression) values and signal:log ratios were calculated from the GeneChip fluorescence intensity data. The software was also used to determine whether each gene was present, marginal, or absent (Detection) and whether the signal:log ratio represented a genuine change in mRNA accumulation (Change; no change, increased, or decreased). Signal:log ratios and change calls were determined for vte1 and vte2 compared with the corresponding wild-type sample of the same age. Microsoft Access was used as the database management software and to filter and query the data. Probe sets that met the following criteria were selected for further analysis. Those determined to be upregulated in the vte mutants were selected as having, at any time point, an absolute call of present in all three samples, difference calls of increase for all three Change comparisons, and signal:log ratios of at least 1.3 for all three Change comparisons. Those determined to be downregulated in the vte mutants were selected as having, at any time point, an absolute call of present in all three wild-type samples, difference calls of decrease for all three Change comparisons, and signal:log ratios of at least −1.3 for all three Change comparisons. Statistically significant changes in mRNA abundance were determined using the statistical package with GeneSpring 7.2 (Silicon Genetics). The GeneChip data were imported into GeneSpring and normalized to respective control samples as specified by the manufacturer for single-color array data. Statistical significance was determined by analysis of variance using a P value of 0.05 as the cutoff. The Benjamini and Hochberg false-discovery rate was used as the multiple comparison correction for type I family-wise error.
The expression data for the 160 induced genes in vte2 under different stress conditions and treatments (Figure 8) were obtained from the meta-analyzer feature of GENEVESTIGATOR (https://www.genevestigator.ethz.ch), which was imported into GeneSpring 7.2 to perform hierarchical clustering (standard correlation) including vte1 and vte2 3-d data sets. For Figure 9, the Affymetrix ATH1 data for MeJA-treated seedlings (NASCARRAYS-174) and ozone-treated plants (NASCARRAYS-26) were obtained from the public database (NASCArray Affywatch; http://affymetrix.Arabidopsis.info/AffyWatch.html). The GeneChips were analyzed as described above. The lists of induced genes for MeJA and ozone treatments were determined by the following criteria: the probe set were called present in all replicates of the treatment (ozone or MeJA), called increased for all replicates relative the untreated controls, and the signal-log ratio was ≥1.3 (2.5-fold) for all replicates relative to untreated controls. The statistical analysis was performed as described above.
RNA Gel Blot Analysis
RNA gel blots with 10 μg of total RNA per lane were prepared, transferred, and hybridized as described previously (Eccles et al., 1992). Probes were prepared from PCR products: for PAD3, forward (5′-CCGGTGAATCTTGAGAGAGCC-3′) and reverse (5′-GATCAGCTCGGTCATTCCCC-3′) (Zhou et al., 1999); for OPR1, forward (5′-TACAGCTCAAGGATATCAAGA-3′) and reverse (5′-GAAACTTATTACATCTTATATAA-3′) (He and Gan, 2001); for AOS, forward (5′-GGGAGCGATTGAGAAAATGG-3′) and reverse (5′-CGACGAGAAATTAACGGAGC-3′); for CORI1, forward (5′-GCCGCCGGGAGGGCAAGTGG-3′) and reverse (5′-CACATGCAACTTAGGACATG-3′); for OPR3, forward (5′-GGGCTCGAGATGACGGCGGCACAAGGGAA-3′) and reverse (5′-CCCACTAGTTCAGAGGCGGGAAAAAGGAG-3′); and for At VSP1, forward (5′-GTTGGTGTGACAAAATGG-3′) and reverse (5′-GCGGTGAAGATATATGC-3′) (Berger et al., 1995). Probes were randomly labeled with [α-32P]dCTP according to the manufacturer's protocol (Invitrogen), and detection of radioisotope-labeled probes was as described previously (Vogel et al., 2005). Blots were stripped and probed with eIF4A to assess equal loading of RNA samples (Taylor et al., 1993).
Accession Numbers
The Affymetrix GeneChip data were deposited at Gene Expression Omnibus (accession number GSE4847; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE4847) in compliance with MIAME standards.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Table 1. Genes Increased in Expression in the vte Mutants Relative to the Wild Type.
Supplemental Table 2. Genes Decreased in Expression in the vte Mutants Relative to the Wild Type.
We thank Jeff Landgraff, Heather Van Buskirk, and Jonathan Vogel for their advice on transcript profiling and data analysis and Annette Thelen of the Research Support Technology Facility at Michigan State University for performing GeneChip hybridization and scanning. We also thank Ray Hammerschmidt for providing a camalexin standard. E.E.F. was supported by the Swiss National Science Foundation National Center of Competence in Research Plant Survival program and by National Science Foundation Grant 3100A0-101711. M.J.M. was supported by the Deutsch Forschungsgemeinschaft Sonderforschungsbereich 367 (Project A2). This work was supported by National Science Foundation Grant MCB-023529 to D.D.P.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Dean DellaPenna (dellapen{at}msu.edu).
↵1 Current address: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824.
↵[W] Online version contains Web-only data.
- Received May 12, 2006.
- Revised November 20, 2006.
- Accepted November 30, 2006.
- Published December 28, 2006.


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text:
Another French interview in promotion of Miracle Workers: Le Parisien.
Translation by Daniel J Radcliffe Holland.
Did you expect to play with Steve Buscemi in this series?
No ! I loved Simon Rich's book, so I committed myself long before I knew who else would play in it. Today I can not imagine anyone else in this role.
Who is Craig, your character?
It's an angel who works in the prayer department to which he tries to answer. He is terrified of failure so he takes no big risks. Then a new colleague arrives who opens his eyes. He realizes he's not going to change the world if he's happy with that.
You present yourself as an atheist. What do you think of the poor image of God and the angels that is given in the series?
I am agnostic or atheist, I lean in any case on this side. The series corresponds to the world around us. If we showed a heaven that worked perfectly, we would wonder why the world is so chaotic. So we try to show a heaven consistent with the state of the world today.
Do you already think of a season 2 of Miracle Workers?
We do not have an official order yet, but I really hope it will happen. If it takes place, it will not be in heaven but in a different universe, with new characters and a whole new story, while keeping the same actors in different roles. A bit like the American Horror Story series.
You play an angel who can influence events on Earth, isn't it a bit like having magical powers?
I did not see it like that, but now that you say it, maybe it looks like it. But fortunately I do not have a magic wand in Miracle Workers, it would have been too weird.
Could you participate in a new project related to Harry Potter?
I can not say never, but it's not something I think about and by the way I do not think anyone thinks about it today. At this point in my career, no, I do not see myself coming back to that universe. Now, in twenty years, I do not know what I will think.
You seem to make very dark films in the cinema (The Woman in Black, Victor Frankenstein), but comedies on television (A Young Doctor's Notebook, Miracle Workers). It's a choice ?
In fact I love both. But the difference between film and series is not deliberate. I grew up watching mostly comedies. On a shoot it is a genre that is very pleasant. When you go to work, you know that you are going to have fun with your friends. It's quite relaxing not to be covered in blood at the end of the day.
What is your view of your career so far?
I'm pretty happy. I just finished a play in New York, I hope to go back on the stage again in the next years. I've been involved in some odd projects lately like a film shot in New Zealand and Germany titled Guns Akimbo that I wanted to do for a long time. It's an independent film, which is always difficult to achieve, so when it's done it's a small triumph. If my career continues like this, I will be satisfied.
Where do you live today?
I share myself between New York and London. I'm based in London but I try to spend as much time as possible in New York, because that's where my girlfriend lives and it's a city I love.
source: leparisien.fr
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Le Parisien interview
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Làbora, more labour insertion to combat social exclusionMore work contracts signed, more people helped and more job guidance given. The positive balance of the Làbora programme in its third year since being set up shows the solidity of this labour insertion project for people in vulnerable situations, more often than not women.
The report for 2017 shows a rise in all indicators for this cooperative initiative. The number of requests dealt with grew 37.6% compared to the first year and totalled 9,300 people. Out of these, 6,505 took part in the insertion programme offering personalised support and 58% were women, a majority repeated in all stages of the programme. In addition, 80% of candidates were over 35.
The labour insertion figures show that 1,893 people found work thanks to the Làbora programme, representing 17.36% more than in 2016. In all, 2,362 labour contracts were signed, a number exceeding the number of people as the same candidate can sign more than one contract. This is largely due to the precarious and temporary nature of the labour market at present.
In terms of activity, company services accounted for the most labour insertions, with 43.3% of contracts signed, followed by tourism and hotels and catering (13.8%) and social services (10%).
What is Làbora?
The Làbora programme advises, trains and supports people in vulnerable situations who have difficulty accessing the labour market. People on the programme get personalised advice enabling them to improve their skills and access recruitment processes which match their profiles. Companies and entities offering positions via the job pool also get guidance on selecting candidates who suit their needs best.
The initiative is part of the Barcelona Employment Strategy 2016-2020 and is promoted by the Municipal Social Services Institute (IMSS), the Catalan Social Action Entities (ECAS), the Catalan Federation of Insertion Companies (FEICAT) and the Catalan Red Cross. The programme has annual funding of 3.2 million euros, 90% of which comes from the Area of Social Rights.
Tags associated with the news item
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© 2006-2020 Radicore Software Ltd
Latest news
RADICORE v2.16.0 released03 June 2020
RADICORE v2.15.0 released02 April 2020
RADICORE v2.14.0 released08 January 2020
Knowledge Base
How Radicore deals with CSRF attacks08 October 2017
How Radicore's Hook System Works16 September 2017
The use of Cascading Style Sheets within Radicore22 April 2008
Support for PHP4 dropped, support for PHP7 started01 October 2016
Why you should build your web application back-to-front06 January 2013
What is the 3-Tier Architecture?14 October 2012
Other Stuff
Pop Quiz on OOP09 May 2020
RE: Improving PHP's Object Ergonomics02 April 2020
Changing Fundamental Language Behaviors16 August 2019
Having installed the toolkit you will want to know how to create an application with it. This tutorial will take you through all the procedures step by step, and show you how all the basic functions can be created without having to write a single line of code. These steps include:
- Creating a directory to contain all the files for each application.
- Importing a database structure into the Data Dictionary.
- Using the Data Dictionary to define relationships for the benefit of the application.
- Exporting that structure to the application by creating a class file for each table.
- Creating transactions by choosing which transaction pattern to use against which table.
Having used the Radicore toolkit to create transactions, you can then immediately run them to see the results. This detailed tutorial will provide working examples by building actual transactions for a test database. These transactions deal with single database tables, one-to-many relationships, and even many-to-many relationships.
It also contains examples of customising screen layouts, and of customising the table classes to implement business rules or task specific behaviour.


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Properties for sale in surrounding suburbs
CommercialVIEW takes the work out of finding the right warehouse or factory for your business. Whether you're looking to buy, sell or lease, we provide the most relevant, current and comprehensive inventory of industrial properties available in the Burleigh Heads area and surrounds. With our extensive range of listings, you're sure to find a property which caters to your unique specifications.
We know that finding the right warehousing premises is crucial to the success of your business, so give yourself the best chance of finding that perfect place by using CommercialVIEW's comprehensive database of commercial property listings. Our easy to use property portal provides access to the most relevant, up-to-date retail listings in Burleigh Heads to help you acquire the best premises for your business at the right price.
warehouses, factory spaces, other industrial properties and much more! We have the industrial and commercial property listings that will best fit your needs, so start browsing now! And what's more, it's completely free to browse!


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CRAWFORD COUNTY, Kan. — Authorities have launched an investigation following the discovery of decomposed human remains in rural Crawford County.
Crawford County Sheriff's Office deputies and detectives located the remains in the area north of K-126 on 80th Street, which is in the western part of the county between McCune and Greenbush, after receiving a call from someone who had discovered them Monday while out for a walk.
The remains found were in the late stages of decomposition. After confirming they were in fact human skeletal remains, the sheriff's office notified the Kansas Bureau of Investigation and requested its assistance with the case.
KBI agents and members of the bureau's Crime Scene Response Team responded to the scene and the remains were collected and sent to Frontier Forensics in Kansas City for examination.
Forensic examiners will attempt to identify the remains, likely through DNA or dental records and at least initially through comparison with any known missing persons reported in the area, Sheriff Danny Smith said.
"Hopefully we can go from there and see what next steps need to be taken," he said.
The cause of death and identity of the deceased person were still unknown as of Wednesday morning, and authorities had no timeline of how soon the forensic examination would be completed, Smith said.


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World Energy: From the «Shale Revolution» to Healthy Pragmatism? (I)Por Camilo Arcaya • 12 jun, 2013 • Category: Economía
The leading European companies approach the problem of extracting shale gas critically, seeing in it an indirect ecological threat for the entire continent. The development of shale gas fields in North America led to a drastic breakdown of the energy system in Europe: the U.S. placed its bets on shale gas, which caused prices in the coal industry to drop; Europeans rushed to buy up the cheap coal, and Europe literally started «smoking». And the Americans themselves, after trying to become pioneers in shale gas extraction, are still cautious in evaluating prospects. Test drilling caused massive protests in the U.S., as the method of hydraulically fragmenting the shale (fracking) used in such cases is hazardous to groundwater and the atmosphere. In addition, leading companies and analytical institutes vary widely in their evaluations of the prospects of shale gas extraction in the U.S., which does not justify affirmations of a «shale revolution» either.


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Hitachi ECLOS CT Scanner
June 12th 2019
Hitachi's ECLOS CT Scanner is designed with the concept of a well-balanced CT which serves the needs of every user. The scanner provides excellent image quality in both high pitching scanning and sub-millimeter scanning. This cross-sectional imaging device offers great features like better diagnosability, high throughput, and easy operability. It aims to meet all needs … Continue reading Hitachi ECLOS CT ScannerRead More


text: JP1 Remotes :: View topic - Atlas 1056 shows up as urc-2035 JP1 Remotes FAQ Search 7 Days Memberlist Usergroups Register Profile Log in to check your private messages Log in File Section File Search Lookup Tool Wiki Atlas 1056 shows up as urc-2035 JP1 Remotes Forum Index -> JP1 - Beginners View previous topic :: View next topic Author Message bt101Joined: 25 Feb 2008Posts: 13 Posted: Fri Feb 15, 2019 11:19 pm Post subject: Atlas 1056 shows up as urc-2035 Hi - I bought an Atlas 1056 from Ebay (says 1056B04) on the back. If I go into remotemaster, I can find this remote as "Atlas OCAP URC-1056 JP1.3 (Black)". I think they're pretty common (at least I see them everywhere). However when I go into rmir and download from device, it shows up as signature 356801 "URC-2025B2". The picture of the device looks a bit like the Atlas 1056 but it is quite different and is missing a lot of buttons. Is there a way I can get rmir to recognise it as the an Atlas 1056 (or perhaps I'm doing something wrong and I can force it or something). Thanks Back to top bt101Joined: 25 Feb 2008Posts: 13 Posted: Sat Feb 16, 2019 10:14 pm Post subject: Never mind, I think I've worked around the problem. I'm just continuing to use the urc-2025 rdf. It appears that the extra keys on this remote show up as phantomX keys. I can't assign keys using the graphical picture, but luckily there is another tab in the upgrade editor where the keys can be assigned in a table. As some posts suggested, you can figure out what the phantom keys are by jamming them into a macro and viewing the results. I notice this remote does not have key moves or efcs, so I'm doing as other posts have suggested and just jamming everything I need into the upgrades. Kudos to those who have developed all of the jp1 software. It was impressive enough years ago, but I was over the moon when I took a second look after several years to see it runs on linux. I do have a suggestion... To solve the problem, I did a lot of searching on the topic of creating your own rdf for a remote. From what I can see, it is recommended to upload the data and have experts look at it. eg: http://www.hifi-remote.com/forums/viewtopic.php?p=59583&sid=119c317df4a7e32e8e3b0376874e0ca2 That's very gracious for someone to do that. However it may be beneficial to have a tutorial/instructions for creating rdfs. A lot of people like myself would like to do it, and it may result in a community effort such as the device upgrades. Back to top Display posts from previous: All Posts1 Day7 Days2 Weeks1 Month3 Months6 Months1 Year Oldest FirstNewest First JP1 Remotes Forum Index -> JP1 - Beginners All times are GMT - 5 Hours Page 1 of 1 Jump to: Select a forum General----------------Non-JP1Code SearchCode Search (Closed)Discrete CodesSlingbox JP1 Forums----------------Nevo and Xsight RemotesJP1 - New Remotes & RDFsJP1 - BeginnersJP1 - General ForumJP1 - Protocol DecodesJP1 - ExtendersJP1 - SoftwareJP1 - KeyboardsJP1 - Hardware Miscellaneous----------------X10 and Home AutomationElectronics RepairMarket PlaceMarket Place ArchiveWeb Site IssuesReplayTVHall of Shame (and other funny stuff) You cannot post new topics in this forumYou cannot reply to topics in this forumYou cannot edit your posts in this forumYou cannot delete your posts in this forumYou cannot vote in polls in this forum Powered by phpBB © 2001, 2005 phpBB Group


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Located in Wuhan Biolake with 30 mu planning land, will be built to "an institution, a center and a base", namely "non-human primate institute, medical translational research center and drug products pilot base", mainly in the research of drug screening and evaluation characterized by humanized animal (disease) models, and is committed to completing standard formulation of Chinese laboratory monkey physiological indicators (initiative), as well as developing the business of gene editing and tumor and disease animal model preparation.
Founded in 2018, Topgene-the United States biotechnology research center is located in Houston, Texas, USA. Taking the rich medical and related technology research and development at the university of Texas medical center as the background, the research center assembles the high-end talents of American medicine, pharmacology and biotechnology, combining with resources superiority and advanced experimental technology characterized by non-human primate laboratory animals in China's parent company and is committed to building CRO enterprise with international advanced level for the biomedical technology innovation in China and the United States.
——Brief introduction to microbiologist, academician Deng Zixin
Deng Zixin, was born in 1957, member of Chinese Academy of Sciences (Member, CAS), fellow of the third world academy of sciences (TWAS), fellow of the American academy of microbiology (AAM). Currently, he is the dean of Wuhan University Pharmacy School, the director of Wuhan Institute of Biotechnology and the chief of State Key Laboratory of Microbial Metabolism.
He is the present of the Chinese Society for Microbiology, the vice present of Chinese Agricultural Biotechnology Society, the chairman of International Committee for Genetics of Industrial Microorganisms (GIM-IC), the member of Intemational Symposium on Biolody of Actinomycetes (ISBA), the chief scientist of National 973 Project, the expert group member of National 863 Plan, the associate editor of the American Chemical society journal ACS Chemical Biology, and the journal editors of Cell series Cell Chemical Biology, American Society for Microbiology Applied and Environmental Microbiology and other domestic and foreign journals.
He is mainly engaged in the molecular biology study of microbial metabolism, especially in chemical biology of actinomycetes genetics and antibiotic biosynthesis, and uncovers the new areas of DNA sulfur modification, publishing more than 400 papers in domestic and international academic journals.
Workstation focuses on malignant tumors, cardiovascular and cerebrovascular diseases, neurodegenerative diseases, diabetes and other diseases to develop the mechanism research in new targets and new function and establish systematic drug screening models, providing technical services of drug screening for new drug research and development departments and enterprises in China. Close cooperation with other domestic screening platforms is undertaken to enhance communications in new technologies and new methods, continually improving the integrity and scientificity of early evaluation system of drug properties and gradually establishing drug efficacy evaluation system and platform.


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ACCIÓ (the Catalan Agency for Business Competitiveness) has opened the second call of the TECNIOspring PLUS programme, which will be closed on 14th September 2017.
The programme provides financial support to individual mobility proposals presented by experienced researchers in liaison with any R&D entity around the world. Fellows are offered two-year employment contracts to develop an applied research project with a focus on technology transfer.
More information on the programme and details about the open call can be found at www.tecniospring.eu.
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Players from different countries enjoying Sánchez-Casal training System during this week.
As everyday you see on court Sergio Casal, Stefan Ortega, Angel Gimenez, Pep Plasín, Lorenzo Fargas among all the staff who always is great creating positive energy. We are really prouf of our Staff!!
The Weekly Tennis Program combines High Performance Tennis Training, which includes technical exercises and tactical strategy and competition, with specific physical training.
Players works on the technical and tactical aspects of the game as well as footwork, mobility and positioning, based on the Sánchez-Casal system in order to enhance their tennis and develop a better game from all areas of the court.
Each player is assigned to a coach, who is responsible for monitoring the development of the player. Players are training in all surfaces (Clay, Hard, Grass) and whatever is your pattern, your level, Sánchez-Casal System works!!
Leading by Daniel Romero and Eduardo Garcia, the Science Sports Area has a clear objective for this week. Improving in this short period of time… how? the student-athlete trains all the physical abilities such as resistance, strength, speed, agility, coordination, and flexibility. Special emphasis in injury prevention.
The Academy has on premises a physiotherapy area, with experienced staff (Nacho Quixano, Aleix Borras) in treating sports injuries, and works with the latest technology available for treatment.
Mental Training is another important part of training, thanks to Andrea Crosas, who spoke on tuesday about routines (concentration, motivation…).
Of course, Nutrition is a key part as well, thanks to Sergi Bonillo, so a healthy menu is ready in the restaurant and sports café.
+34 93 479 16 16


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Crested Butte Mountain Resort a place known for hosting the US Extreme Ski Championships each year is one again taking big mountain progression and park riding to the next level by introducing a BagJump for season pass holder and anyone with an adventure park ticket.
With a strong direction to create a more innovative and unique experience for guests, Crested Butte Mountain Resort (CBMR) has just opened the Coke Zero® Gravity BagJump as its latest amenity in the Adventure Park. The 50 feet by 50 feet by 11 feet deep bag is filled with air and acts as a safe and cushioned landing pad for jumpers, tubers, skiers and snowboarders.
BagJump™ is a versatile product that can be used all year round, from beginners to professionals, for Olympic level training or just for recreation; giving the feeling of "landing on a cloud" when jumping into it. In Crested Butte, the BagJump will act as a place for kids and adults to sled into on a tube and for skiers and snowboarders to test their freestyle skills. In the summer months, adrenaline seekers can free fall into the bag off an elevated platform.
"Adding the Coke Zero Gravity BagJump continues to fulfill our goal of adding new attractions to the resort for summer and winter months," says Nick Herrin, the director of resort services for CBMR. "It is a safe, yet exciting experience for any age."
The BagJump will be a part of the Adventure Park ticket for unlimited use and available for Platinum Season Passholders. The BagJump is now open daily from 1 – 5 p.m. Skiers and riders will be required to go through a safety course to receive a stamp of approval in order to utilize the BagJump on their skis and boards.
"The Crested Butte facility is one of the best I've ever worked with," adds Elliott Levitt, US Representative for BagJump. "I look forward to BagJump being a part of the operations in Crested Butte for many years to come."
Heading to Crested Butte this winter? Save on Crested Butte Mountain Resort 3 of 4 day tickets, currently on sale - 35% OFF


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Bäckstedt not happy with Roubaix route changes
Defending Paris-Roubaix champion Magnus Bäckstedt (Liquigas-Bianchi) has reacted to the route...
Defending Paris-Roubaix champion Magnus Bäckstedt (Liquigas-Bianchi) has reacted to the route changes made by race organiser ASO, saying "Arenberg is what Paris-Roubaix is all about! It is the location of the first major selection in the race. If it is being removed so that it can be restored then I can accept that, but if it's being removed because organisers don't think its safe for riders and they might fall, then that is unacceptable."
The Wallers/Arenberg cobblestone section will be removed from the course in 2005 because its first 200 metres have deteriorated and made the "entrance" onto the 2400 km-long pavés too dangerous for the riders. In its press release, race organisers do consider restoring the section to possibly include it as of 2006. Meanwhile, the exclusion of the Arenberg cobbles does not make any sense to Bäckstedt.
"You don't go into Paris Roubaix not thinking about the dangers", he said. "It is a dangerous race and that is what makes it the monumental classic which it is. You come out of the Arenberg section with the contenders generally in the group, you then lose two or three guys a section until you get down to the real potential winners.
"I would say that taking out the tough section of the race is like putting a gear restriction on bunch sprints to limit the speed we sprint at. There are many races including unnecessary dangers, like having the finish line 200 m after a corner that has just had a 2 km straight! As professional riders, we are all concerned about safety, but in the right place and context."
Backstedt now heads to Italy for the presentation of his 2005 Liquigas-Bianchi team, before taking a flight to Doha, Qatar at the end on the month, where he will participate in the Tour of Qatar alongside his teammates Mario Cipollini, Dario Andriotto, Nicola Loda, Marco Milesi, Marcus Ljungqvist, Gianluca Sironi and Marco Zanotti. Liquigas riders Dario Cioni, Danilo Di Luca, Devis Miorin and Charles Wegelius will fly to Toluca, Mexico, to train at altitude until the end of February.
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|"Holy cow!"
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Graceful Calla Lily
A sweet by yet sophisticated way to show your deepest wishes, the Graceful Calla Lily arrangement blooms with gorgeous beauty and elegance. Delightful white open cut calla lilies create an impressive display of tranquility and calmness. Arranged in a clear glass vase, this flower bouquet is the best way to captivate your recipient for an anniversary, to express your heartfelt gratitude, or to offer comfort and sympathy.
|12 calla lilies in vase


text: Mrs. RMN planned a real nice trip to Busan for us. We loaded extra cans of gas in the family van (who wants to pay nearly twice as much as normal?) and set off. First two nights were in Busan on Haeundae Beach. We got a "condotel" which is basically a three-room hotel room (2x bedrooms and a family room/kitchenette). Mrs. RMN had packed some marianated meats so that plus rice made for an inexpensive dinner. Breakfast was cereal and bread with milk and juice. The condotel was at the south end of the beach meaning everything was close (i.e. no driving). The kids loved the beach. Be sure to keep an eye out on my flickr feed to see some of the fun!


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Make It Real, actor Brian Gaskill's debut spoken word album, brings us into a world of wolves, artists, dancers, femme fatales. We have Hollywood, we have bars, we have houses full of ghosts, maps on the kitchen floor leading to heaven. We have Janis Joplin and Houdini and Van Gogh. We have a punk-rock mermaid who is a healer, a woman with a body made to hold all that's soft, private.
With the spoken word, Brian explores the importance of moment, decision, choice, wish. Of running, falling. Of rhythm and music. His voice is strong, determined, vulnerable.
Brian's album is not just honest, but personal. These recordings bring listeners in to a certain element of human that is intrinsically private, an interior complexity we feel that we shouldn't be allowed to navigate because it doesn't belong to us. We are brought into birth here, ocean. Abandonment. Creation.
But yet the truth is, she didn't come back, Brian reads in the opening poem, "M Street." But this is my pencil and my paper and my art—and yes even my rules—and whether it's life or art I don't care, it's still alive and the only me I know today so that will have to do for now. And that's the way our story begins.
Ashley Inguanta: Tell me about the beginnings of this album. When did you start writing, recording?
Brian Gaskill: The poetry is a mix of stuff from mostly the last 8 years, though much of it I wrote in December or last year, as I forced myself to write every day because I felt very raw, and I wanted to get that out and see where it took me. There is much that is not on this album; it will be on another album, or maybe a book eventually, once I figure out the whole publishing thing. I recorded the whole album in about 5 hours on a Saturday afternoon in February. I wanted to "make it real," make it happen, and let it be as raw as the writing felt.
AI: What is the biggest difference between writing your poems and recording them? Describe the connection you receive with words as you write them and as you speak them.
BG: For me there is little difference. When I am writing I hear it. I say it out loud. I want to like the way it sounds. It's inner monologue, maybe in a way. I am a slow reader for the same reason. I read with my mouth moving and say every word. I love first-person narrative for that reason. I escape; it becomes my story. Of course this album is my story, so it's not so much an escape as a finally facing myself and not censoring myself.
AI: In terms of your journey recording this album, what was the most freeing poem to write? Record? The scariest?
BG: All of the newer ones were all of these things, but the title track, "Make it Real," it's brutally honest in terms of looking in the mirror and showing that reflection to others, and because of the details it surprised me; when I read it back it chokes me up. "Fall into Heaven" was the most freeing to record, in terms of sort of living out rapper fantasies (haha), but frankly releasing this at all was all pretty scary. I wanted to finally admit in public that I am a writer, and a creative soul, and I really had nothing left to lose. The only thing I haven't tried is just being myself in my creations, so I figured it was time to start
AI: What do you hope your writing will do for the world? Where do you plan on going next with your poetry?
BG: For the world? I cant possibly pretend it will reach the world, but whoever it does reach I hope it inspires them to create, to open up, to be themselves, to face their faults and face what is great about them, and inspire them to be fully human for better or worse, and accept themselves and others. I guess that would be my goal in whatever art I pursue. I hope I can taste some of that.
AI: If you could collaborate with any artist (actor, musician, poet) who would it be? What would you create together?
BG: Tough one. If I could travel back to the early 60's I might collaborate with Jean Luc Goddard on a film that would be the story of an American poet in Paris struggling to be understood, and who finds himself while getting wrapped up in a crime syndicate. Soundtrack by Miles Davis. And also starring both Anna Karina and Brigitte Bardot.
AI: Is there anything else you would like to say?
BG: I said what I needed to say for now on the album. I'm still just out here trying to "make it real." I hope people will listen. I hope some will be inspired and make some dream real in their own life. As for me, I've barely begun.


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GenChem ABOR Learner-Centered Education 2009 General Chemistry Redesign Department of Chemistry University of Arizona
GenChem Developers Chemical Education Committee Steve Brown Andy Grall Anne Padias John Pollard Vicente Talanquer Wayne Wesolowski Support Graduate Teaching Assistants Teaching Service Office Prep Room
GenChem In the summer of 2007, our team started working in the redesign of the two-semester General Chemistry sequence for science and engineering majors to address the following problems: The Problems Weak correlation between the lecture and the laboratory courses. Lack of consistency among the different instructors. Limited contact between TAs and lecture instructors. Use of traditional lecture format to teach the courses. Very large discussion sections (~150 students). Lack of systematic, valid, and reliable assessment tools.
GenChem 1. Combine the lecture-lab courses into one. Course (4 credits) ~288 Lab/ Discussion Instructor TA Better correlation (same students in lecture and labs; clear association of TAs and instructors); More effective discussions; More efficient use of instructors and TAs time; The Proposal
GenChem 2.Homogenize the curriculum and the assessment practices among all course sections. Unit 1 Unit 2 Unit 3 Unit 4 Common Diagnostic Exam Common Final Exam Common Partial Exams Lecture Lab Common report structure, practicals, and rubrics. The Proposal Common on- line homework system Common Guiding Notes
GenChem 3.As mandated by the ABOR-LCE initiative, create a more leaner-centered environment in which students take more ownership in their own learning (active participation). The Proposal Collaborative small group work; Guided inquiry activities and experiments; Whole class interactions.
GenChem Timeline Pilot Test Spring 2008 Off-sequence First Semester CHEM 151 (3 lecture sections). Full Phase I Fall 2008 On-sequence First Semester CHEM 151 (7 lecture sections). Off-sequence Second Semester CHEM 152 (2 lecture sections). Full Phase II Spring 2009 CHEM 151/CHEM 152
GenChem Products Redesigned sequence; trimmed content; Common power point guides for first semester; On-line homework problem sets; Common diagnostic and four midterm exams; Lecture Revised/Adapted experiments; Common power point lab/discussion guides; Additional experiment notes; Revised lab manual; Common midterm and final practical exams; Laboratory
GenChem Impact on Learning CHEM 151 Total # of Students DiagnosticFinal Exam ACS Traditional 2006-2007 370346.7% (SD = 17.7) 54.0% (SD = 16.3) Reformed Spring-Fall 08 212843.8% (SD = 14.8) 59.3% (SD = 15.1) CHEM 152 Total # of Students Final Exam ACS Traditional 2006-2007 (off-sequence) 54045.2% (SD = 7.69) Reformed-Pilot Fall 2008 (off-sequence) 34649.9% (SD = 11.6)
GenChem Impact on Performance Average Grade: 67.1% to 70.7%
GenChem Impact on Performance Average Grade: 64.4% to 68.1%
GenChem Improved Retention From 11.4% to 6.5% in CHEM 151. From 14.4% to 4.8% in CHEM 152. Failing Rates Withdrawal Rates From 6.49% to 4.96% in CHEM 151. From 8.86% to 6.31% in CHEM 152. We have not been able to assess the impact of the transformation on drop rates in a reliable way given the types of enrollment data collected by the UA.
GenChem Impact on Cost Savings Major cost savings were associated with the reduction in the number of course planning and student contact hours for faculty and lecturers. Planning and assessment time reduction: Common set of lecture notes, lab presentations, and multiple choice exams. Contact hours reduction: Graduate teaching assistants are now responsible for teaching the discussion sessions for the General Chemistry courses (integrated into the laboratory time).
GenChem Pedagogical Improvements A variety of on-line interactive simulations were made available to instructors and students to facilitate in-class activities and discussions. Laboratory activities were modified to create more opportunities for students to design experiments. In-classroom collaborative group activities were created/adapted and incorporated into the common weekly lecture and laboratory notes. Development and implementation of common midterm and final exams based on a common set of learning objectives for the two courses.
GenChem Team work highlighted the importance of having regular academic meetings to discuss different ideas and points of view about how to best teach the different topics. The development team has faced challenges in how to train and better support the work of new instructors and graduate teaching assistants who were not involved in the development of the project. The team recognized the importance of assigning course leaders who could help support the work of everyone involved. Implementation Issues
GenChem One of the major lessons learned during the implementation of the project is the central role that graduate teaching assistants play in the implementation and success of the new model. Observations of student work in the laboratory suggest that the seamless integration of experimental work and discussion sessions is a challenge for most graduate teaching assistants. The development team is currently discussing and exploring different options to solve this problem. Implementation Issues
GenChem Sustainability The Department of Chemistry at the University of Arizona fully supports the changes that were implemented and is committed to provide the resources needed to sustain the project. At this moment, all of the General Chemistry courses offered by the Department are being taught following the new format.
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ERROR: Build failed with: exit status 1
On several runners i'm getting this kind of problem:
DEBU[0018] 6a22da4b 499 Build log updater finished.
DEBU[0018] 6a22da4b 499 Build log: gitlab-ci-multi-runner 0.5.0 (c38415a)
Using Shell executor...
Running on Emil-MacBook-Pro.local...
Fetching changes...
ERROR: Build failed with: exit status 1
How to debug this kind of problems?
I was following this installation guid: https://gitlab.com/gitlab-org/gitlab-ci-multi-runner/blob/master/docs/install/osx.md