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- © 2010 American Society of Plant Biologists
Systemic acquired resistance (SAR) is a form of defense that provides resistance against a broad spectrum of pathogens in plants. Previous work indicates a role for plastidial glycerolipid biosynthesis in SAR. Specifically, mutations in FATTY ACID DESATURASE7 (FAD7), which lead to reduced trienoic fatty acid levels and compromised plastidial lipid biosynthesis, have been associated with defective SAR. We show that the defective SAR in Arabidopsis (Arabidopsis thaliana) fad7-1 plants is not associated with a mutation in FAD7 but rather with a second-site mutation in GLABRA1 (GL1), a gene well known for its role in trichome formation. The compromised SAR in gl1 plants is associated with impairment in their cuticles. Furthermore, mutations in two other components of trichome development, GL3 and TRANSPARENT TESTA GLABRA1, also impaired cuticle development and SAR. This suggests an overlap in the biochemical pathways leading to cuticle and trichome development. Interestingly, exogenous application of gibberellic acid (GA) not only enhanced SAR in wild-type plants but also restored SAR in gl1 plants. In contrast to GA, the defense phytohoromes salicylic acid and jasmonic acid were unable to restore SAR in gl1 plants. GA application increased levels of cuticular components but not trichome formation on gl1 plants, thus implicating cuticle, but not trichomes, as an important component of SAR. Our findings question the prudence of using mutant backgrounds for genetic screens and underscore a need to reevaluate phenotypes previously studied in the gl1 background.
Plants have evolved a large array of defense mechanisms to resist infection by pathogens. Upon recognition, the host plant initiates one or more signal transduction pathways that activate various plant defenses and thereby prevent pathogen colonization. In many cases, resistance is associated with increased expression of defense genes, including the pathogenesis-related (PR) genes and the accumulation of salicylic acid (SA) in the inoculated leaf. Induction of these responses is accompanied by localized cell death at the site of pathogen entry, which can often restrict the spread of pathogen to cells within and immediately surrounding the lesions. This phenomenon, known as the hypersensitive response, is one of the earliest visible manifestations of induced defense responses and resembles programmed cell death in animals (Dangl et al., 1996; Gray, 2002; Glazebrook, 2005; Kachroo and Kachroo, 2006). Concurrent with hypersensitive response development, defense reactions are triggered in sites both local and distal from the primary infection. This phenomenon, known as systemic acquired resistance (SAR), is accompanied by a local and systemic increase in SA and jasmonic acid (JA) and a concomitant up-regulation of a large set of defense genes (Durrant and Dong, 2004; Truman et al., 2007; Vlot et al., 2009).
SAR involves the generation of a mobile signal in the primary leaves that, upon translocation to the distal tissues, activates defense responses resulting in broad-spectrum resistance. The production of the mobile signal takes places within 3 to 6 h of avirulent pathogen inoculation in the primary leaves (Smith-Becker et al., 1998), and the inoculated leaf must remain attached for at least 4 h after inoculation for immunity to be induced in the systemic tissues (Rasmussen et al., 1991). Mutations compromising SA synthesis or impairing SA, JA, or auxin signaling abolish SAR (Durrant and Dong, 2004; Truman et al., 2007, 2010). SAR is also dependent on the SALICYLIC ACID-BINDING PROTEIN2 (SABP2)-catalyzed conversion of methyl SA to SA in the distal tissues (Kumar and Klessig, 2003). Recent studies have suggested that methyl SA is the mobile signal required to initiate SAR in distal tissues in tobacco (Nicotiana tabacum; Park et al., 2007) and Arabidopsis (Arabidopsis thaliana; Liu et al., 2010), although another group reported a disparity in their findings related to the role of methyl SA in Arabidopsis (Attaran et al., 2009). Notably, the time point of requirement of SABP2 activity (between 48 and 72 h post inoculation; Park et al., 2009) does not coincide with the early generation and/or translocation of the mobile signal into distal tissues (within 6 h post inoculation).
The mutations acyl carrier protein4 (acp4), long-chain acyl-CoA synthetase2 (lacs2), and lacs9, which are impaired in fatty acid (FA)/lipid flux (Schnurr et al., 2004; Xia et al., 2009), also compromise SAR (Xia et al., 2009). Detailed characterization has shown that the SAR defect in acp4, lacs2, and lacs9 mutants correlates with their defective cuticles. Analysis of the SAR response in acp4 plants has shown that these plants can generate the mobile signal required for inducing SAR but are unable to respond to it. It is likely that the defective cuticle in these plants impairs their ability to perceive the SAR signal, because mechanical abrasion of cuticles disrupts SAR in wild-type plants (Xia et al., 2009). This SAR-disruptive effect of cuticle abrasion is highly specific, because it does not alter local defenses and hinders SAR only during the time frame during which the mobile signal is translocated to distal tissues.
SAR is also compromised in plants that contain a mutation in glycerol-3-phosphate dehydrogenase (Nandi et al., 2004). The glycerol-3-phosphate dehydrogenase (GLY1) reduces dihydroxyacetone phosphate to generate glycerol-3-phosphate, an obligatory component and precursor for the biosynthesis of all plant glycerolipids. Consequently, a mutation in GLY1 results in reduced carbon flux through the prokaryotic pathway of lipid biosynthesis, which leads to a reduction in the hexadecatrienoic (16:3) FAs (Miquel et al., 1998; Kachroo et al., 2004). Carbon flux and SAR are also impaired in plants containing mutations in FATTY ACID DESATURASE7 (FAD7; Chaturvedi et al., 2008). The FAD7 enzyme desaturates 16:2 and 18:2 FA species present on plastidial lipids to 16:3 and 18:3, respectively. Consequently, the fad7 mutant plants accumulate significantly reduced levels of trienoic FAs (16:3 and 18:3). Compromised SAR in mutants affected in certain plastidial FA/lipid pathways has prompted the suggestion that plastidial FA/lipids participate in SAR (Chaturvedi et al., 2008). Such a tempting conclusion is also favored by the fact that SAR requires the DIR1-encoded nonspecific lipid transfer protein, which is required for the generation and/or translocation of the mobile signal (Maldonado et al., 2002). In addition, azelaic acid, a dicarboxylic acid, was recently shown to prime SA biosynthesis and thereby SAR (Jung et al., 2009). The fact that azelaic acid is derived from oleic acid, a FA well known for its role in defense (Kachroo et al., 2003, 2004, 2005, 2007, 2008; Chandra-Shekara et al., 2007; Jiang et al., 2009; Venugopal et al., 2009; Xia et al., 2009), further suggests that FA/lipids might participate in SAR.
This study was undertaken to reexamine the role of the FA/lipid pathways in SAR and to determine the nature of the FA/lipid species mediating SAR in fad7-1 plants. Our results show that impaired FA/lipid flux is not associated with compromised SAR in fad7-1 plants but, rather, with an abnormal cuticle, which is the result of a nonallelic mutation in the GLABRA1 (GL1) gene. Besides GL1, other mutations affecting trichome formation also compromised cuticle and thereby SAR. A compensatory effect of exogenous GA on gl1 plants suggests that GA might participate in resistance to bacterial pathogens by restoring cuticle formation.
SAR Is Compromised in the fad7-1 Plants But Not in Other fad Mutants
To determine which, if any, of the desaturated FA species contributed to SAR, we first evaluated SAR in all fad mutants that are defective in the desaturation of various FA species present on plastidial or extraplastidial membrane lipids (for the respective FAD-catalyzed activities, see Supplemental Fig. S1; for review, see Kachroo and Kachroo, 2009). The fad2-1, fad3-2, fad4-1, fad5-1, fad6-1, and fad7-1 mutants were first inoculated with MgCl2 or avirulent bacteria (avrRpt2) followed by a second inoculation with virulent bacteria on distal tissues at 48 h post primary inoculation. The growth of the virulent bacteria was monitored at 0, 3, and 6 d post inoculation (dpi; Fig. 1). As expected, MgCl2-infiltrated leaves of wild-type (ecotype Columbia [Col-0]) plants supported more growth of the secondary virulent pathogen than the plants that were preinfected with the avrRpt2 strain, indicating appropriate induction of SAR (Fig. 1). Similar to wild-type plants, the fad2-1, fad3-2, fad4-1, fad5-1, and fad6-1 mutants also showed proper induction of SAR. In agreement with a previous report (Chaturvedi et al., 2008), the fad7-1 mutant plants showed compromised SAR; plants infiltrated with MgCl2 or avrRpt2 bacteria were equally susceptible to the secondary virulent bacteria in the distal tissues. Together, these data suggested that the FAD7-catalyzed desaturation of 18:2 to 18:3 on plastidial membrane lipids might be important for the proper induction of SAR.
fad7-1 Plants Are Compromised in the Pathogen-Induced Accumulation of SA
Since SA plays a critical role in SAR, we first tested if the fad7-1 mutant plants were competent in pathogen-responsive accumulation of SA. SA levels in wild-type and fad7-1 plants were determined before and after inoculation of Pseudomonas syringae containing avrRpt2. As expected, wild-type plants inoculated with avirulent pathogen showed a significant increase in both free SA and salicylic acid glucoside (SAG) in their primary as well as systemic tissues. Although the fad7-1 plants also showed an increase in SA and SAG levels in the primary tissues, levels of SA/SAG were significantly lower in their distal leaves in comparison with wild-type plants (Fig. 2A). Thus, impaired SAR in fad7-1 plants correlated with their inability to accumulate SA in the distal tissues. To confirm this, we tested the effect of exogenously supplied SA on SAR in fad7-1 plants. The wild-type and fad7-1 leaves were infiltrated with MgCl2 (mock) or avrRpt2 bacteria. Vascular exudates collected from these infiltrated leaves were mixed with water or the SA analog benzo(1,2,3)thiadiazole-7-carbothioic acid (BTH) and injected into the leaves of a fresh set of wild-type and fad7-1 plants. The distal leaves of this second set of plants were then inoculated with virulent bacteria, and proliferation of the virulent bacteria was monitored at 0 and 3 dpi (Fig. 2B). Notably, BTH-containing exudate from MgCl2-infiltrated Col-0 plants conferred SAR on Col-0 plants but not on fad7-1 plants. In comparison, the BTH-containing exudate from MgCl2-infiltrated fad7-1 was unable to confer SAR on either Col-0 or fad7-1 plants. Interestingly, the exudate from avrRpt2-infiltrated fad7-1 plants conferred SAR on Col-0 plants but not on fad7-1 plants. The BTH-containing exudate from avrRpt2-infiltrated Col-0 or fad7-1 plants produced better SAR on Col-0 but not on fad7-1, suggesting that exogenous BTH had an additive effect on SAR in Col-0. Together, these results suggested that exogenously supplied BTH was unable to restore the defective SAR in fad7-1 plants.
To determine if pretreatment of the whole plant with BTH restored SAR in fad7-1 plants, we treated Col-0 and fad7-1 plants with BTH for two consecutive days followed by inoculation of primary leaves with MgCl2 (mock) or avrRpt2 bacteria. The virulent bacteria were then infiltrated into the distal leaves 48 h after primary infiltration (MgCl2/avrRpt2), and growth of virulent bacteria was monitored at 3 and 6 dpi (Fig. 2C). Whole plant application of BTH conferred enhanced resistance to virulent pathogen in both wild-type and fad7-1 plants. However, the BTH-treated fad7-1 plants continued to support higher growth of virulent bacteria compared with BTH-treated Col-0, suggesting that BTH application on fad7-1 enhanced resistance but not to wild-type-like levels. To determine if this was due to a partial insensitivity to SA, we compared the levels of the SA-inducible marker, PR-1, in fad7-1 plants. Similar levels of PR-1 transcript were induced in response to BTH in both Col-0 and fad7 plants (Fig. 2D), suggesting that fad7-1 plants are able to induce SA-derived marker gene expression.
fad7-1 Plants Are Compromised in Pathogen-Induced Accumulation of JA
The FAD7 enzyme catalyzes the desaturation of 18:2 to 18:3 FA on membranous lipids, and a mutation in FAD7 reduces trienoic FA levels in the plant (Fig. 3A). Notably, the fad7-1 plants accumulated reduced levels of basal and pathogen-induced JA (Fig. 3B), which correlated well with reduced levels of the JA precursor 16:3 and 18:3 FAs. To determine if reduced accumulation of JA was responsible for compromised SAR, we tested if exogenously supplied JA restored SAR in fad7 plants. The wild-type and fad7-1 leaves were infiltrated with MgCl2 (mock) or avrRpt2 bacteria. Vascular exudates collected from the MgCl2- or avrRpt2-infiltrated leaves were mixed with water or JA and injected into the leaves of a fresh set of wild-type and fad7-1 plants. The distal leaves of this second set of plants were then inoculated with virulent bacteria, and their proliferation was monitored at 0 and 3 dpi (Fig. 3C). Unlike BTH, addition of JA to exudates from MgCl2-infiltrated Col-0 or fad7-1 plants did not reduce the growth of virulent bacteria. Furthermore, addition of JA to exudate from avrRpt2-infiltrated Col-0 plants also did not increase the potency of SAR in Col-0. Application of exudate on the fad7-1 plants did not induce SAR, regardless of the source of the exudate (Col-0 or fad7-1 plants). Together, these data suggested that the inability to accumulate JA likely did not contribute to the compromised SAR in fad7-1 plants. This was further confirmed by whole plant application of JA (Fig. 3D). The Col-0 and fad7-1 plants were treated with JA for 2 d followed by inoculation of primary leaves with MgCl2 (mock) or avrRpt2 bacteria. The virulent bacteria were inoculated on distal leaves 48 h after primary infiltrations, and their growth was monitored at 3 and 6 dpi. JA-treated fad7-1 plants showed a marginal reduction in the growth of the virulent pathogen but they did not show SAR. Additionally, similar to results obtained in the exudate-related experiment (Fig. 3C), whole plant pretreatment with JA did not improve SAR in either mock- or avrRpt2-inoculated wild-type plants. Together, these results argue against reduced JA as the cause of defective SAR in fad7-1 plants.
A gl1 Mutation in the fad7-1 Plants Is Responsible for Their Defective SAR
In addition to FAD7, the Arabidopsis FAD8 enzyme also catalyzes the desaturation of 18:2 to 18:3 on plastidial lipids. This is evident in the fad7-2 fad8-1 double mutant plants, which are more severely reduced in their trienoic FA levels. To test if trienoic FAs contribute to SAR, we assayed SAR in the fad7-2 fad8-1 double mutant plants. As expected, the fad7-2 fad8-1 plants showed negligible levels of 16:3 and a significant reduction in 18:3 levels (Fig. 4A). Surprisingly, the fad7-2 fad8-1 plants showed normal SAR (Fig. 4B). One possibility was that the fad8 mutation restored SAR in the fad7-2 fad8 double mutant. However, this was rather unlikely, given the fact that both FAD7 and FAD8 catalyze the same desaturation event (18:2–18:3). It was also not easily testable, since the fad8 single mutation has no detectable effect on overall FA composition (McConn et al., 1994). Therefore, we retested the requirement of FAD7 in SAR induction by evaluating SAR in the fad7-1 allelic mutant, fad7-2. Both fad7-1 and fad7-2 showed similar profile of FAs and resulted in a similar decrease in the levels of trienoic FAs (Fig. 4A). However, unlike fad7-1, the fad7-2 plants showed normal SAR (Fig. 4B). Together, these data suggested that compromised SAR in fad7-1 plants was not associated with their reduced trienoic FA levels or their altered FA/lipid profile.
A comparison of the genetic backgrounds used for isolating fad7-1 and fad7-2 mutants revealed that fad7-1 was isolated in the gl1 background whereas fad7-2 was isolated in the wild-type background. The GL1 gene encodes the R2R2-MYB transcription factor, which is required for trichome differentiation in Arabidopsis (Oppenheimer et al., 1991). A mutation in the GL1 gene results in a glabrous phenotype due to a lack of trichomes on leaves. Indeed, similar to leaves from gl1 plants, fad7-1 gl1 leaves were devoid of any trichomes (Fig. 4C). On the other hand, wild-type, fad7-2, and fad7-2 fad8-1 leaves showed normal trichomes (Fig. 4C). Quantitative FA profiling showed a wild-type-like profile of FAs in gl1 plants (Fig. 4A), suggesting that, unlike fad7-1, the gl1 mutation was not associated with an altered FA profile. However, similar to fad7-1, the gl1 plants were compromised in SAR (Fig. 4B). Together, these data suggested that the gl1 mutation was responsible for compromised SAR in fad7-1 plants.
To confirm this further, we expressed a genomic copy of the FAD7 gene in fad7-1 gl1 plants. All the T1 plants showed wild-type-like levels of trienoic acid but were still devoid of trichomes, suggesting that these transgenic plants were complemented for fad7-1 but not for the gl1 mutation. Three independent T1 lines were analyzed in the T2 generation, and these segregated for the fad7-1 mutation in an approximately 3:1 wild-type-like:fad7-like manner; the fad7-1 plants were scored based on their genotype as well as FA profile (Fig. 4, D and E). However, all the T2 plants lacked trichomes (Supplemental Fig. S2), further confirming that the FAD7 transgene complemented only the fad7-1 and not the gl1 mutation. The fad7:FAD7 transgenic plants showed compromised SAR, like fad7-1 and gl1 plants (Fig. 4F), further reiterating the fact that the fad7-1 mutation was not responsible for compromised SAR.
gl1 Plants Are Impaired in the Perception, But Not the Generation, of the Mobile Signal
We next assessed whether GL1 contributed to mobile signal production or perception in the SAR response. We evaluated the response of wild-type, gl1, and fad7-1 gl1 plants to vascular exudates collected from pathogen-inoculated wild-type, gl1, or fad7-1 gl1 leaves. The wild-type, gl1, or fad7-1 gl1 leaves were infiltrated with MgCl2 or avrRpt2 bacteria, and vascular exudates collected from the inoculated leaves were injected into the leaves of a fresh set of wild-type, gl1, or fad7-1 gl1 plants. Distal leaves of the exudate-infiltrated plants were then inoculated with virulent bacteria, and proliferation of virulent bacteria was monitored at 0 and 3 dpi (Fig. 5A). As expected, exudates from avrRpt2-infected wild-type plants conferred protection against virulent pathogen in wild-type plants. Similar to the wild type, exudates from avrRpt2-infected gl1 or fad7-1 gl1 plants also conferred resistance against virulent pathogen in wild-type plants. In comparison, the same exudates (wild-type, gl1, or fad7-1 gl1 plants) conferred only marginal SAR on gl1 or fad7-1 gl1 plants. These data also correlated with PR-1 gene expression; the fad7-1 gl1 plants showed basal levels of PR-1 transcript in response to exudate application compared with wild-type plants (Fig. 5B). Together, these results suggested that gl1 plants are competent in generating the mobile SAR signal(s) but are defective in its perception.
The gl1 Plants Are Defective in Their Cuticle
The inability of the fad7-1 gl1 plants to perceive SAR signal(s) was reminiscent of phenotypes associated with acp4 plants (Fig. 5A; Xia et al., 2009), which are defective in cuticle formation. These similarities prompted us to investigate whether the fad7-1 gl1 and gl1 plants also contained defective cuticles, similar to acp4 plants. The fad2-1, fad3-2, fad4-1, fad5-1, fad6-1, fad7-2, and fad7-2 fad8-1 mutants were also included in this analysis. The known cuticle-impaired mutant gpat4 gpat8 was included as a positive control (Fig. 6A; Li et al., 2007). Toluidine blue, a hydrophilic dye that only penetrates leaves with permeable cuticles (Tanaka et al., 2004), stained leaves in fad7-1 gl1, gl1, and gpat4 gpat8 mutants. Furthermore, fad7-1 gl1 and gl1 also showed increased water loss (Fig. 6B) and chlorophyll leaching (Fig. 6C), further supporting their defective cuticle phenotype.
We also analyzed the outermost cell wall of the epidermis of fad7-1 gl1 and gl1 leaves by transmission electron microscopy (TEM). As expected, the cuticle of the wild-type leaf appeared as a continuous and regular electron-dense osmiophilic layer outside the cell wall (Fig. 6D). In comparison, fad7-1 gl1 mutants showed electron-opaque cuticles (Fig. 6D, arrow). Comparison of the scanning electron micrography (SEM) results of wild-type, fad7-1 gl1, and gl1 leaf surfaces showed that the mutants each had an uneven surface, which was greatly folded compared with that of wild-type leaves (Fig. 6E; data not shown for gl1).
To determine if this defect in cuticle structure was associated with alterations in the content and/or composition of cuticular waxes or cutin polyester monomers, we compared levels of waxes and cutin monomers between wild-type, fad7-1 gl1, and gl1 leaves (Supplemental Fig. S3). The fad7-1 gl1 and gl1 leaves showed reduction in several FAs, alkane and primary alcohol species, compared with wild-type plants (Supplemental Fig. S3A). The gl1 leaves showed an approximately 32% overall decrease in cutin aliphatic monomer content. In the case of gl1, the decrease was more pronounced in two major monomers (16:0-, 18:1-, and 18:2-dicarboxylic acid and 16-OH-16:0 [Supplemental Fig. S3B]). Taken together, these results show that GL1 is required for the biosynthesis of the cuticular wax and cutin polymers in leaves.
The gl1 Plants Show Compromised Resistance to Fungal Pathogens
Since cuticle plays an important role in defense against fungal pathogens (Kurdyukov et al., 2006; Bessire et al., 2007; Chassot et al., 2007; Li et al., 2007; Mang et al., 2009), we next evaluated the response of fad7-1 gl1 and gl1 plants to the necrotrophic pathogen Botrytis cinerea and the hemibiotrophic fungal pathogen Colletotrichum higginsianum. Interestingly, both fad7-1 gl1 and gl1 plants showed enhanced susceptibility to B. cinerea and C. higginsianum; spray and spot inoculations showed significantly larger lesions on fad7-1 gl1 and gl1 leaves (Fig. 7; Supplemental Fig. S4). Unlike fad7-1 gl1 and gl1 plants, the fad7-2 and fad7-2 fad8 plants showed wild-type-like response to B. cinerea and C. higginsianum, arguing against a role for trienoic FAs in resistance to necrotrophic or hemibiotrophic fungal pathogens.
Mutations in GL3 and TTG1 Impair the Cuticle and Compromise SAR
Since the GL1 gene is involved in trichome formation, we next tested if trichome formation was generally associated with a defective cuticle and thereby impaired SAR. The cuticular phenotypes were assessed in two known mutants, gl3 and transparent testa glabra1 (ttg1), which are affected in the differentiation of trichomes (Supplemental Fig. S5). The GL3 and TTG1 genes encode a basic helix-loop-helix protein and WD40 repeat protein, respectively. GL3 and TTG1 along with GL1 are thought to form a combinatorial regulatory complex during trichome differentiation (Walker et al., 1999; Payne et al., 2000; Zhang et al., 2003; Morohashi et al., 2007). Indeed, similar to gl1, both gl3-1 and ttg1 mutants do not form any trichomes on their leaves (Supplemental Fig. S5). Interestingly, the gl3 and ttg1 leaves rapidly stained with toluidine blue (Fig. 8A) and also showed increased chlorophyll leaching (Fig. 8B). These results suggest that gl3 and ttg1 mutants contain a defective cuticle. Consistent with the proposed role for cuticle in SAR, both gl3 and ttg1 plants were unable to induce proper SAR (Fig. 8C).
GA application is known to stimulate trichome formation (Chien and Sussex, 1996; Perazza et al., 1998). Since our results indicated that trichome differentiation was associated with cuticle formation, we next tested whether GA can restore trichome and/or cuticle formation on gl1 leaves. Exogenous application of GA to wild-type and gl1 plants caused a significant enhancement in their growth (Supplemental Fig. S6A). Although GA application was unable to induce trichome formation on gl1 leaves (Supplemental Fig. S6A), the GA-treated gl1 plants showed a significant reduction in the toluidine blue-stained areas (Fig. 9A). Furthermore, GA-treated wild-type and gl1 plants showed a significant reduction in chlorophyll leaching from these leaves (Supplemental Fig. 6B). These data suggested that GA application led to enhancement of the cuticle in both wild-type and gl1 plants. Consistent with toluidine blue-staining and chlorophyll-leaching phenotypes, the GA-treated wild-type and gl1 plants showed a pronounced increase in the levels of cuticular components (Fig. 9B). Interestingly, GA-treated wild-type plants showed significantly better SAR compared with water-treated plants (Fig. 9C). Exogenous application of GA also improved SAR on gl1 plants, but the effect was less pronounced compared with wild-type plants, likely because the cuticle might not be completely restored. Nonetheless, unlike SA or JA, GA was able to partially restore defective SAR in gl1 plants.
The gl1 mutant was first isolated based on its glabrous phenotype (Koornneef et al., 1982), and this phenotype has served as a useful marker for numerous mutant isolations and/or screenings. A cursory database search identified over 200 articles in which the gl1 background has been used to study molecular, genetic, and biochemical aspects of diverse aspects of plant physiology (Liu and Zhu, 1997; Aarts et al., 1998; Bender and Fink, 1998; Cecchini et al., 1998; Xie et al., 1998; Yi and Jack, 1998; Bieza and Lois, 2001; Ellis and Turner, 2001; Laby et al., 2001; Devoto et al., 2002; He et al., 2002; Collins et al., 2003; Quiel and Bender, 2003; Barkan et al., 2006; Zhu et al., 2007; Speth et al., 2009; Jung and Niyogi, 2010; Rowe et al., 2010). Notably, many of the mutants that were isolated or crossed into the gl1 background have been studied for the defense physiologies. Interestingly, most studies were conducted with the assumption that the gl1 mutant itself does not affect any phenotype but the loss of trichomes. Our work shows that this is clearly not the case and that gl1 plants are compromised in defense to both bacterial and fungal pathogens. Additionally, the gl1 mutation affects cuticular development. Our findings question the prudence of using mutant backgrounds to design genetic screens and underscore the need to reevaluate phenotypes that were previously studied in the gl1 background.
This is clearly the case for the fad7-1 mutant, which was originally isolated in the gl1 background (Browse et al., 1986). Subsequent studies suggested a role for glycerolipid synthesis in SAR due to the inability of the fad7-1 (fad7-1 gl1) plants to induce SAR (Chaturvedi et al., 2008). However, normal SAR phenotype in allelic fad7-2 and the fad7-2 fad8 double mutant argue against a role for trienoic FA/lipid metabolism in SAR. This is further reiterated by the fact that trienoic FA levels but not the ability to induce SAR were restored in fad7-1 transgenic lines expressing a wild-type copy of FAD7. Characterization of the fad7-1 mutant showed that its impaired SAR is associated with a second-site mutation in the GL1 gene. Notably, a mutation in the GL1 gene impaired cuticle development, thereby compromising the plant's ability to induce SAR. Interestingly, besides GL1, two other mutations affecting trichome formation (gl3 and ttg1) also contained deformed cuticles and were defective in inducing SAR. These data suggest that trichome differentiation and cuticle development may involve an overlapping biochemical basis. However, a correlation between the lack of trichomes and the presence of a defective cuticle was not observed in transgenic plants expressing the FATTY ACID ELONGATION1 gene specifically in the epidermis; these plants do not contain trichomes but have a normal cuticle (Reina-Pinto et al., 2009).
Is there a connection between trichome formation and cuticle development? To begin with, epidermal cells are involved in both of these processes. Trichomes differentiate from the pluripotent epidermal cells in a patterned manner, which is determined by genetic interactions between trichome-promoting and trichome-repressing factors (for review, see Schellmann and Hülskamp, 2005; Ishida et al., 2008). Proteins encoded by GL1, GL3, and TTG1 act as positive regulators, and mutations in these genes affect trichome development. Both GL1 and GL3 act as transcriptional regulators and are known to regulate genes involved in various other processes besides trichome development (Morohashi and Grotewold, 2009). Interestingly, one of the direct targets of GL3/GL1 includes FIDDLEHEAD (FDH), which encodes a protein related to β-ketoacyl-CoA synthase involved in cuticle development (Pruitt et al., 2000; Voisin et al., 2009). Notably, the fdh mutant shows a significant reduction in the number of trichomes (Yephremov et al., 1999). Similarly, several other mutants, including lcr (for LACERATA), bdg (for BODYGUARD), yre (for YORE-YORE, allelic to WAX2/CER3), and dso (for DESPERADO/AtWBC11), which are defective in cuticle formation, exhibit abnormal differentiation of trichomes and reduced trichome numbers (Wellesen et al., 2001; Kurata et al., 2003; Kurdyukov et al., 2006; Panikashvili et al., 2007). Suppression of ketoacyl reductase activity, which catalyzes the first reduction step leading to the synthesis of very-long-chain FAs, also results in abnormal trichomes and correlates with reduced cuticular wax levels (Beaudoin et al., 2009). These findings, together with our results, support the notion that overlapping pathways involving GL1, GL3, and TTG1 might regulate cuticle development as well as trichome formation. This is further supported by the fact the defective trichome phenotypes of the yre gl2 and yre ttg1 double mutant plants are more severe than the yre, ttg1, and gl2 single mutants (Kurata et al., 2003).
The ability of exogenous GA to complement the SAR defect in gl1 plants but not restore trichome formation suggests that trichomes might not play a role in SAR. This is further supported by the fact that compromised SAR in acp4 plants is not associated with trichome formation; acp4 plants show wild-type-like trichomes on their leaves (Supplemental Fig. S7). It appears that GA might participate in systemic immunity by affecting cuticle development. This is based on the fact that GA application reduced chlorophyll leaching by increasing the levels of cuticular components. Increased incorporation of palmitic acid and oleic acid into cutin monomers was also seen in GA-treated rice (Oryza sativa) and pea (Pisum sativum) plants (Bowen and Walton, 1988; Hoffmann-Benning and Kende, 1994), suggesting that GA-mediated increased carbon flux into cuticle might be a common phenomenon in plants. GA application also induced stronger SAR in wild-type plants. GA application is known to derepress its signaling pathway by inducing the degradation of DELLA proteins (Achard and Genschik, 2009), which negatively regulates the accumulation of reactive oxygen species (ROS; Achard et al., 2008). Notably, GA treatment, in the absence of pathogen inoculation, did not alter ROS levels; ROS levels in uninoculated, GA-treated wild-type or gl1 plants were similar to those in water-treated plants. This suggests that the GA-triggered increased carbon flux into cuticle is not mediated via ROS. However, it does not rule out a role for ROS in GA-triggered SAR. The fact that exogenous application of hydrogen peroxide is unable to improve the resistance response of wild-type plants to Pseudomonas discounts a role for ROS in GA-triggered gl1 plants. This is further supported by the result that exogenous application of SA, which potentiated a defense response leading to increased accumulation of ROS (Shirasu et al., 1997), was unable to trigger SAR in gl1 plants. Similarly, SA and JA treatments were also unable to complement defective SAR in another cuticle-deficient mutant, acp4 (Xia et al., 2009). Together, these observations support the notion that GA-mediated restoration of cuticle might be responsible for the restored SAR in gl1 plants. More importantly, these and our earlier results (Xia et al., 2009) reconfirm the fact that cuticle-deficient mutants are impaired in their ability to perceive mobile signal.
Plant Growth Conditions and Complementation Analysis
Arabidopsis (Arabidopsis thaliana) plants were grown in MTPS 144 Conviron walk-in chambers at 22°C, 65% relative humidity, and a 14-h photoperiod. For complementation, an approximately 3.5-kb region encompassing the FAD7 coding region was amplified from Col-0 genomic DNA using BamHI and XbaI linkered primers, 5′-AAATTCATGCGGAATCAGAGAACG-3′ and 5′-TCTATGTTTCCGATACTGAAGC-3′, respectively, and cloned into the pBAR1 binary vector. After confirmation of the DNA sequence, the binary vector was transformed into fad7-1 plants using the floral dip method. The transgenic plants were selected on soil sprayed with the herbicide Basta. The complementation was confirmed by FA and genotype analysis of the T1 plants and by analyzing the segregation of FA phenotypes in the T2 generation. The fad7-1 mutation was identified as a C-to-T transition that converted the amino acid at position 253 from Pro to Leu. The coding region of FAD7 was amplified using 5′-ATGGCGAACTTGGTCTTATCAGAA-3′ and 5′-GAGGTCAAAGTAAGAGCAGATTGA-3′ primers. Genomic cleaved-amplified polymorphic sequence analysis for the fad7-1 was performed by amplifying a 481-bp fragment using primers 5′-GAGGAGTCTCCATTGGAGGAA-3′ and 5′-CATGTTGCTAGTAGACCAACCC-3′, which was digested with MspI.
RNA Extraction and Northern Analyses
Small-scale extraction of RNA from one or two leaves was performed in the TRIzol reagent (Invitrogen) following the manufacturer's instructions. Northern analyses and synthesis of random primed probes was carried out as described before (Kachroo et al., 2005).
SA, FA, and JA Quantifications
SA and SAG were extracted and measured from approximately 0.3 g fresh weight of leaf tissue as described before (Chandra-Shekara et al., 2006). Measurements were performed in triplicate, and the experiment was repeated twice.
FA extraction was carried out by placing leaf tissue (n = 6) in 2 mL of 3% H2SO4 in methanol. After 30 min of incubation at 80°C, 1 mL of hexane with 0.001% butylated hydroxytoluene was added. The hexane phase was then transferred to vials for gas chromatography (GC) analysis. One-microliter samples were analyzed by GC on a Varian FAME 0.25-mm × 50-m column and quantified with flame ionization detection. For quantification of FAs, leaves (50 mg) were extracted together with an internal standard, 19:0, and the FA levels were calculated based on the detected peak areas corresponding to the FA retention time relative to the areas of the internal standard. FA quantifications were repeated three times.
For JA estimations, leaves (1 g) were ground in liquid nitrogen and extracted in 100% methanol using dihydro-JA as an internal standard. The extract was acidified to pH ≤ 4 with 1 m HCl and passed through C-18 Sep-Pak columns (Waters; 500 mg, 3 mL), which were preequilibrated with 75% methanol containing 0.2% acetic acid. The column-purified extract was saturated with sodium chloride and reextracted with diethyl ether. The ether extract was completely dried under a stream of nitrogen gas and methylated with diazomethane. The oxylipins were solubilized in 0.5 mL of hexane and dried to approximately 10 μL under a gentle stream of nitrogen gas, and 1 μL was injected for GC-mass spectrometry (MS) as described above. The JA peaks were identified using mass spectrometry. For quantitative analysis of JA, the mass spectrometer was run in selective ion monitoring mode for mass-to-charge ratio (m/z) 224 and 226. The ratio of m/z 224 to 226 in the peak area (retention time between 9.5 and 11.0 min) was used to calculate the JA levels based on the abundance of m/z 224 and 226 generated by standard JA, dihydro-JA, and their equal-level mixture. Measurements were performed in triplicate, and the experiment was repeated twice.
For JA levels in vascular exudates, samples were extracted using a solution containing glacial acetic acid, methanol, chloroform, and potassium chloride (0.9%; 1:4:8:8, v/v) and 17:0 as an internal standard. The lower phase was removed and dried under a stream of nitrogen gas, and samples were derivatized with diazomethane, dried and reconstituted in methyl tert-butyl ether, transferred to a glass insert, dried again under a stream of nitrogen gas, and reconstituted in a minimum volume of acetonitrile. Samples (1 μL) were analyzed with GC-MS as described above. Measurements were performed in triplicate, and the experiment was repeated twice.
SA, JA, and GA Treatments
SA, JA, and GA (GA4 and GA7) treatments were carried out by spraying 500, 50, or 100 μm solutions, respectively. JA-treated plants were covered with a transparent plastic dome to maximize exposure to JA.
Pathogen Infections
Inoculations with the bacterial pathogen Pseudomonas syringae were conducted as described before (Kachroo et al., 2005). The bacterial cultures were grown overnight in King's B medium containing rifampicin and/or kanamycin. The cells were washed and suspended in 10 mm MgCl2. The bacterial suspension was injected into the abaxial surface of the leaf using a needleless syringe. Three discs from the inoculated leaves were collected and homogenized in 10 mm MgCl2. The extract was diluted, and appropriate dilutions were plated on King's B medium. For analysis of SAR, the primary leaves were inoculated with MgCl2 or the avirulent bacteria (107 colony-forming units [CFU] mL−1), and 48 h later the systemic leaves were inoculated with virulent bacteria (105 CFU mL−1). Unless noted otherwise, samples from the systemic leaves were harvested at 3 dpi. All SAR experiments were repeated at least three times.
Colletotrichum higginsianum (IMI 349063) and Botrytis cinerea were maintained on potato dextrose agar and V8 medium, respectively. Four-week-old Arabidopsis plants were used for both spray and spot inoculations. Spore suspensions at concentrations of 104 to 106 spores mL−1 were used for various experiments. For spot inoculations, 10 μL of spore suspension was used to inoculate Arabidopsis leaves. After inoculations, the plants were transferred to a PGV36 Conviron walk-in chamber and covered with a plastic dome to maintain high humidity. Disease symptoms were scored between 4 and 11 dpi. A digital Vernier caliper was used to measure lesion size in spot-inoculated leaves. Each experiment was repeated at least twice, and each included 30 to 50 individual plants. Statistical significance was determined using Student's t test.
Collection of Phloem Exudate
Leaf exudate was collected as described earlier (Maldonado et al., 2002). In brief, plants were induced for SAR by inoculation with P. syringae containing avrRpt2 (106 CFU mL−1). Twelve to 24 h later, petioles were excised, surface sterilized in 50% ethanol and 0.0006% bleach, rinsed in sterile 1 mm EDTA, and submerged in approximately 1.9 mL of 1 mm EDTA and 100 μg mL−1 ampicillin. Exudates were collected over 48 h and infiltrated into healthy plants. Infiltrated leaves were harvested after 2 d for PR-1 gene expression studies. For SAR studies, virulent pathogen was inoculated in the distal leaves 2 d after infiltration of exudate.
Microscopy, Chlorophyll Leaching, and Water Loss
For SEM analysis, both abaxial and adaxial surfaces of the leaf samples were mounted on sample holders with 12-mm conductive carbon tabs (Ted Pella, Inc.), sputter coated with gold-palladium, and observed on a Hitachi S-3200 SEM device with and without a backscatter detector at 5 and 20 kV.
For TEM analysis, leaves were fixed in paraformaldehyde and embedded in Epon-Araldite. Leaves were sectioned on a Reichert-Jung Ultracut E microtome with a Diatome diamond knife and observed with a Philips Tecnai Biotwin 12 TEM device.
For chlorophyll leaching assays, 100 mg of leaves was weighed and gently agitated, in the dark at room temperature, in tubes containing 80% ethanol. Absorbance of each sample was measured at 664 and 647 nm, and micromolar concentrations of total chlorophyll per gram fresh weight were calculated using the following formula: total micromoles of chlorophyll = 7.93 (A664) + 19.3 (A647).
For water loss measurements, 4-week-old plants were either subjected to drought or kept moist. The leaf weight was measured from approximately 50 leaves.
Analysis of Wax and Cutin Components
For analysis of the wax component, 500 mg of 4-week-old leaves was immersed in 10 mL of chloroform for 10 s. The leaves were rinsed once more with 10 mL of chloroform. An internal standard (100 μg of n-tetracosane) was added, and the sample volume was evaporated under a gentle steam of nitrogen. The samples were dried under a stream of nitrogen gas and methylated with diazomethane, dried again, and derivatized with 100 μL of acetic anhydride in 100 μL of pyridine, and the sealed tubes were incubated for 60 min at 60°C. The samples were again dried under a stream of nitrogen and dissolved in 1 mL of acetonitrile. Samples (1 μL) were injected into an HP-5 column of the GC device equipped with a flame ionization detector. The same samples were also run on an HP-5 column (30-m × 0.25-mm × 0.25-mm film thickness) on a GC device equipped with a mass spectrometer. Various components were identified based on their retention times as compared with standards and by MS analysis. Quantification was based on flame ionization detector peak areas as compared with the peak areas of the internal standard n-tetracosane added prior to derivatization. Measurements were performed in triplicate, and the experiment was repeated twice.
Cutin monomer composition and content were determined using the sodium methoxide-catalyzed transmethylation method followed by acetylation of the hydroxyl groups with acetic anhydride and GC-MS slightly modified from a previous description (Bonaventure and Ohlrogge, 2002; Molina et al., 2006). After methanolysis, the methylene dichloride extract of cutin monomers was washed with 0.9% potassium chloride instead of 0.5 m sodium chloride. For GC-MS analysis, the Varian FAME capillary column used was as described in wax analysis with helium carrier gas at 1 mL min−1. The mass spectrometer was run in scan mode over 35 to 450 atomic mass units (electron impact ionization). Measurements were performed in triplicate, and the experiment was repeated twice.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. An abbreviated scheme for fatty acid and lipid biosynthesis.
Supplemental Figure S2. Leaves from the indicated genotypes showing the presence or absence of trichomes.
Supplemental Figure S3. Cuticular wax and cutin monomer profiles.
Supplemental Figure S4. Response of gl1 to the necrotrophic pathogen B. cinerea.
Supplemental Figure S5. Leaves of the indicated genotypes showing the absence of trichomes on gl3 and ttg1 plants.
Supplemental Figure S6. Effects of GA treatment on trichome formation, leaf size, and chlorophyll leaching.
Supplemental Figure S7. Scanning electron micrographs of leaves from Nö and acp4 plants.
We thank Ljerka Kunst, David Smith, and David Hildebrand for useful comments. We thank John Johnson for help with GC, Ludmila Lapchyk for technical help, Larry Rice for help with SEM, and Mary Gail Engle for help with TEM. We thank Bruce Downie for GA, John Ohlrogge for gpat4 gpat8 seeds, and the Arabidopsis Biological Resource Center for fad7-1 gl1, gl1, gl3, and ttg1 seeds.
↵1 This work by supported by the National Science Foundation (grant nos. MCB 0421914 and IOS 0749731) and the Kentucky Science and Engineering Foundation (grant no. KSEF–09–RDE–012).
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Pradeep Kachroo (pk62{at}uky.edu).
↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.
↵[W] The online version of this article contains Web-only data.
↵[OA] Open Access articles can be viewed online without a subscription.
- Received June 18, 2010.
- Accepted August 4, 2010.
- Published August 10, 2010.


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(a–f). Fold changes in expression of IL10, IFNγ and IFNβ genes in tissues of mice after RVFV infection.
2011-09-16T01:18:03Z (GMT) by
RecNP immunized mice (n = 3 per time point) are indicated by solid black bars, adjuvant control mice (n = 3 per time point) by grey bars and PBS control mice (n = 3 per time point) by white bars. The horizontal dotted lines indicate the cut-off values for upregulation (+2) or downregulation (−2). The asterisk (*) indicates where the P-value is smaller than or equal to 0.05 (statistically significant results). Standard error values are indicated by the error bars. Note the differences in the Y-axis scales. The following time points are indicated: 3, 6, 12, 24, 72 and 120 hours.


text:
Emu Loader v8.8 is released. Emu Loader was created to manage an arcade games emulator called M.A.M.E. With this frontend, you can change practically any option that is available in the emulator, like the screen resolution, sound quality, game options, etc. There are lots of extra features that makes your life much more easier, like the snapshots viewer where all captured images from M.A.M.E. games can be viewed when selecting games.
Emu Loader Changelog:
Fixed a bug in "TCustomColorBox" Delphi 7 class, "SetSelected()" procedure
- it was not repainting the control properly, and it was not caused by the frontend (damn you Delphi 7!)
- my custom TColorBoxEx component was also affected due to this "Delphi 7" bug
- the fix was taken from "Code Gear Delphi 2010" source code ("vlc\ExtCtrls.pas")
Event "OnSelect()" was not properly executed on TColorBoxEx components (color dropdown buttons)
A few default settings in "EmuLoader.ini" and "nightmode.ini" were causing problems on a clean install
Colors for "Game Docs Status Bar" not updated when switching between light mode and night mode
Fixes for the night mode
- search bar background had gradient always enabled even if the gradient setting was not ticked
- floating panel gradient background colors were not properly set, specially at startup
- TColorBoxEx were resetting to default colors at frontend startup when viewing the "2 pages" in night mode screen
Hint box colors and style not updated when switching between light mode and night mode
Control "Buttons" was listed twice in "Game Details" screen when a game have multiple controls + buttons
Game selection bar colors and font colors were not correctly set in several screens, including in "Customize Game Fonts"
Font of column header was not properly set when night mode is enabled (MAME Machines Filter Panel)
My custom component TEditEx was not repainting the frame color when required
Pressed button custom colors were not applied to buttons in night mode
Font "Terminal" is used in a few places and if it's not installed in Windows, it will be replaced by "Lucida Console" (Windows 7 users don't have to worry about this)
App crash when trying to show game images if image category folders are not successfully loaded
Updates to my TAdvCheckBoxEx and TAdvRadioGroupEx components
- fixed icons positions as they were not drawn at the same position on Windows 7 and Windows 10
- RadioGroup icon updated with a 13x13 sized image when working with Delphi 7 in design time (easier to align controls)
- added a couple commands to get the correct CheckBox/RadioGroup icon sizes from Windows theme
Metadata files are no longer used when mounting images on a "virtual drive" app (.cue, etc...) even if "use .cue" tool bar button is enabled (console/computer games)
Game image files failed to mount on a virtual drive, returning an error to the user (console/computer games)
Night mode is now enabled by default on a clean install or if "EmuLoader.ini" file is missing
Shadow texts are disabled in light mode for panel texts, tool bar panels, floating panels; it looks better this way
Improvements to night mode settings (night mode screen)
- added "Shadow Enabled" checkboxes for texts in "Search Games Panel Colors", so you can disable shadow texts
- added "Shadow Enabled" checkboxes for texts in "Panels Colors", so you can disable shadow texts
- added preview panels (click the "Preview" buttons) so you can see your color customizations
- Games List Selection Bar
- Panels Colors
- Image Hint Box
- Search Games Panel Colors
- tweaks to the layout
Rounded corners in GroupBox frames are disabled for night mode
The overlay folder "Dark Gray" is no longer needed, these icons are now the default for night mode ("\resources\main_icons\overlay\Dark Gray\" folder can be deleted)
Blue overlay icons moved to a new "Blue" folder ("\resources\main_icons\overlay\Blue\")
If overlay files of a sub-folder are not found, the "Tool Bar Overlay Icons Folder" setting will reset to "Default" (night mode screen)
Improvements to "Image Hint Box" (preferences screen, night mode screen)
- setting "Show Unzipped/Zipped Icon" is gone, the icon is now mandatory
- added a "Italic" font style checkbox, so you can disable the italic style (disabled by default)
- text font size reduced to 10 pixels
- added a new "image resolution (image type)" text below the image category title; only font color can be customized
- added a "Larger Font Size" checkbox, for a 14 pixels font size like it was before (disabled by default)
- added a "Box Position" setting to choose where the hint box will appear; default position is "Middle/Left"
- fixed "middle vertical" position, it was not taking the hint box height into account
Selection bar of menus/popup menus is a little darker, around 10%
Moved settings panel to a popup menu ("Favorites Manager" settings button)
Game details screen changes
- removed shadow of texts in the left panel (night mode)
- removed dual border from ROMs list
- minor improvements to make sure border spaces are pixel perfect
Removed shadow of texts in system title bars and replaced yellow font color by orange, on all screens (night mode only)
Removed shadow of texts from labels and checkboxes in almost all screens (night mode)
Color tweaks to texts font colors in "Delete/Rename Game Image File" screens (night mode)
Several source code optimizations to remove redundant duplicates, mostly for the night mode
Modified my TShadowLabel component to not show the shadow text if component is in "disabled" state and the "ShadowEnabled" property is unchecked
If a "Label", "CheckBox", "RadioButton", "GroupBox" component is in disabled state and the "ShadowEnabled" property is unchecked, the shadow text is no longer displayed
- it looks a lot better in night mode
- in light mode, the "3D effect" no longer applies, but texts still look good
Games list in "Delete Multiple Games Files" screen now uses the same colors / background image as the main games list
Replaced "Consolas" font by "Calibri" in "MAME Game Docs" panel
Tons of interface tweaks, specially texts and their colors
New interface for preferences screen
- cleaner and easy to navigate, also to prevent waste of blank space
- page 1 is for frontend settings, page 2 is mostly for colors
- tweaks to existing settings so they are better aligned with panels
Several settings moved from "EmuLoader.ini" to a new "\ini_files\LightMode.ini" file (preferences screen, mostly)
- they are all exclusive to the light mode
- now you can do a clean install and restore these settings easily
- most key names in .ini file were renamed to match key names from night mode preset files, you must modify your light mode settings again (sorry)
- some "MAME Game Docs" settings moved from "el_extras.ini" file into "LightMode.ini"
The following is removed to simplify the interface
- frames of games list panel, images panel and game docs panel is kaput!
- setting "Games List Frame Colors" (preferences screen, night mode settings screen)
- setting "Games List Status Bar, Inner/Outer Frame" (preferences screen, night mode settings screen)
- setting "Games List Status Bar, Font Shadow Color" (preferences screen, night mode settings screen)
- setting "Game Docs Splitter Style / Colors"; same style/colors as image splitters is used, as it was before (preferences screen, night mode screen)
- setting "Image Panel Frame Colors" (preferences screen, night mode settings screen)
- setting "Enable Edit Box Custom Frame Color" is no more; custom frame is always enabled in night mode (night mode screen)
- setting "Games List Splitter" style is gone for night mode; only a single color is supported (night mode screen)
- setting "Tool Bar Inner/Outer Frame" is no more (night mode screen)
New games list background images, for both night mode and light mode ("\resources\images\")... thank you Fabian for the new images
New setting "Disable Aspect Ratio", to stretch the image on the entire image panel; requires "Image Stretch" setting to be enabled (popup menu "Images")
New setting "Round Corners" to "Games List Selection Bar" colors (preferences screen, night mode screen)
Night mode colors support for arcade emulator setup screens: MAME, HBMAME, Supermodel, Daphne, SEGA Model 2, ZiNc
New "Left Align Emulators Info and Games Info" setting to show message box texts aligned to the left instead of centered (preferences screen, "General" panel)
New setting to customize games filters tool bar in light mode (preferences screen)
- you can use gradient or solid colors
- new "Windows Theme" checkbox, so you can disable Windows themed tool bar painting
Support for multiple night mode profiles
- change profiles with the new "Night Mode Profiles" box ("Customize Night Mode" screen, access thru main menu)
- all profiles are now stored in "frontend_path\nightmode\" folder
- file "nightmode.ini" moved to the new "\nightmode\" sub-folder and renamed to "Default.ini"
- setting "Tool Bar Overlay Icons Folder" can be different for each profile
- setting "Menus / Popup Menus" frame color can be different for each profile
- current profile is automatically saved to .ini file when switching between profiles or when closing the "Customize Night Mode" screen
New "RGB Quick Edit" for color dropdown boxes "TColorBoxEx" (popup menu, mouse right-click)
- for an easy and quick way to select a RGB color manually
- screens that support this new popup menu
- Customize Night Mode
- Image Category Settings
- Customize Game Fonts
- Customize Thumbnails
- Preferences screen
Added "Show Font Name" setting in "Customize Game Fonts" screen
- access in popup menu (disabled by default)
- useful for high resolution screens (1080p or higher)
Added a custom game fonts set for night mode, split from light mode
- have a set of game fonts for light mode and another for night mode
- light mode filename: "\ini_files\sysgamecustomfont.ini"
- night mode filename: "\ini_files\sysgamecustomfont_nightmode.ini"
- updated "Customize Game Fonts" screen to load the night mode font settings if this mode is active (games popup menu, main menu "Games List")
- if file "sysgamecustomfont_nightmode.ini" doesn't exist, it is generated from "sysgamecustomfont.ini" at startup (useful for night mode)
New "Use A Single Background Color" setting for image categories
- select a background color that will be used for all image categories ("Image Category Settings" screen)
- you no longer need to select the same color on each category if you want to use the same color for all of them
- toggle setting in main menu "Images", images popup menu or in "Image Category Settings" screen (disabled by default)
Added options to customize games list header colors in night mode
- text font
- column background color, hot color, disabled color, pressed color
- settings in Night Mode screen, "Games List Column Header" panel
MAME v0.212 support
Download: EmuLoader v8.8
Source: Here
Emu Loader v8.8 is released. Emu Loader was created to manage an arcade games emulator called M.A.M.E. With this frontend, you can change practically any option that is available in the emulator, like the screen resolution, sound quality, game options, etc. There are lots of extra features that makes your life much more easier, like the snapshots viewer where all captured images from M.A.M.E. games can be viewed when selecting games.
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Sasser swizz
78% of those polled by antivirus firm Sophos say the sentence for the Sasser virus writer was not harsh enough. The poll indicates that the PC users wants virus writers jailed.
A web poll of more than 550 business PC users, conducted by Sophos, has revealed that 78% believe that last Friday's sentencing of Sven Jaschan, the German teenager found guilty of writing and distributing the Netsky and Sasser worms, was not harsh enough.
A further 17% of respondents agreed with the North Germany court decision, which sentenced Jaschan to one year and nine months on probation and 30 hours community service. A mere 5% felt this sentence was too severe.
"With almost 80% of those surveyed saying Jaschan's sentence was too lenient, it seems that many computer users aren't convinced justice has been served," says Mohammad Noraiee, managing director at Al Adeeb IT, which is the distributor for Sophos in the UAE.
"But perhaps even more interesting, is Microsoft splashing out US$250,000 to the two unidentified people who helped track Jaschan down - especially when speculation hints that these people are teenagers who may have had some involvement with Jaschan. It's good to see Microsoft taking strong action against such crimes, but it might struggle if it has to shell out big bucks for every virus writer who gets arrested," he adds.
Microsoft offers bounties for information leading to the successful prosecution of cyber criminals who attack its software.


text:
Working within the Federal Reserve System, the New York Fed implements monetary policy, supervises and regulates financial institutions and helps maintain the nation's payment systems.
Do you have a Freedom of Information request? Learn how to submit it.
See the world's largest accumulation of gold as you learn about the New York Fed and Federal Reserve System on a free tour.
The latest Annual Report chronicles the impact of Federal Reserve policies and includes data on the New York Fed's operations.
Our economists engage in scholarly research and policy-oriented analysis on a wide range of important issues.
The Weekly Economic Index provides an informative signal of the state of the U.S. economy based on high-frequency data reported daily or weekly.
The Center for Microeconomic Data offers wide-ranging data and analysis on the finances and economic expectations of U.S. households.
Our model produces a "nowcast" of GDP growth, incorporating a wide range of macroeconomic data as it becomes available.
As part of our core mission, we supervise and regulate financial institutions in the Second District. Our primary objective is to maintain a safe and competitive U.S. and global banking system.
The Governance & Culture Reform hub is designed to foster discussion about corporate governance and the reform of culture and behavior in the financial services industry.
Need to file a report with the New York Fed? Here are all of the forms, instructions and other information related to regulatory and statistical reporting in one spot.
The New York Fed works to protect consumers as well as provides information and resources on how to avoid and report specific scams.
The Federal Reserve Bank of New York works to promote sound and well-functioning financial systems and markets through its provision of industry and payment services, advancement of infrastructure reform in key markets and training and educational support to international institutions.
The New York Fed provides a wide range of payment services for financial institutions and the U.S. government.
The New York Fed offers the Central Banking Seminar and several specialized courses for central bankers and financial supervisors.
The New York Fed has been working with tri-party repo market participants to make changes to improve the resiliency of the market to financial stress.
The New York Fed engages with individuals, households and businesses in the Second District and maintains an active dialogue in the region. The Bank gathers and shares regional economic intelligence to inform our community and policy makers, and promotes sound financial and economic decisions through community development and education programs.
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The College Fed Challenge is a team competition for undergraduates inspired by the working of the Federal Open Market Committee.
September 12, 2000
Overall, U.S. consumers benefited from cheaper imports in the wake of the Asia crisis, while the nations domestic production and employment were largely unhurt, according to James Harrigan, a senior economist at the New York Fed.
Harrigan states that the large currency devaluations experienced by Korea, Malaysia, Thailand, and Indonesia beginning in mid-1997 raised concerns that imports from these countries would soar while demand for U.S. exports weakened, causing U.S. industries to suffer. However, his industry-level analysis of the devaluations impact reveals that these industries generally experienced little or no pain.
Harrigan also finds that:
Harrigans article--The Impact of the Asia Crisis on U.S. Industry: An Almost-Free Lunch?--will be published in the Banks Economic Policy Review.
Contact: Douglas Tillett or Steven Malin


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Feature Paper and Cover of Energies published by CNR-IMM and EGP researchers, entitled "Reversible Efficiency Variation of Tandem Amorphous/Microcrystalline Si Photovoltaic Modules in Outdoor Operation", by FR Galluzzo, C Gerardi, A Canino, and S. Lombardo.
Amorphous Si multijunction PV modules are subjected to instability phenomena due to the Staebler-Wronski effect. We performed outdoor tests to study the effect of operating temperature and incident solar spectrum. Experimental results show clear correlation between performance improvements and PV module thermal history.
Open access at:


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Listeners Club
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about two years ago
Mark Chase
It's time for another Mix Pet Of The Week and a couple more furry cuties from the Butte Humane Society who are looking for forever homes!
Meet Clementine
Age: 2 years
Gender: Female
Breed: Domestic Shorthair/Mix
Color: Black
Meow! My name is Clementine. I am the most gorgeous black cat you will ever set eyes on! I absolutely love attention from all the staff and volunteers here. I am only two years old so I have lots of time to give all my love! I am okay around other feline companions so I think I could go home with one as long as they respect my space. I would prefer to go to a home with no dogs because I can tend to be pretty sassy towards them. Come down to the Cat Adoption Center at 587 Country Dr. and meet me! I will greet you with sweet kisses and lots of purring.
Say Hello To Cinnamon
Age: 4 years
Breed: Terrier, American Pit Bull/Mix
Color: Tan/Blue
Hi there! My name is Cinnamon! I am a very happy and friendly gal. I am very accepting of any handling and love to be hugged! I have a ton of energy but am very gentle so I think I would be a great addition to a family that had some kids for me to run around with! I am hoping that I don't have to stay here in the kennels for too long because a girl like me needs space to run and people to love on! If you are looking for someone to take on all your adventures, please come down to the dog adoption at 2579 Fair St.
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text:
Note: We will only be reading Sections I and II of this paper this week!
The .pdf file can be found here (Note you need to be logged in to use this link)
An external link to the paper is here.
The DOI is 10.1103/RevModPhys.78.537.
Wiki's explanation of "cosmic time" was rather terse. Is there a good way to understand how this relates to time in our frame? –
A: As far as I understand, it is just a fancy word. It is time as a coordinate, and in a local Mincowski spacetime (any local system can be reduced to this), this "cosmic time" t is equivalent to time we use. The only concern is that the coordinate time may not be a physical quantity in some cases (it may not be a good "clock"). For example, during inflation, what drives and determines the inflation is inflaton field. In this context, the inflaton is the "clock," and t is usually considered a rather auxiliary quantity. In any case, t is a coordinate time. –
Any hope for me to understand "tachyonic instability"? –
A: I am not completely sure about all the implications it makes, but the basic idea is fairly simple (as far as I know). You can look at Fig. 3 in the paper. The field starts from up in the potential and the system ("universe") rolls down the hill toward . At any given , the minimum in potential is at at the beginning, and so the system oscillates around it. However, once is achieved, the potential bifurcates and is no longer a stable equilibrium. The system becomes unstable and rolls down toward the new minima relatively fast. Since the effective mass term for gets negative sign (the curvature of the potential becomes negative in direction), it is called "tachyonic." –
Back to papers


text:
Getting Beyond Hype: Four Questions to Predict Real Impact. REBEL8 2014 Spring Lookbook. REBEL8 is back with its latest lookbook for Spring 2014.
Shot by Dylan Bartolini-Volk and modeled by Mark Doble, the lookbook exhibits a selection of hoodies and pullovers emblazoned with the label's signature script and logo, while denim jackets draw inspiration from vintage motorcycle apparel and button-up shirts see updated "zeus" prints. Flannel shirts and khaki pants are reoccurring staples each season, while trademark headwear designs further the label's gritty aesthetics. What Drug Dealers Can Teach Struggling Young Creatives.
Alexa Clay writes and speaks about underground and grassroots innovation, technological advancements, economic shifts, and the power of individuals whom she empathetically labels as misfits.
These are the people living on the fringes of society, ranging from drug dealers to hermits. She's profiling many of these mavericks in The Misfit Economy, a book co-written by Kyra Maya Phillips, that will be published by Simon & Schuster in May 2015. HOW TO: Make a $5 wind-powered phone charger for your bike How To Make a $5 Cell Phone Bike Charger. Oglala tribal member going to court in attempt to end injunction barring him from growing hemp. FARGO, North Dakota — Alex White Plume thought his decade-long wait to produce industrial hemp on a South Dakota Indian reservation was ending when the federal government softened its stance on marijuana enforcement and lawmakers expanded the development of hemp under certain circumstances.
But federal prosecutors in South Dakota refuse to lift an injunction against White Plume that prevents the enrolled member of the Oglala Sioux Nation from growing the crop. "One thing that really hurts my feelings is to get treated as minorities," White Plume told The Associated Press. "We have always been here. We have superior standing. " Hemp can be used to make clothing, lotion and many other products, but growing it has been illegal under federal law because it is a type of cannabis plant and looks like marijuana. White Plume's attorney, former U.S. Purdon said he's disappointed with the DOJ's position and says he plans to take legal action against the government. Welcome to Forbes. The Easy Solution To Getting Around Cities Might Have Been Developed Back In The 1930s. Car manufacturers still can't figure out why Millennials aren't buying vehicles.
Workplace Democracy in Nonprofit Organizations - Sustainable Economies Law Center. At our favorite local worker-owned cafe, Alchemy Collective Cafe in Berkeley, we recently asked the question: What do you get when you cross a worker cooperative with a 501(c)(3) nonprofit?
A worker self-directed nonprofit! We've started defining this as an organization in which all workers have the power to influence the realms and programs in which they work, the conditions of their workplace, their own career paths, and the direction of the organization as a whole. Watch the webinar "Worker Self Directed Nonprofits: Implementing Workplace Democracy in Nonprofit Organizations" "The nonprofit system has tamed a generation of activists. Building Creative Economies.
Creative Entrepreneurs and Economies Our mission is to accelerate the success of creative entrepreneurs and economies.
At Creative Startups we believe in the power of creative entrepreneurs to create dynamic and prosperous communities. To support individuals and communities working in the creative industries we: Launch successful startups through Creative Startups Accelerator, the nation's first accelerator dedicated to creative entrepreneurs,Build community-based regional economic development strategies for success in the creative economy,Create dynamic partnerships to build capacity and resources for creative entrepreneurs and communities. Creative entrepreneurs drive global change, create economic value and promote cultural tradition and innovation. History of Creative Startups. Eddie Huang & Elena Bergeron: Pop Culture: Our Most Powerful Weapon. Shark Tank Episode Tree T Pee Full HD. Collective Impact. Large-scale social change requires broad cross-sector coordination, yet the social sector remains focused on the isolated intervention of individual organizations.
S ee also: " Roundtable on Collective Impact " " Channeling Change: Making Collective Impact Work " " Understanding the Value of Backbone Organizations in Collective Impact " The "Four Cs" of 21st Century Education. Most of us know that "there is a profound gap between the knowledge and skills most students learn in school and the knowledge and skills they need in typical 21st century communities and workplaces."
So states the Partnership for 21st Century Skills, a national organization comprised of both business (Apple, Intel, Adobe, HP) and education (National Education Association, Pearson, Scholastic) leaders, committed to "fusing the three Rs and four Cs." As an advocate for the skills of innovation, I'm thrilled to see attention now placed on these Four Cs, with 14 states, including Illinois, having signed on to adopt the Partnership framework as a way to ready K-12 students for the 21st century. Most of us know the three Rs are reading, writing and arithmetic, but what are the Cs? Start up lessons from Patron Billionaire John Paul DeJoria. Identifies Attributes of the Global, Socially-Conscious Consumer.
Half of Consumers under Age 40 Willing to Pay Extra for Products and Services from Socially-Responsible Companies CONTACT: Jennifer Frighetto, email@example.com, 847.605.5686 NEW YORK –March 27, 2012 – Sixty three percent of global, socially-conscious consumers are under age 40, they consult social media when making purchase decisions and are most concerned about environmental, educational and hunger causes, according to a new study from Nielsen, a leading global provider of information and insights into what consumers watch and buy.
Nielsen's Global Corporate Citizenship Survey of more than 28,000 Internet respondents in 56 countries shows that 46 percent of global consumers are willing to pay extra for products and services from companies that have implemented programs to give back to society. Nielsen defines these consumers as socially-conscious consumers. Business Model Canvas Explained.


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Shelf Life
First Love Monster
by Paul Jensen, Gabriella Ekens,
Is it possible to hug an entire anime series? I ask because that's exactly what I want to do with How To Keep A Mummy this season. That show is unreasonably, absurdly adorable and I love it. Welcome to Shelf Life.
Jump to this week's review:
First Love Monster
On Shelves This Week
Attack on Titan - Season 2 BD+DVD, Limited Edition
Funimation - 300 min - Sub+Dub - MSRP $49.98|$84.98
Currently cheapest at: $29.96 Amazon|$63.74 Right Stuf
Synopsis: Eren Jaeger and the surviving soldiers of humanity have little time to rest in the aftermath of the battle agains the Female Titan, as a new group of Titans appear dangerously close to home.
Extra: We have episode reviews for this season, along with a catch-up guide to the first season. You'll find this season streaming on Crunchyroll, Funimation, and Hulu.
Cinderella Boy - Complete Collection DVD
Discotek - 300 min - Sub - MSRP $34.95
Currently cheapest at: $22.26 Amazon
Synopsis: Private detectives Ranma and Rera end up switching bodies after a run-in with the mafia, and must work together to discover the cause of their unusual problem.
Extra: We don't have much coverage of this series, though it does come from the same creator as Lupin the 3rd. Our user ratings have an average of 5.9 out of 10.
Flip Flappers - Complete Collection BD, Limited Edition
Sentai - 325 min - Sub+Dub - MSRP $69.98|$149.98
Currently cheapest at: $45.49 Right Stuf|$85.69 Amazon
Synopsis: As middle school student Cocona searches for what she wants to do with her life, she is whisked away to a world of fantasy by a mysterious girl named Papika.
Extra: We have both episode reviews and a full series review for this show, which I remember mainly for its very cool art style. It's available streaming on Crunchyroll, HIDIVE, and Hulu.
Izetta: The Last Witch - Complete Collection BD+DVD
Funimation - 300 min - Sub+Dub - MSRP $64.98
Currently cheapest at: $48.74 Right Stuf
Synopsis: When the forces of an imperialist nation threaten the small country of Elystadt, Princess Ortfine calls on a witch named Izetta to help turn the tide of the war.
Extra: Much like Flip Flappers above, we have episode reviews and a full series review for this one. You'll find it streaming on Crunchyroll and Funimation.
Library War - Complete Collection BD
Discotek - 300 min - Sub - MSRP $39.95
Currently cheapest at: $25.97 Right Stuf
Synopsis: In a dystopian future where expression is tightly controlled by the government, groups called Library Forces are organized to protect local libraries.
Extra: We have a review of a previous DVD release of this series, and it's available streaming on Crunchyroll.
NieA_7 - Complete Collection BD+DVD
Discotek - 300 min - Sub+Dub - MSRP $39.95
Currently cheapest at: $24.99 Amazon
Synopsis: College student Mayuko Chigasaki already had enough problems making ends meet, and her troubles only get stranger when she takes in a roommate from another planet.
Extra: We have some old reviews of this series from back in 2002, which you'll find here and here.
Scar-Red Rider XechS - Complete Collection DVD
Funimation - 300 min - Sub - MSRP $44.98
Currently cheapest at: $33.74 Right Stuf
Synopsis: Teenage scientist Akira Asagi is recruited to lead a special combat unit that defends the world from invading monsters called Nightfly O'Note.
Extra: This show's first episode didn't get a particularly warm reception in our Preview Guide, and that's about as far as our coverage goes. You'll find it streaming on Funimation.
Urusei Yatsura: Beautiful Dreamer BD, DVD
Discotek - 107 min - Sub+Dub - MSRP $29.95|$19.95
Currently cheapest at: $19.34 Amazon|$12.97 Right Stuf
Synopsis: As Ataru, Lum, and their classmates rush to prepare for the school festival, they slowly realize that they're repeating the same day over and over.
Extra: While we don't have any formal reviews for this entry in the classic Urusei Yatsura franchise, our user ratings are pretty strong with an average of 7.9 out of 10. This movie is available streaming on Crackle.
Shelf Life Reviews
Nothing this week.
Nothing this week.
First Love Monster
We're nearly free from our post-holiday backlog of Perishable releases, but a couple of them are still lurking here and there in the review schedule. Speaking of which, here's Gabriella's review of First Love Monster.
So this is exactly the type of high-octane trash that you'd expect given its premise. Kaho moves into a house filled with manboys, gets involved with one somehow, and finds herself both charmed and disgusted by his immature antics. To be honest, the object of her accidental affections – 5th grader Kanade – seems more like an unusually dumb guy than a little kid, but I digress. This is the kind of anime where you think you already know where you stand just from hearing the premise, and the show proceeds to do nothing to change your mind. However, if there's a potential saving grace here, it's that self-aware horny anime for girls have a pretty decent track record for being funny if they strike the right balance between their parodic elements and sincere celebration of female desire. From Ouran High School Host Club to Kiss Him, Not Me!, First Love Monster is already part of a genre known for spinning comedy out of problematic premises, so its obvious comedic bent gave me a fragment of hope that this thing wouldn't be a total trashfire.
Oh, who am I kidding – this show sucked and I didn't like it. The Perishable rating is right at the top of the review. While First Love Monster is immensely self-aware, its flirtations with parody do nothing to counter its extremely niche fetishism; if anything, the comedic tone helps it plant its feet even more firmly within those questionable sands. If there's anything unique about First Love Monster, it's definitely the tone. As an occasional fan of this genre myself, I wouldn't be making "fujoshi trash" jokes if this show didn't 100% invite them. First Love Monster's main appeal comes from embracing the same self-deprecating mode that some fujoshi use to refer to themselves: equal parts ironic and cynical, but sincerely thirsty. Multiple characters (including another love interest) exist just to insult Kaho, the show constantly constructs scenarios around how reprehensibly dumb its protagonist is supposed to be, that sort of thing. The point is that you – the person choosing to watch this of your own volition – embrace and acknowledge that you have an alarming fetish, so you deal with this by turning the ensuing shame into part of the kink. It's cynical and off-putting to anyone who isn't in on the joke, but it's also true to an aspect of fujoshi culture that I hadn't seen portrayed by anime before. Strangely enough, this show's closest tonal relative is probably WataMote, or No Matter How I Look at It, It's You Guys' Fault I'm Not Popular!, but that show was more about the type of girl who would be into First Love Monster, not a straightforward transmission of the fantasy wrapped with a self-deprecating wink. (Also, WataMote was a good show that you should watch, and this is not.)
My slight interest in this show as a curiosity has nothing to do with whether First Love Monster is watchable if you're foreign to the depths of the 'rotten girl' sensibility. The show isn't that funny, looks pretty bad, and it's frequently offensive enough to the point where I can't recommend it to anyone who wasn't ready to devour this whole thing based on its premise. For one thing, the show trades in much of the same hacky humor employed by bad fanservice anime in general, such as a "comical pedophile" side character. When they're not just gross, the jokes are largely lazy, consisting of pseudo-elementary schoolers shouting the word "wiener" for minutes on end. Now I've been known to appreciate the occasional dick joke, but the mere acknowledgment of the member's existence isn't enough to get yuks out of me. It's the same for just shouting the word "poop", "balls", etc. So even when the show wasn't being all-out offensive (either fetishizing or making light of pedophilia), it was just unfunny. Visually, the only nice thing that I can say about the show is that it's colorful. Otherwise, it's yet another minimally animated affair with generic bishonen character designs. So if you're just into raw manservice, there are hundreds of alternate avenues that won't make you ogle mysteriously post-pubescent twelve-year-olds.
Extras include the regular slate of trailers and clean theme songs that are barely worth mentioning. Other than that, there's an episode commentary from the English voice actors that mostly consists of them goofing off. I'll admit that I enjoyed the dub for this show – it's pretty dumb and hyperactive but the voice actors seem like they're having fun, and their diffident attitudes were the source of most of the laughs that I did get out of the show. For once, this flippancy actually matched the show's intended tone, so I just let myself enjoy it.
The nicest thing I can say for First Love Monster is that it's one of the less boringly terrible shows I've had to endure throughout my tenure on Shelf Life. The self-loathing fujoshi trash angle was certainly unique, and the show does occasionally manage to be innovative in its commitment to crass stupidity. That's not to say that its clever by any stretch—you won't find any Pop Team Epic-style transcendent idiocy here. It was just loud and offensive enough to stand out in the pantheon of shows rated Perishable. It was different enough to hold my attention without pain (a rare luxury at this point). What I'm saying is that if we had separate ratings for the "bad shows for nobody" and the "truly execrable wastes of space," First Love Monster would fall into the higher echelon.
At least I got my roommate to laugh by showing him the Blu-Ray box, just like Super Lovers – although he had to read the back-of-the-box description instead of just glancing at the cover. On the bright side, this review concludes my current foray into the realm of trash, since my slate is looking decent for the near future. But I won't be gone for long – there will always be trashy anime to review, and I can never resist the siren's call for long.
That wraps up the review section. Since we don't have a Shelf Obsessed entry this week, that's it for this week. Thanks for reading, and remember to send photos of your anime collection to [email protected] if you'd like to see them featured in the next installment of Shelf Obsessed!
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Ardex X32 fastener (fix) for most of our stones.
about 3,3 kg / sqm with thin bed method and 8x8x8 mm comb
The calculations are very individual depending on how much the craftsman wants to put in and should be seen as a recommendation. The calculation even requires the basis.
Product Description
- Cement based with ARDURAPID effect.
- Hardening with full water bonding.
- For laying of natural stone, cement stone, slate and granite ceramics.
- Used for plates with varying thickness.
- Prevents discoloration and deposits.
- Water repellent and weather resistant.
Indoor and outdoor.
Area of use
For mounting natural stone such as marble, jam marble, granite, quartzite, slate, and tiles, terracotta, granite ceramics and the like.
For mounting of Calibrated natural stone made with the thin bed method with a toothpaste with a tooth size of at least 8 mm.
For laying of calibrated and uncalibrated natural stone made in the medium bed method in layer thicknesses between 5 and 20 mm.
For laying of large natural stone slabs of varying thickness with the thick bed method in layer thicknesses between 10 and 30 mm.
For flushing on walls and floors before installation of tiles, tiles and natural stone.
On concrete and masonry (cementputs) older than 3 months.
|Smooth, creamy, plastic.
|For smooth surfaces: Approx. 3,3 kg / m² powder at comb size 8x8x8 mm.
In the medium or thick bed method: Approx. 1,2 kg powder / m² and mm.
|Working time
|Approx. 1 hr at 20 ° C.
|About 20 min at 20 ° C.
|Fitting (floor)
|After about 3 hr at 20 ° C.
|Fitting (wall)
|After about 3 hr at 20 ° C.


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Mosgiel resident Evan Sadler wants access to a non-fluoridated water supply in his town.
Mr Sadler, speaking in the public forum at a Mosgiel-Taieri Community Board meeting last week, said the Dunedin City Council should have held a referendum before switching Mosgiel to town water supply from the Mount Grand treatment plant in 2017, exposing its residents to fluoride.
He requested the board ask the council to provide a water supply without fluoride for residents to collect, similar to the water available outside Speight's Brewery in central Dunedin.
He hoped the water would be made available soon, while the debate about the side effects of fluoride continues.
"There is no use us sitting in old people's homes having our diapers changed and saying we should have done this and should have done that – now is the time to do something."
Later in the meeting board chairwoman Sarah Davie-Nitis said the board would request the council investigate the feasibility of providing a water supply without fluoride in Mosgiel.


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What are widgets/today extensions?
They are extensions that show in the Notification's center Today view, and whose purpose is to display a short amount of relevant information. When we add a Today extension to our projects we aim to allow the user to obtain information in a quick manner (a single look), without forcing him to open the App, and navigate through various screens to obtain that information.
Adding a Today Widget to our project
To illustrate how to add a Today widget to an existing project I will use as a starting point the project I used some posts ago to show how to do add left swipe actions to an UITable.
In that project we were using Alamofire to fetch data (a list of todos) from a rest service and display them as a list. If we have an App that displays a list of Todos, we can improve the functionality by adding a Today Extension that lists the top most important pending todos, or the todos due today or this week.
The name Today Extension/ Today Widget is a bit misleading: we are not coerced into displaying data for "today": the information that we will consider valuable to display in the today View will vary from one application to another.
In this example, to make things easier, I will be simply displaying the same Todos that I display in the main App, limited to the 2 top ones.
Steps to add a today widget
1. Select File/New/Add Target, and select TodayExtension.
2. Give a name to your extension, and add it to your project.
XCode will show a warning offering to activate the extension scheme. Since this will allow us to debug the extension, we will agree to Activate the scheme.
4. Notice that in our project has appeared now a new folder with the name we had given to the extension (TodoExtension), and that there are already some files inside: a storyboard, a controller, and the Info.plist file.
5. We will select the storyboard, and start to design the screen for our extension. For starters, I'll change the size to work more comfortably.
6. Add a table view controller to display our todos, and set the constraints so that the table view takes the whole of the view controller.
7. Next we should select the table view, and add a protoype cell.
8. We will assign an identifier to the cell: todoTodayCell
9. Lets take a look at our TodayViewController: this is the default code created automatically when we add the extension:
class TodayViewController: UIViewController, NCWidgetProviding { override func viewDidLoad() { super.viewDidLoad() // Do any additional setup after loading the view from its nib. } override func didReceiveMemoryWarning() { super.didReceiveMemoryWarning() // Dispose of any resources that can be recreated. } func widgetPerformUpdate(completionHandler: (@escaping (NCUpdateResult) -> Void)) { // Perform any setup necessary in order to update the view // If an error is encountered, use NCUpdateResult.Failed // If there's no update required, use NCUpdateResult.NoData // If there's an update, use NCUpdateResult.NewData completionHandler(NCUpdateResult.newData) } }
The function widgetPerformUpdate it is called to update the widget, so we will put the code to fetch the Todos to display here. But before that we are going to finish to set up the Table View.
Lets create a data source:
var items : [Todo] = []
Add and IBOutlet for the Table View:
And implement the datasource, and delegate extensions:
extension TodayViewController : UITableViewDelegate, UITableViewDataSource { func tableView(_ tableView: UITableView, numberOfRowsInSection section: Int) -> Int { return items.count } func numberOfSections(in tableView: UITableView) -> Int { return 1 } func tableView(_ tableView: UITableView, cellForRowAt indexPath: IndexPath) -> UITableViewCell { let cell = tableView.dequeueReusableCell(withIdentifier: "todoTodayCell", for: indexPath) cell.textLabel?.text = items[indexPath.row].title return cell } func tableView(_ tableView: UITableView, didSelectRowAt indexPath: IndexPath) { /* So far empty, but we could implement this function so that we open the App and jump to the detail, when clicked */ } }
10. We will reuse the service we use in the main App to fetch the Todos.
TodoService.sharedInstance.getTodos().then { todos -> Void in self.items = Array(todos.prefix(2)) self.reloadTable() }.catch { error in print("Error: could not load todos") }
In this example I am using the TodoService I had defined in the main App to fetch the data, and filtering the 2 top results. In a real application I might have in my TodoService a specific function to fetch the specific data relevant to the Today window.
We need to remember also to update the Target Membership of the files from our main project that we want to use in our extension to include our TodoExtension.
All in all our TodayViewController will look like this:
class TodayViewController: UIViewController, NCWidgetProviding { var items : [Todo] = [] @IBOutlet weak var tableView: UITableView! override func viewDidLoad() { super.viewDidLoad() } override func didReceiveMemoryWarning() { super.didReceiveMemoryWarning() } func reloadTable() { self.tableView.reloadData() } func performUpdate() { TodoService.sharedInstance.getTodos().then { todos -> Void in self.items = Array(todos.prefix(2)) self.reloadTable() }.catch { error in print("Error: could not load todos") } } func widgetPerformUpdate(completionHandler: (@escaping (NCUpdateResult) -> Void)) { performUpdate() completionHandler(NCUpdateResult.newData) } }
Before deploying the extension we can set the name it will display in the TodayView by modifying the attribute Bundle display name int the Info.plist of our extension folder.
And with that we have a basic Today Extension for our todos ready to use :-).
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