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- Copyright © 2000 American Society of Plant Physiologists
Leaf metabolites, adenylates, and Rubisco activation were studied in two transgenic tobacco (Nicotiana tabacum L. cv W38) types. Plants with reduced amounts of cytochrome b/f complex (anti-b/f) have impaired electron transport and a low transthylakoid pH gradient that restrict ATP and NADPH synthesis. Plants with reduced glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) have a decreased capacity to use ATP and NADPH in carbon assimilation. The activation of the chloroplast NADP-malate dehydrogenase decreased in anti-b/f plants, indicating a low NADPH/NADP+ ratio. The whole-leaf ATP/ADP in anti-b/f plants was similar to wild type, while it increased in anti-GAPDH plants. In both plant types, the CO2 assimilation rates decreased with decreasing ribulose 1,5-bisphosphate concentrations. In anti-b/f plants, CO2 assimilation was further compromised by reduced carbamylation of Rubisco, whereas in anti-GAPDH plants the carbamylation remained high even at subsaturating ribulose 1,5-bisphosphate concentrations. We propose that the low carbamylation in anti-b/f plants is due to reduced activity of Rubisco activase. The results suggest that light modulation of activase is not directly mediated via the electron transport rate or stromal ATP/ADP, but some other manifestation of the balance between electron transport and the consumption of its products. Possibilities include the transthylakoid pH gradient and the reduction state of the acceptor side of photosystem I and/or the degree of reduction of the thioredoxin pathway.
Plants are subjected to fluctuating environmental conditions, which result in changes in the supply of light and CO2 for photosynthesis. Regulatory mechanisms exist in the chloroplasts, which match the rates of electron transport with carbon assimilation to ensure the coordination of the photosynthetic reactions. The PCR and photorespiratory cycles consume ATP and NAD(P)H produced by chloroplast electron transport and the availability of these compounds affects their functioning. Several photosynthetic enzymes are regulated by metabolites, either by their end products or intermediates occurring later in the PCR cycle (for review, see Stitt, 1996). Linear electron transport also leads to alkalization and an increase in the Mg2+concentration in the stroma (Heldt, 1979; Portis, 1981). Many PCR cycle enzymes have a pH optimum around 8.0 and require Mg2+ as a cofactor, so these light-induced ionic movements promote their activation. In addition, many chloroplast enzymes are light regulated via the ferredoxin-thioredoxin pathway (Buchanan, 1984).
The activity of Rubisco also changes in vivo. The activation of Rubisco generally increases as light intensity increases (Mächler and Nösberger, 1980; von Caemmerer and Edmondson, 1986) and deactivation is sometimes observed when stromal inorganic phosphate is depleted (Sharkey, 1990). Rubisco is active only when a specific lysyl residue within its catalytic site is carbamylated (complexed with CO2) and bound with Mg2+. This carbamylation process is considered to be enhanced by the light-dependent stromal alkalization and increase in Mg2+ concentration (Lorimer et al., 1976; Andrews and Lorimer, 1987). However, it is evident that, in vivo, the changes in carbamylation state are mediated by another stromal protein, Rubisco activase (Somerville et al., 1982; Salvucci et al., 1985).
Activase requires ATP hydrolysis to function and is inhibited by ADP, so presumably it is sensitive to the stromal ATP/ADP ratio (Robinson and Portis, 1988; Wang and Portis, 1992). This may not be the only mechanism regulating Rubisco activase. The transthylakoid proton gradient and electron transport may also be involved in mediating the response of its activity to light (Campbell and Ogren, 1990a, 1992). Additionally, the activity of Rubisco is affected by chloroplast metabolites. Phosphorylated compounds, including the substrate ribulose 1,5-bisphosphate (RuBP), can bind to uncarbamylated sites preventing activation (Portis, 1995). Rubisco activase facilitates the detachment of these compounds and thus promotes carbamylation and catalysis. The relationship between Rubisco and RuBP concentration is complicated because under some conditions, the carbamylation may be promoted by the presence of RuBP (Mate et al., 1996).
In this study we used two different transgenic tobacco (Nicotiana tabacum) types to investigate the interactions between chloroplast light reactions and carbon metabolism. Plants with reduced amounts of the chloroplast cytochrome (Cyt) b/f complex (anti-b/f plants) have a decreased electron transport capacity, which should lead to a low transthylakoid pH gradient (ΔpH) and a reduction in the ATP and NADPH synthesis (Price et al., 1995b,1998). In contrast, plants with a reduction in the activity of glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH plants) have a decreased capacity to use ATP and NADPH in carbon assimilation and have a high ΔpH (Price et al., 1995a). We studied the energy status of these contrasting plant types by measuring the whole-leaf adenylate levels. In addition, we used the activation state of chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) as an indicator of the stromal NADPH/NADP+ ratio. These measurements enabled us to evaluate how the electron transport rate, transthylakoid pH gradient, ATP/ADP ratio, and RuBP concentration interact in determining the degree of Rubisco activation and how the activity of Rubisco activase may be regulated in response to the light signal in chloroplasts.
Plant Material and Growth Conditions
The transformation of tobacco (Nicotiana tabacum L. cv W38) with an antisense construct directed against the Rieske FeS subunit of the chloroplast Cyt b/f complex (FeS) (referred to as anti-b/f plants) has been described previously (Price et al., 1995b). Antisense plants in this study were raised from selfed T3 seeds of the line B6F-2.2-513-16, producing a range of phenotypes in respect to FeS protein content and CO2 assimilation rates (Price et al., 1998). Both untransformed W38 and antisense plants were grown in 5-L pots in a growth cabinet and fertilized with Hewitt's complete nutrient solution (Hewitt and Smith, 1975) three times a week. The light intensity was 100 to 120 μmol quanta m−2s−1, the photoperiod was 20 h, and the temperature was kept constant at 25°C. The low growth irradiance and long days were used to minimize the instability of the antisense phenotype (Price et al., 1995b). The anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) plants were grown from T1 seeds of plant GAP-R (Price et al., 1995a), and had a variety of GAPDH activities. Anti-GAPDH and untransformed W38 tobacco plants were grown in an air-conditioned greenhouse where the peak photon flux density was 700 to 900 μmol m−2 s−1. The plants were used at 8 to 16 weeks after germination, and young fully expanded leaves were selected for the measurements.
Gas Exchange Measurements and Rapid Freeze-Clamping of Leaves
Leaf gas exchange was measured in a chamber attached to a rapid freeze-clamping apparatus (Badger et al., 1984) using a portable gas exchange system (LI 6400, LI-COR, Lincoln, NE) as described in Ruuska et al. (1998). CO2 assimilation was measured at an irradiance of 1,000 μmol quanta m−2s−1 at either 350 to 380 microbars (μbar) or 700 μbar CO2 in air, and the leaf temperature was kept at 25°C. For the measurements conducted at 350 μbar, the leaf was stabilized for at least 40 min before it was rapidly freeze-clamped. For the 700-μbar CO2measurements a section of the same leaf was first kept at 350 μbar CO2 in air, 1,000 μmol quanta m−2 s−1 until stomatal opening was nearly complete (about 20 min), after which the incoming CO2 partial pressure was increased to 700 μbar. The gas exchange characteristics were recorded after 30 to 40 min, and a leaf disc was freeze-clamped. Additional leaf discs were collected for chlorophyll and Cyt f measurements and GAPDH activity assays.
Enzyme Assays
For Rubisco assays, one-half of the freeze-clamped leaf disc (2.7 cm2) was homogenized in 1.4 mL of ice-cold CO2-free extraction buffer containing 50 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH, pH 7.8, 1 mm Na-EDTA, 5 mmMgCl2, 10 mm dithiothreitol (DTT), 1% (w/v) polyvinylpolypyrrolidone, and 1 mmphenylmethylsulfonyl fluoride (PMSF). The extract was centrifuged for 15 to 20 s (10,000g), and the initial activity of Rubisco was measured immediately at 25°C by adding a 10-μL aliquot of the supernatant into 262 μL of the reaction mixture containing 55 mm 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (HEPPS)-KOH, pH 8.0, 22 mmMgCl2, 0.27 mm Na-EDTA, 13 mmNaH14CO3 (specific radioactivity approximately 1,500 cpm/nmol), and 0.4 mm RuBP. Total activity was assayed after 10 μL of the supernatant was incubated at 25°C for 5 min in 55 mm HEPPS-KOH, pH 8.0, 22 mmMgCl2, 0.27 mm Na-EDTA, and 14.5 mmNaH14CO3. The assay was initiated by adding 0.4 mm RuBP. In both assays the reaction was terminated after 60 s with formic acid, samples were dried and acid-stable 14C was measured by liquid scintillation. The exact specific radioactivity of NaH14CO3 was determined for each set of assays by measuring the amount of 14C rendered acid stable by allowing a reaction containing 10 nmol of RuBP (measured spectrophotometrically according to the method of He et al. [1997]) go to completion with excess Rubisco using the procedure described for total activity.
The Rubisco catalytic site concentration was determined by the stoichiometric binding of [14C]CPBP (an unresolved mixture of 2′-carboxy-d-arabinitol-1,5-bisphosphate and 2′-carboxy-d-ribitol-1,5-bisphosphate), and the carbamylation level was measured by exchanging loosely bound [14C]CPBP at non-carbamylated sites with an excess of [12C]CPBP (Butz and Sharkey, 1989) as described in Ruuska et al. (1998). The catalytic turnover rates were calculated by dividing the initial or total in vitro activities by the number of carbamylated or total Rubisco sites.
Total GAPDH activity was determined spectrophotometrically as described in Stitt et al. (1989) and Ruuska et al. (1998).
The activation state of chloroplast NADP-MDH was assayed according to the method of Scheibe and Stitt (1988) with minor modifications. One-half of a freeze-clamped leaf disc was extracted in 900 μL of ice-cold buffer (sparged with humidified nitrogen) containing 50 mm Na-acetate, pH 6.0, 1% (w/v) bovine serum albumin (BSA), 4 mm DTT, 0.1% (w/v) Triton X-100, 0.5 mm benzamidine, 0.5 mm ε-aminocaproic acid, and 0.5 mm PMSF. The crude extract was centrifuged at 10,000g for 5 min at 4°C, and the initial activity of NADP-MDH was assayed immediately in a total volume of 1.5 mL containing 100 mm Tris-HCl, pH 8.0 (sparged with humidified nitrogen), 1 mm Na-EDTA, 1 mm DTT, 0.2 mm NADPH, and 100 μL of supernatant.
The reaction was initiated by adding 2 mm oxaloacetic acid and the decline in A 340 was monitored. Reductive activation of NADP-MDH was achieved by incubating an aliquot of the supernatant in 250 mm Tris-HCl, pH 9.0 (sparged with nitrogen), and 125 mm DTT under nitrogen atmosphere at room temperature for 15 min. Time course experiments confirmed that 15 min was sufficient to fully activate tobacco NADP-MDH. The low DTT concentration in the extraction buffer (4 mm) did not cause activation of the enzyme. NADP-MDH activities were corrected for the side activity of NAD-MDH, which was assumed to be 0.2% of the total NAD-MDH activity (Scheibe and Stitt, 1988; Grace and Logan, 1996). The total NAD-MDH activity was measured in 1.5 mL in 100 mm Tris-HCl, pH 8.0, 10 mm MgCl2, 0.2 mm NADH, and 10 μL of diluted supernatant. The reaction was initiated with 1 mm oxaloacetic acid (Scheibe and Stitt, 1988). With this procedure, activation levels below 5% were obtained from dark- or low-light-adapted tobacco.
Western-Blot Quantitation of Cyt f and Rubisco Activase Content
Anti-b/f plants have been characterized previously by measuring the amount of FeS polypeptide in leaves by western blotting. In this study an antibody directed against the Cyt f protein was used (Barkan et al., 1986) because of its higher specificity. A close correlation between the amounts of FeS and Cyt fprotein has been demonstrated (Anderson et al., 1997; Price et al., 1998), indicating that the content of the whole Cyt b/fcomplex is reduced by the antisense construct. The western blotting and protein quantitation were performed as described previously (Price et al., 1995b). The amount of Rubisco activase in leaves was measured as in Mate et al. (1993).
Metabolite and Adenylate Assays
Extraction Procedure
One-half of the rapid-kill leaf disc was quickly weighed (while frozen) and ground into powder in a mortar cooled with liquid nitrogen. Five-hundred microliters of 5% (w/v) perchloric acid containing 10 mm EDTA was added and the mixture was homogenized until melted. The crude extract was transferred to a microcentrifuge tube and heated for 5 min in a 60°C water bath to inactivate nucleotide phosphohydrolase activity not destroyed by acid alone (Ikuma and Tetley, 1976; Biotto and Siegenthaler, 1994), cooled on ice, and centrifuged at 4°C for 10 min at 10,000g. An aliquot of the supernatant was neutralized to pH 6.0 to 7.0 using 3mK2CO3 and kept on ice before KClO4 was removed by centrifugation at 10,000g for 10 min at 4°C. The sample was aliquoted, snap-frozen, and stored at −80°C.
Luminometric Determination of ATP and ADP
A sensitive luminometric procedure was chosen to enable all metabolites and adenylates to be measured with a single half-leaf disc derived from the freeze-clamping apparatus. ATP content was measured using a luminometer (TD 20/20, Turner Designs, Sunnyvale, CA) fitted with an autoinjector. The assay mixture contained reaction buffer (10 mmN-[2-hydroxy-1,1-Bis{hydroxymethyl}ethyl]glycine-KOH [Tris-KOH], pH 7.75, 8.75 mm Mg-acetate, 0.5 mm EDTA, and 20 mm KCl) and diluted sample in a total volume of 80 μL. The measurements were initiated by injecting 100 μL of appropriately diluted luciferin/luciferase reagent (ATP Bioluminescence Assay Kit CLS II, Boehringer Mannheim/Roche, Basel, or ATP assay mix, Sigma Chemicals, St. Louis) into the assay mixture, and the total light signal emitted was integrated for 5 s after a 3-s delay. Internal standardization was carried out by measuring each sample twice, once alone and once with 10 pmol of ATP added (Wulff and Doppen, 1985).
For ADP determinations, 200 μmphosphoenolpyruvate (PEP) was added to the reaction buffer. The enzymatic conversion of ADP to ATP was started by adding 0.2 unit of pyruvate kinase to each reaction mixture. After 25 min at room temperature, ATP was assayed by injecting the luciferin/luciferase reagent as described above. The internal standardization with ATP was the same as for the ATP assays.
Luminometric Determination of 3-Phosphoglycerateolate (PGA) and RuBP
We expanded the assay method of Lilley et al. (1985), which uses several glycolytic enzymes to convert PEP, 2-phosphoglycerate (2-PGA), and 3-phosphoglycerate (3-PGA) to pyruvate, coupled with stoichiometric conversion of ADP to ATP. A third step was incorporated in which Rubisco and NaHCO3 were added to the assay mixture to convert RuBP to 3-PGA. Three separate assays were performed for each sample. First, the combined background amount of ATP, PEP, and 2-PGA were assayed by including enolase and pyruvate kinase in the assay mixture. In the second step, phosphoglycerate mutase was added and the sum of ATP, PEP 2-PGA, and 3-PGA was determined. Finally, by including Rubisco and NaHCO3, the sum of RuBP, 3-PGA, ATP, PEP, and 2-PGA was measured. The actual amounts of 3-PGA and RuBP were then obtained by subtraction.
The metabolite assays were performed in the same manner as the ADP assays but with 3 μm ADP and 5 mmNaHCO3 substituted for PEP in the preliminary incubation buffer. The reaction for the measurement of [ATP +PEP + 2-PGA] was initiated by adding 0.4 unit of pyruvate kinase and 0.04 unit of enolase to the mixture. For assaying the total amount of [3-PGA + ATP + 2-PGA + PEP], 0.003 unit of phosphoglycerate mutase was also included, and when measuring [RuBP + 3-PGA + ATP + 2-PGA + PEP], 0.008 unit of tobacco Rubisco purified according to the method of Servaites (1985) was also present. After 25 min, ATP produced was measured by injecting the luciferin/luciferase reagent as described above. The samples were diluted so that <20 pmol of ATP was produced. Internal standardization with ATP was the same as for the ATP assays.
For metabolite assays, 2,3-biphosphoglycerate-independent phosphoglycerate mutase was purified from wheat germ as described byRuuska (1998) using methods based on those of Grisolia and Carreras (1975), Leadlay et al. (1977), Smith and Hass (1985), and Grana et al. (1989), with an additional step to remove adenylate kinase by adsorption to a HiTrap Blue (Pharmacia Biotech, Piscataway, NJ) affinity column.
It was noted by Lilley et al. (1985) (and also by us) that there is a background formation of ATP in this system due to ATP production from ADP by traces of adenylate kinase present in the pyruvate kinase and tobacco Rubisco preparations. The effect of this background activity was reduced by keeping the substrate ADP concentration and the amounts of the coupling enzymes as low as possible. With these precautions, the background ATP content measured in the absence of metabolite samples was 0.5 to 1 pmol per assay.
Recovery Tests
The recoveries of adenylates and metabolites were checked by adding 20 to 30 nmol of ATP, ADP, 3-PGA, and RuBP (together or separately) to perchloric acid extracts of leaf discs before assay by the above procedures. The exact concentrations of the added adenylates or metabolites were determined by spectrophotometric assays. ATP was determined with phosphoglycerate kinase-glyceraldehyde phosphate dehydrogenase and ADP with pyruvate kinase-lactate dehydrogenase (Bergmeyer and Grassl, 1985). 3-PGA and RuBP were measured as described by He et al. (1997). The recoveries (averages ± se) were: ATP, 99% ± 2% (n = 6); ADP, 92% ± 4% (n = 3); 3-PGA, 86% ± 7% (n = 4); and RuBP, 98% ± 4% (n = 5).
CO2 Assimilation Rates
Individual tobacco plants with a variety of Cyt fcontents were screened initially by measuring chlorophyll fluorescence (Price et al., 1995b). Plants with Cyt f contents ranging between 60% to 5% of the average wild-type level were obtained (Fig.1, A and C). These plants had to be grown under low light to minimize the instability of the antisense phenotype (Price et al., 1995b). However, the extended daylength ensured that CO2 assimilation rates of the growth-cabinet-grown wild-type plants were similar to the wild-type plants grown together with the anti-GAPDH plants in the greenhouse (Fig. 1, A and B). The CO2 assimilation rate decreased when the amount of Cyt f was reduced (Fig. 1A). The most severely effected anti-b/f plants had assimilation rates below 3 μmol m−2s−1, while the average rate for the wild-type plants was 12.8 μmol m−2s−1. When the CO2assimilation rates were measured at 700 μbar CO2, the relationship between leaf Cytf content and assimilation rate at high light became strikingly linear (Fig. 1C), whereas at 350 μbar CO2 the relationship was more curvilinear (Fig.1A).
Anti-GAPDH plants had a variety of GAPDH activities from about 50% to less than 5% of wild-type level and assimilation rates ranging to below 10% of the average wild-type rate when measured at 350 μbar CO2 (Fig. 1, B and D). There was no effect on the CO2 assimilation rate at 350 μbar CO2 until GAPDH activity was reduced to below 30% of wild-type level. The relationship between the assimilation rate and GAPDH activity was similar at 700 and 350 μbar CO2.
The Contents of Rubisco, 3-PGA, and RuBP
The average Rubisco contents of leaves are presented in TableI. Rubisco content was on average 25% lower in low-light-grown plants than in greenhouse-grown plants. The soluble protein content, on a leaf-area basis, was also lower in low-light-grown plants (data not shown). Consequently, the Rubisco content as a fraction of soluble proteins was similar at the two light intensities, being on average, 28% ± 2% for all low-light grown plants and 31% ± 3% for all greenhouse-grown plants. The decrease in either the Cyt b/f content or GAPDH activity did not cause significant changes in the content of Rubisco of the leaves compared with their respective control plants (Table I).
The growth conditions had a strong impact on the RuBP content of wild-type leaves. When measured from leaves kept at 1,000 μmol quanta m−2 s−1, the low-light-grown wild-type plants had approximately 50% less RuBP than greenhouse-grown wild-type plants, regardless of the measuring CO2 concentration (Table I). Because both RuBP and Rubisco contents were lower in low-light-grown wild-type plants, the ratio of RuBP to Rubisco sites was only slightly less than in the greenhouse-grown wild-type plants. The reduction in both the Cytb/f content and GAPDH activity reduced the contents of RuBP in the transgenic plants. When measured at 350 μbar CO2, the RuBP content declined to close to the Rubisco site concentration in both plant types, but not below it. The amount of 3-PGA was comparable to wild-type levels in both of the antisense plant types at 350 μbar CO2. As a consequence, the RuBP/3-PGA ratio fell significantly below wild-type levels in both transgenic plant types.
When the leaves were exposed to 700 μbar CO2, the RuBP content decreased in all plants (Table I). This was associated with an increase in 3-PGA pool sizes, so the RuBP/3-PGA ratio was lower than at 350 μbar CO2 in all plants. Despite the decrease in RuBP content at elevated CO2, the ratio between RuBP and Rubisco sites remained above 1 in most of the wild-type and anti-b/f plants. At 700 μbar CO2, however, the RuBP content in almost all anti-GAPDH plants decreased below the Rubisco site concentration, and the average RuBP per site ratio was 0.72.
Whole-Leaf Adenylate Contents
The amounts of ATP and ADP in the leaves were measured to assess changes in the adenylate pools due to the shifted balance between ATP production and consumption in the chloroplasts (Fig.2; TableII). The reduction in Cyt fcontent did not affect the whole-leaf adenylate content: both the total amounts of ATP and ADP and the ratio between the two remained similar to wild-type levels, even in the most severe antisense plants (Fig. 2, A, C, E, and G). On average, the low-light grown plants had 14.8 ± 2 μmol ATP and 10.9 ± 1 μmol ADP m−2 and the ratio of ATP/ADP was 1.43 ± 0.1. In contrast, when the activity of GAPDH was reduced below 50% of the wild-type level, the amount of ATP increased and, simultaneously, ADP content decreased. This led to an approximately 2-fold rise in the ATP/ADP ratio compared with wild-type plants (on average, 1.55 ± 0.34 in the wild type and up to 3.1 in the most severe anti-GAPDH plants), whereas the sum of ATP + ADP remained constant, being 44 ± 6 μmol m−2 in all greenhouse-grown plants (Fig. 2, B, D, F, and H).
When the CO2 concentration was increased to 700 μbar, it had little effect on the ATP/ADP ratios in the wild-type and anti-b/f tobacco plants compared with the ratios at 350 μbar (Table II). There was also no difference in the ATP/ADP ratio between the low-light-grown wild-type and anti-b/f plants at 700 μbar CO2. At elevated CO2, however, the average ATP/ADP ratio in the anti-GAPDH plants decreased and become similar to the wild-type ratio. It is worth noting that at 700 μbar CO2 the ATP/ADP ratio was higher in the anti-GAPDH plants with the lowest GAPDH activity than in wild-type plants and transgenic plants with intermediate GAPDH activity (data not shown).
NADP-MDH Activation State
The activation state of the chloroplast NADP-MDH was measured as the ratio between the initial (non-activated) activity and the total activity (after 15 min of reductive activation, see Scheibe and Jacquot [1983]) from leaf samples collected from 350 μbar CO2 and 1,000 μmol quanta m−2 s−1. The activation state decreased dramatically when the Cyt b/f content was reduced (Fig. 3A), indicating a strong decline in the NADPH/NADP+ ratio. At 1,000 μmol quanta m−2 s−1 the NADP-MDH activation in low-light-grown wild-type plants was 76% ± 4%, and decreased to 5% in the most severely affected antisense plants. The reduction in the activation level was due to a decrease in the initial activity of the enzyme (data not shown). In contrast, lowered GAPDH activities did not affect the NADP-MDH activation level compared with the wild-type plants (Fig. 3B). The average activation level in greenhouse-grown wild-type and anti-GAPDH plants was 53% (±4%) when measured at 1,000 μmol quanta m−2 s−1.
Rubisco Carbamylation State and in Vitro Activities
At high light and 350 μbar CO2, the carbamylation state of Rubisco was high (80%) in all wild-type and anti-GAPDH plants (Fig. 4B; TableIII). However, as the amount of Cytb/f content reduced, the Rubisco carbamylation status declined, being less than 50% in the most severe anti-b/fplants (Fig. 4A). In Figure 4, C to F, the catalytic turnover rates of Rubisco sites are presented as a proportion of the average rate in control plants, and the mean values are given in Table III. There was no significant difference in the turnover rate of the carbamylated sites (initial activity per carbamylated sites) in either of the transgenic plant types compared with control plants. The total turnover rates (total activity per total sites) in anti-GAPDH plants were identical to controls as well. Nevertheless, in anti-b/fplants there was a significant decrease in the total activity per total Rubisco sites as the Cyt f content declined, such that the most severe anti-b/f plants had about half the turnover rate of the control plants. Similar results were obtained when the leaves were measured at 700 μbar CO2 (Table III).
RuBP Content and Rubisco Turnover Rates in Vivo
Figure 5, A and B, illustrates the changing relationships between RuBP and Rubisco sites in both anti-b/f and anti-GAPDH plants. As summarized in Table I, the RuBP/Rubisco site ratio at 350 μbar CO2declined from wild-type-levels (between 4 and 7) down to around 1. As the RuBP content declines, the in vivo turnover rates of Rubisco decrease due to substrate limitation. Figure 5, C and D, present the in vivo turnover rates of carbamylated Rubisco sites (calculated from the gas exchange measurements and Rubisco assays) at 350 μbar CO2. The average rates were 1.18 ± 0.08 s−1 and 0.97 ± 0.06 s−1 for the low-light and greenhouse-grown wild-type plants, respectively. In the most severe transgenic plants with the lowest RuBP content, the turnover rates were more than 50% lower than in the wild types. Figure 5, E and F, shows the in vivo turnover rates of carbamylated Rubisco sites as a function of RuBP content.
CO2 Assimilation Decreases in Anti-b/fand Anti-GAPDH Plants
The decrease in Cyt b/f content caused the CO2 assimilation rate to decline (Fig. 1, A and C), as demonstrated earlier (Price et al., 1995b, 1998). At 350 μbar CO2, the relationship between the Cytb/f content and the CO2 assimilation rate was curvilinear, but as the leaves were exposed to 700 μbar CO2, the relationship became linear. This was expected, since at high CO2 and high light CO2 assimilation is limited solely by the electron transport capacity (Farquhar et al., 1980). The curvilinear relationship between the assimilation rate and the Cyt b/fcontent at 350 μbar CO2 indicates that at ambient CO2, wild-type plants are close to the transition from Rubisco carboxylation-limitation to RuBP-regeneration limitation (von Caemmerer and Farquhar, 1981). As reported previously (Price et al., 1995a), the activity of GAPDH could be reduced to about 30% of the average wild-type activity before the CO2 assimilation rates at 350 μbar CO2 were affected. The activity of GAPDH does not appear to be limiting for photosynthesis even at elevated CO2 because, again, the CO2assimilation rates decreased only in plants with less than 30% of the wild-type GAPDH activity.
As reported previously (Price et al., 1995a, 1998), the decreased CO2 assimilation rate in both anti-b/fand anti-GAPDH plants was associated with reduced RuBP concentration (Table I; Fig. 5, A and B). In anti-GAPDH plants, the reason for the lowered RuBP concentration was straightforward: the reduced amount of GAPDH forms a bottleneck in the regeneration pathway. In anti-b/f plants, however, there are two reasons. First, the electron transport rate has decreased in these plants, leading to decreased rate of ATP and NADPH synthesis. Second, there is a possibility that, in anti-b/f plants, the ferredoxin-thioredoxin-mediated activation of some of the PCR cycle enzymes has also decreased, further impairing the RuBP regeneration capacity. This assumption is supported by the large decrease in NADP-MDH activation state (Fig. 3A), which indicates a severe limitation of the NADPH synthesis and ferredoxin reduction, as discussed below.
At high CO2 and high light, CO2 assimilation in C3plants is determined by the RuBP regeneration rate. The transfer to elevated CO2 decreased the RuBP pool sizes in all plants (Table I), but the RuBP content in most anti-b/fplants and all wild-type plants remained above the Rubisco site concentration. This is similar to previous studies on the response of chloroplast metabolites to changes in CO2 showing that even at high CO2, RuBP contents tend to exceed the Rubisco site concentration (Badger et al., 1984; Dietz and Heber, 1984; von Caemmerer and Edmondson, 1986; Seemann et al., 1987). The RuBP pool sizes were only reduced below the Rubisco site concentration in the anti-GAPDH plants when exposed to 700 μbar CO2. However, it should be kept in mind that, in vivo, the conditions in the chloroplast might be such that the maximal carboxylation efficiency requires RuBP concentrations well above the Rubisco site concentrations. Assuming that the Rubisco site concentration in the chloroplasts is 2 mm and theK m for RuBP is 0.2 mm (allowing for competition by other phosphorylated compounds, but ignoring chelation by Mg2+), the approximate fractional activity of Rubisco is 0.95 when the RuBP/site ratio equals 2, and decreases to 0.75 when this ratio reaches 1 (for details, see Ruuska et al. [1998]). Reduction of the free RuBP concentration by sequestration with divalent metals will reduce activity further.
Whole-Leaf ATP/ADP Does Not Change in Anti-b/f Plants, But the Activation of NADP-MDH Decreases
The balance between chloroplast electron transport and carbon assimilation, and therefore the production and consumption of ATP and NADPH, has been altered in opposite directions in anti-b/f and anti-GAPDH plants. The energy status of the leaves was studied by measuring whole-leaf adenylate levels. In addition, the activation state of the chloroplast NADP-MDH was measured and used as an indicator of the stromal NADPH/NADP+ ratio (Scheibe and Jacquot, 1983;Harbinson et al., 1990; Foyer, 1993).
The adenylate ratios in anti-b/f plants did not differ from wild-type plants irrespective of the CO2conditions (Table II). However, the severely decreased activation state of NADP-MDH in these plants (Fig. 3A) suggests that the rate of linear electron transport, and presumably also proton translocation, were reduced markedly. Chlorophyll fluorescence measurements have shown that the transthylakoid pH gradient is low in anti-b/f plants (Price et al., 1995b). Since only the whole-leaf adenylates were measured, it is possible that the cytosolic and mitochondrial pools may have masked the changes in the chloroplast ATP/ADP ratio. In a study conducted with protoplasts of wheat leaves, it was found that 47% of the total adenylates were located in the chloroplasts; 44% were located in the cytosol and 9% in the mitochondria (Stitt et al., 1982). The ATP/ADP ratio in the cytosol is higher than that in the chloroplasts, whether measured in the light or the dark, because ATP is transported there from chloroplasts and mitochondria (Stitt et al., 1982; Gardeström and Wigge, 1988; Heineke et al., 1991).
The observed reduction in the activation level of NADP-MDH in anti-b/f plants (Fig. 3A) resembles the results of Harbinson et al. (1990) and Krall et al. (1995). These authors measured NADP-MDH activation in leaves at different light intensities and noted a strong correlation between light intensity, electron transport rate, and NADP-MDH activation. The low activation state of NADP-MDH in anti-b/f plants indicates that the reduction state of the stromal NADP+ pool is significantly lowered. Decreased NADPH/NADP+ ratio has two main implications for carbon fixation. First, the lack of reducing equivalents limits the function of the PCR cycle and RuBP regeneration. Second, the low stromal reduction status reduces the activity of the chloroplast ferredoxin-thioredoxin pathway, and therefore the enzymes regulated by this system (for example Fru 1,6-bisphosphatase) are likely to be less active (Holfgrefe et al., 1997). The decreased activity of the PCR cycle enzymes may explain why the ATP/ADP ratio did not change in anti-b/f plants. The consumption of ATP by the PCR cycle may be severely restricted, thus compensating for the decreased rate of ATP synthesis. The same mechanism may also explain why light intensity generally has little effect on leaf adenylate levels (Dietz and Heber, 1984; Brooks et al., 1988a; Sage et al., 1990).
The activation level of NADP-MDH in the most severe anti-b/fplants at high light (Fig. 3A) resembles that seen in wild-type plants in darkness or very low light (data not shown), suggesting thatb/f complex depletion impairs perception of the light signal by the thioredoxin pathway. When leaves were exposed to 1,000 μmol quanta m−2 s−1, NADP-MDH in the low-light-grown wild-type plants activated to over 80%. This activation level is high compared with, for example, 35% in pea leaves measured in air and 1,000 μmol quanta m−2s−1 (Harbinson et al., 1990) or 53% in the greenhouse-grown wild-type and anti-GAPDH plants (Fig. 3B), which experienced growth light intensities close to measuring conditions. Greenhouse-grown plants had about 1.5 times greater total NADP-MDH activities (on an area basis) than low-light-grown plants (legend to Fig. 3), and this greater capacity may explain the different activation states observed in wild-type plants.
ATP/ADP Increases in Anti-GAPDH Plants But Not the Activation of NADP-MDH
The increase in the whole-leaf ATP/ADP ratio in anti-GAPDH plants at 350 μbar CO2 (Fig. 2H) is comparable to the observations of other transgenic tobacco plants with impaired capacity for carbon assimilation, due to either the reduced amount of Rubisco (Quick et al., 1991) or of phosphoribulokinase (Paul et al., 1996). This effect of decreased carbon fixation capacity on the adenylate pools is analogous to the increase in ATP/ADP ratio when photosynthesis is limited by low CO2 (Gardeström, 1987;Gilmore and Björkman, 1994), since the majority of ATP synthesized in chloroplasts is used in carbon metabolism. This was clearly the case in the anti-GAPDH plants, since increasing the CO2 concentration caused the average ATP/ADP ratio to decline to close to wild-type levels (Table II).
Interestingly, the reduction state of the stromal NADP+ pool at 350 μbar CO2 (estimated from the NADP-MDH activation level, Fig. 3B) in anti-GAPDH plants was similar to the wild-type plants. It might be expected to rise in the antisense plants with decreased capacity to use reducing equivalents in carbon fixation. These results are comparable to data obtained by Lauerer et al. (1993), who measured the activation state of NADP-MDH at growth irradiance in plants with reduced amounts of Rubisco. The maintenance of a wild-type-like NADPH/NADP+ ratio in anti-GAPDH plants indicates that PSII efficiency is well regulated in these plants, as had previously been demonstrated with measurements of chlorophyll fluorescence (Price et al., 1995a). Transport of reducing equivalents from chloroplasts to cytosol via the "malate shuttle" (Scheibe, 1987) may contribute to this regulation.
Reduction in the Electron Transport Capacity Affects Rubisco in Anti-b/f Plants
The Rubisco carbamylation state in all wild-type and anti-GAPDH plants was on an average 80% at 350 μbar CO2and high light (Fig. 4, A and B; Table II), which is typical for tobacco (Mate et al., 1993, 1996; Price et al., 1995a). However, the carbamylation level of Rubisco decreased when the amount of Cytb/f was reduced (Fig. 4A), as previously reported (Price et al., 1998). The reason for the low carbamylation state could be that conditions in the chloroplasts are such that they directly inhibit the carbamylation process, or the decreased carbamylation could be mediated by Rubisco activase. It is possible that the stromal pH is lower in anti-b/f plants due to restricted electron transport and reduced transthylakoid proton gradient. The low pH can impair the carbamylation process, perhaps in concert with a reduction in the concentration of free Mg2+ in the stroma, as discussed previously (Price et al., 1998). However, the stromal buffering capacity is quite high (estimated as 30 mm), which should prevent large changes in pH (Hauser et al., 1995a, 1995b).
Alternatively, the decrease in carbamylation of Rubisco in anti-b/f plants is somewhat similar to the effect of reduced Rubisco activase content (Mate et al., 1993, 1996). However, the amount of Rubisco activase was the same in wild-type and anti-b/fplants (data not shown). This raises the interesting possibility that the activity of activase may be reduced in anti-b/f plants. The restriction in electron transport could decrease the stromal ATP/ADP ratio, which could inhibit the activase function. The decrease in carbamylation in anti-b/f plants would then be analogous to the suggested mechanism for Rubisco deactivation observed in phosphorus-depleted plants, in which ATP synthesis is limited by a lack of inorganic phosphate (Brooks et al., 1988b; Sharkey, 1990).
However, as discussed above, the ATP/ADP ratio in the anti-b/f plants was similar to the wild-type plants (TableII). There are several studies suggesting that, although Rubisco activase needs ATP to function, changes in the stromal ATP/ADP ratio may not be the only means by which activase activity and Rubisco carbamylation are modulated. Brooks et al. (1988a) suggested that the minimal changes in the stromal ATP/ADP ratio that occur when irradiance is varied are insufficient to account for the observed changes in Rubisco activation. Studies by Campbell and Ogren (1990a, 1990b, 1992) suggested that activase senses the light intensity via the electron transport rate through PSI rather than through stromal adenylates, and that the presence of a ΔpH is also required. Our results agree with this notion: the decrease in Rubisco carbamylation in anti-b/f plants can be interpreted as a consequence of activase being unable to detect the light signal.
The low carbamylation in anti-b/f plants was remarkably similar to the decrease in carbamylation in wild-type tobacco leaves when the light intensity was reduced (data not shown): in both cases the carbamylation decreased from a maximum of 80% down to 40%. In anti-GAPDH plants, on the other hand, in which the photosynthetic electron transport rate was also reduced, the carbamylation state remained high (Fig. 4, Price et al., 1995a). These plants have a high ΔpH (Price et al., 1995a), a high ATP/ADP ratio (Table II), and the degree of reduction of pyridine nucleotides remained similar to that of wild-type plants (Fig. 3).
Our results indicate that the light regulation of Rubisco activase in vivo is not mediated by the stromal ATP/ADP ratio or the electron transport rate per se, but rather by some manifestation of the balance between electron transport rate and the consumption of its products. Possibilities include the ΔpH or the degree of reduction of the acceptor side of PSI. The mechanism by which activase activity could respond to these factors is unclear. A recent report of regulation of activase activity via the thioredoxin pathway (Zhang and Portis, 1999) would provide a ready explanation for our observations. However, this regulation is a property only of the longer isoform of activase produced by alternate splicing in some species. Tobacco lacks this isoform and does not show this kind of regulation in vitro (Zhang and Portis, 1999). Nevertheless, our data indicate that the activity of activase in tobacco may also be regulated by the thioredoxin system by an as-yet-unknown mechanism.
Role of RuBP
RuBP modulates Rubisco activity in vivo. It binds tightly to uncarbamylated active sites, blocking the carbamylation process, and removing RuBP from these sites is a primary function of activase. On the other hand, it has also been suggested that Rubisco carbamylation is directly modulated by RuBP (Mate et al., 1996). There are observations showing that a low steady-state RuBP concentration may cause Rubisco to decarbamylate, such that the rate of RuBP consumption is matched with its regeneration rate (Mott et al., 1984; Sage, 1990). In our study, as well as the previous one (Price et al., 1995a), Rubisco carbamylation remained high in anti-GAPDH plants despite low RuBP concentrations. Even at 700 μbar CO2, when the RuBP pools in anti-GAPDH plants decreased below the Rubisco site concentration (Table I), the Rubisco carbamylation state remained high (Table II).
A recent in vitro study demonstrated that subsaturating CO2 combined with subsaturating RuBP causes Rubisco to decarbamylate, but Rubisco activase can prevent this decarbamylation (Portis et al., 1995). It is therefore possible that the maintenance of the high Rubisco carbamylation state in the anti-GAPDH plants was due to a high activity of Rubisco activase counteracting the tendency of Rubisco to decarbamylate when RuBP is scarce. This is supported by the high concentration of ATP, the elevated ATP/ADP ratio (Table II), and the high ΔpH (Price et al., 1995a), all of which are thought to promote the functioning of Rubisco activase (Portis, 1992). Alternatively, the high carbamylation state maintained in anti-GAPDH plants may also indicate that, in the absence of RuBP, binding of other chloroplast metabolites such as 3-PGA to Rubisco sites can prevent decarbamylation (Badger and Lorimer, 1981).
In some cases the manipulation of chloroplast metabolism has actually increased the activation of Rubisco. Elevated Rubisco activation levels (measured as the ratio between initial and total activities) have been observed in tobacco plants with reduced amounts of Rubisco (Quick et al., 1991) and phosphoribulokinase (Paul et al., 1996). However, those measurements were made at lower irradiances. The anti-phosphoribulosekinase plants are comparable to the anti-GAPDH plants, since both have high ATP/ADP ratios and reduced RuBP contents. The activation state of Rubisco in anti-GAPDH plants was not higher than in control plants, whether measured as carbamylation state (Fig.4B) or as a ratio between initial and total activity of Rubisco (data not shown). This can probably be explained by the high light intensity used in our measurements. It may be that the 80% carbamylation state routinely measured in tobacco leaves at high light is the upper limit for carbamylation.
In Vitro Activity
Phosphorylated sugars can bind tightly either to uncarbamylated Rubisco sites, preventing activation, or to carbamylated sites, inhibiting catalysis. In vivo, Rubisco activase facilitates the dissociation of these compounds from active sites. The presence of tightly binding inhibitors in Rubisco sites can be detected as a decrease in the catalytic rate measured under substrate saturation (Seemann et al., 1985; Keys et al., 1995; Parry et al., 1997). We measured the carbamylated and total Rubisco site concentrations, as well as initial and total activities, and calculated the catalytic turnover rates of Rubisco sites. The turnover rates of the carbamylated sites in both anti-b/f and anti-GAPDH plants were comparable to control values (Fig. 4, C and D), indicating that the carbamylated sites were free from inhibitory ligands after extraction. However, the total activity per total sites in the anti-b/f plants declined as the Cyt b/f content decreased (Fig. 4E). The reason for this could be that the uncarbamylated sites had tightly bound inhibitory compounds, which did not dissociate during the 5-min activating incubation. Nevertheless, during the carbamylation assays these inhibitory ligands were displaced by [14C]CPBP, which has very high affinity to Rubisco sites.
It is possible that the presence of inhibitors in Rubisco sites in anti-b/f plants is another consequence of the decreased activity of Rubisco activase, in addition to the lowered carbamylation state. Studies with anti-activase tobacco plants discovered a slight impairment in the in vitro turnover rates of both carbamylated and uncarbamylated Rubisco sites (He et al., 1997). A tight-binding inhibitor of Rubisco has recently been found in tobacco leaves in the light (Parry et al., 1997). However, endogenous inhibitors are not the only factors that can decrease Rubisco activities measured in vitro. Rubisco extracted from leaves may be partially degraded by endogenous proteases (Servaites, 1985) or may be inhibited by polyphenolics (Bahr et al., 1981) and polyphenol oxidase (Koivuniemi et al., 1980), all of which are abundant in tobacco leaves. It is therefore possible that the decrease in the total catalytic activity of Rubisco in anti-b/f plants is the result of direct inactivation. The difference in catalytic turnover rates between greenhouse-grown and growth-cabinet-grown wild-type plants may be attributable to the same phenomena (Table III).
In Vivo Catalysis
Studies of transgenic tobacco plants with severely reduced amounts of Rubisco activase show that not only carbamylation, but also in vivo turnover rates of carbamylated Rubisco sites, are impaired by the lack of activase (He et al., 1997). As both RuBP content and RuBP/3-PGA ratios are high in anti-activase plants, the low Rubisco turnover rate can be deduced as resulting from the lack of activase. Since the RuBP concentrations and the RuBP/3-PGA ratio are lowered in both anti-GAPDH and anti-b/f plants, we cannot judge if there is any additional decrease in the catalysis of Rubisco in the anti-b/f plants, other than that which would be expected from the substrate limitation by RuBP (Fig. 5).
We thank Prof. Ross McC. Lilley from the University of Wollongong for the advice with the luminometric assays, Dr. D. Büssis for advice on the GAPDH assays, and Dr. J.R. Evans for helpful discussions.
↵1 This work was supported in part by an Australian National University Doctoral Scholarship, an Overseas Postgraduate Research Scholarship, and a grant from the Academy of Finland to S.A.R.
↵2 Present address: Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824.
↵* Corresponding author; e-mail susanne{at}rsbs.anu.edu.au; fax 61–2–6249–5075.
- Received July 2, 1999.
- Accepted October 12, 1999.


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Barça is now in the Copa del Rey Final. And they've achieved it in a brilliant way, scoring 21 goals and only conceding 8. Review with us all the goals that the team has scored in the 2016/2017 Copa del Rey. Enjoy them!
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subset: commoncrawl


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Let's make March a month of action for REAP!
25 Feb 2014 — 02:09 PM
March is the month when important budget decisions are made at the Statehouse, and so it is a very important time for all of us who support REAP to share our enthusiasm for the program with friends, colleagues, and our elected officials.
Remember, one of the most important opportunities we have to speak out for REAP in March is to come to the statehouse in person on March 18 for REAP Day. Details about the event are available here and you can commit to attend online right now.
To make it easier to spread the word, check out these resources you can share with friends online:
Suggested Facebook posts:
- I'll be at the statehouse March 18 to speak out for clean water and healthy soil in Iowa. Won't you join me? Sign up at http://envirolobbyday.eventbrite.com
- Iowa's REAP Program has provided over $300 million in benefits for Iowans with 25 years of conservation projects in all 99 counties--let's keep this progress going! Join me at the state capitol March 18! Sign up at http://envirolobbyday.eventbrite.com
- Learn about how Iowa's REAP Program benefits all 99 Counties at www.REAPALLIANCE.org.
Suggested Tweets:
Join me at the state capitol to speak out for conservation action on Mar. 18! http://envirolobbyday.eventbrite.com #IowaREAP #EnviroLobbyDay
Hey #IALegis! Let's celebrate 25 years of REAP with $25 million this year! #IowaREAP #EnviroLobbyDay
25 years ago: #IowaREAP bill passes House with 100 votes--the desire to protect our environment is unanimous. #EnviroLobbyDay
- Thank #IowaREAP for over $300 million invested in Iowa's natural beauty! Attend #EnviroLobbyDay 2014.
#IowaREAP: benefiting 99 counties and 100% of Iowans since 1989. #EnviroLobbyDay
37,000 REAP plates on the road shows that Iowans care! Continue supporting REAP at #EnviroLobbyDay 2014.
#IowaREAP's funding formula has stood test of time, supporting a diversity of projects to all parts of the state
Photos to share:
You can share these photos on Facebook, Twitter, and Instagram to let others know why conservation funding matters to you!


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Lamborghini is doing whatever it can to keep its naturally aspirated V10 and V12 engines, even when its rivals are moving to smaller turbocharged engines. Instead of downsizing its engines, Lamborghini has confirmed that it will electrify its V10 and V12 engines.
Speaking to Autocar, Lamborghini's technical director, Maurizio Reggiani confirmed that Lamborghini will use hybrid engines for its next sports cars. "We will continue to choose natural aspiration for the super-sports cars. In the future, we will need to take account of fuel consumption and emissions. I am convinced the naturally aspirated engine coupled with a hybrid system can be the right answer," Reggiani stated.
The next Aventador is expected to get a hybrid powertrain, while the replacement for the Huracán could get a 10-cylinder plug-in hybrid powertrain.
Talking about the next Aventador, Reggiani stated, "We need to reinvent this icon without [losing] the characteristics of the current car: carbon fiber, the V12 naturally aspirated engine and other components. Looking forward, if it is a hybrid then in what ways can we compensate for its weight?"
To help power the new hybrid powertrains, Lamborghini is currently working with the Massachusetts Institute of Technology (MIT) to develop carbon fiber bodywork that can store energy.
Source: Autocar


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Media spreads fake news: No one was barred from AFP HQ
By Priam Nepomuceno
AFP spokesperson Col. Edgard Arevalo said there are no orders to bar the entry of members of the Defense Press Corps (DPC).
He, however, requested DPC members to present theirs IDs when asked by military personnel guarding the gates.
"For the information of DPC members, please present your DPC ID to the gate when demanded. There is no instruction to bar entry of DPC members. Others will clear with Col. (Noel) Detoyato (head of ) the Chief AFP Public Affairs Office," he added.
Arevalo also denied reports that the AFP scheduled a press conference Thursday night.
"Also, there is no (scheduled) press briefing. You will hear it from me (in case there is one), Col. Toods (Detoyato) or Director (Arsenio) Andolong (Defense spokesperson)," he added. (PNA)
Media spreads fake news: No one was barred from AFP HQ Reviewed by AsianPolicy.Press on 4:12:00 AM Rating:


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You know what bugs me on TV cop shows – other than when two very obviously gay characters aren't gay together? When a single woman on a TV cop show comes home alone in the dark and we know even before we know her name that she is doomed to a terrible death by someone lying in wait. It's sort of the oldest trick in the book when it comes to crime procedurals. For extra special been there, seen that points, there's usually a scene of the doomed woman looking at herself happily in a mirror right before the killer strikes. And, alas, Rizzoli & Isles chose to start its summer finale with exactly this tired old trope.
It's morning and a non-single (but unwilling to admit it publicly) Jane Rizzoli barges into Maura Isles' townhouse. Not a knock, not a slowly open to check if the coast is clear – because even though she stubbornly refuses to give up her own apartment, this is clearly Jane's home. Though, come to think of it, how long has it been since we've seen Jane's place anyway? I'm pretty sure she's just renting it out on Airbnb at this point, like I suggested.
Jane tells Maura to hop to it because aforementioned doomed girl is indeed dead. Maura is perplexed she didn't get to do their trademark tandem "Rizzoli" and "Isles" phone answering routine. Her cell was on silent. Then Jane notices a second coffee mug and assumes it is for her. But alas it's for Prof. Beard, who is keeping up appearances by pretending to have lost his pants.
Oh, Jane. This is the price you pay for continuing this charade. Sometimes you have to see the beard in a kimono before your first cup of morning coffee. Jane's face says pretty much all we need to know about her feelings on this situation. Maura scolds Jane for being "a little terse" with Prof. Beard. She replies that, "Men in kimonos make me uncomfortable." Oh, Janie – men in general make you uncomfortable. Maura corrects her as such, but in a secret girlfriend code which sounds to the outsider like she is correcting Jane's grammatical use of the word "kimono."
Jane and Maura arrive at the doomed woman's place, when Jane realizes the beard mentioned something about an "Allie." She inquires because Maura's business is her business, especially any business involving a dude in a kimono. Allie is Jack's seventh-grade daughter, who is going to shadow Maura for a school project.
This worried Jane because the girl might get bored. Yes, yes – we can all read between the lines. "Bored" means "major boundary issues." Since when are they meeting the offspring of beards? Maura says she'll just "vamp" if things get boring/boundary crossing by talking about things that interested her at that age. You know, favorite childhood books and/or the anti-microbial properties of common foods.
Jane gives her girlfriend the, "I love you, you incredible nerd" face and hopes Maura goes the Anne of Green Gables route instead of the broccoli route. Also, all of a sudden I'm envisioning little Jane Rizzoli reading all about Anne Shirley and it is filling my heart with so much joy.
Jane surveys the victim's apartment and knows immediately the woman is in her twenties. It was the "Starry Night" poster that tipped her off. She complains about the law that says all first apartments must be decorated with the same five posters. And then she bets Maura $20 that the accompanying poster in the bedroom will be Marilyn Monroe on the subway grate. Maura takes the bet because taste is an individual's preference based on their specific cultural experience.
Bet made, they storm past Korsak to see who wins. Korsak is always in the middle of dead body flirting. But, unlike Senior Criminalist Susie Chang, he knows better than to interrupt.
Because the two rules of life are never start a land war in Asia and never go against a Rizzoli when $20 is at stake, Jane wins the bet. There's Ms. Monroe in all her white dress glory. Jane demands Maura, "Pay up, baby!" But Maura replies, with a point of her gloved-finger, "Maybe after." Flirting over dead bodies is one thing, exchanging sexual favors over them is entirely another thing.


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WASHINGTON, DC – Another big day for democracy as members of the 113th Congress took their oaths of office in the House and Senate.
House Republicans re-elected Speaker John Boehner over former speaker Nancy Peolosi.
Randy Weber of Alvin and Steve Stockman of Webster are among the eight new House members from Texas. Weber is a former state representative, and Stockman is back in the House after serving one term from 1994 through 1996.
Over in the Senate, Vice President Biden took his place as the presiding officer, then swore in the new and returning senators, including Texans John Cornyn and Ted Cruz.
The folks in Houston we talked with think members of Congress need to grow up and start doing their job.
Carla Garza says members of Congress need to listen. 'Congress really needs to pay attention to the citizens that they represent. They'll find they'll be without jobs.'
Jim McDermott believes they need to compromise. 'Each side has their own agenda. So I think there should be more middle of the road type. But that's not going to be easier.'
And Tina Pilgram thinks they should start playing nicely. 'I think they all just need to get along and play nicely together.'
Before all of the swearing occurred, members of congress did some praying at the traditional prayer service at St. Peter's Catholic Church. They and the nation will need all the help we can get as the new congress tries to get a handle on the debt ceiling and spending cuts.
One of the first items on the agenda will be the massive aid package for victims of Super Storm Sandy, which Speaker Boehner refused to bring to a vote earlier this week.
He better bring it up, or else he may find out what happens when you screw with the folks in Jersey.


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"Into The Parabnormal" is a three-hour weekly radio program hosted by Jeremy Scott discussing topics that are not in the mainstream… as we like to say, somewhere between abnormal and paranormal!
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Hello All
Been reading the forum for a while, and found some really great topics, thanks to all.
I wonder if anyone might be able to tell me how I can navigate the service menu on my Samsung UE40H6400
I can enter the menu using Mute + 1 + 1 + 9 + Enter
But cannot scroll down the options. The only thing I can do is to change the Hospitality Mode to On or Off using the arrow right key then the arrow up. To exit this I press return key.
But I cannot scroll down to the other options of Power On, menu OSD, Music Mode etc
I read someone used the channel up/down keys then made alteration usinf volume up/down
This does not work for me. I have pressed every button on the IR and Wand remotes.
I wonder if I am in the wrong service menu, and this menu needs a different remote?
Appreciate any help on this. All I wanted to check was if this TV does have BlueTooth speaker capabilities as I wold like to fit a sound bar as the internal speakers are very poor. I know I could go with Optical Out, but BlueTooth bars are cheap these days.
Many thanks
Samsung UE40H6400 - Service Menu Navigation
Here for general support for H series TVs, request and problem solve area.
Post Reply
3 posts • Page 1 of 1
Re: Samsung UE40H6400 - Service Menu Navigation
Well, yesterday I've rooted my UE50H6470 (6400) and entered the Service Menu with pressing following combination of buttons without any problems:
Then the TV starts and after a short while the Service Menu is on Screen
Then the TV starts and after a short while the Service Menu is on Screen
My TV's: UE50H6470 and UE75H6470
Re: Samsung UE40H6400 - Service Menu Navigation
Thank you very much for the reply. I tried what you said and got into the menu, found Bluetooth Headphones, enabled it, and now it appears in the main Audio menu. I imagine now I select this option to be able to connect to a Blue Tooth Sound Bar. Really do appreciate your time and help.
Post Reply
3 posts • Page 1 of 1


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The Tueller Drill:
Sergeant Dennis Tueller, of the Salt Lake City, Utah Police Department wondered how quickly an attacker with a knife could cover 21 feet (6.4 m), so he timed volunteers as they raced to stab the target. He determined that it could be done in 1.5 seconds.
Saturday in Seattle.
You sitting, opponent standing. S/he moves to your side. You turn so that the knee nearest opponent is on the mat and your far knee up. Near palm on the mat and far arm in high warding position. Brace against opponent's collarbone and technical lift to get to feet.
Opponent sitting, you standing. You go to hir side and wrap your FAR arm under hir legs as if to pass. S/he pushed your head to the mat. You hop your feet over hir to the other side.
From closed guard: Do a basic guard break and underhook a thigh as if to stack and pass. Then hop to the other side instead. Opponent starts to roll away from you. Get a sash grip (make sure you are UNDER hir ceilingward arm) and stick your hook in under hir hip as she tries to gain hir knees. Take the back. At this point, get a choking lapel grip.
After we had drilled this for a while, we had the backmounted person free a hook and try to crabwalk out. Attacker keeps the sash grip and rolls belly down sprawling. A choke can often be accomplished from here. if it is not happening, loosen up enough to bail opponent to trying to escape, and retake the back.
Rolls with a handful of people I had never met before. Taught an upa to a white belt. Got two straight elbow locks on a blue belt- the same elbow lock I got on Sean yesterday. That is awesome. But I need to work on remembering to stay out of top half guard on people who are good at retaining half guard and/or doing useful things from bottom half guard. That blue belt had a gnarly half guard retention, and I actually commented on it, yet still found myself sliding right back into it repeatedly. I feel so good about my top half guard passing that I tend to just go into it and proceed to pass as a routine part of my game. It usually works, but I need to take note when I draw someone who is not letting me get away with that, and try OTHER things.


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Does the Visual Attention Span Play a Role in Reading in Arabic?
Lallier, Marie
Abu Mallouh, Reem
Mohammed, Ahmed M.
Khalifa, Batoul
Perea, Manuel
Carreiras, Manuel
MetadataShow full item record
Marie Lallier, Reem Abu Mallouh, Ahmed M. Mohammed, Batoul Khalifa, Manuel Perea & Manuel Carreiras (2018) Does the Visual Attention Span Play a Role in Reading in Arabic?, Scientific Studies of Reading, 22:2, 181-190, DOI: 10.1080/10888438.2017.1421958
It is unclear whether the association between the visual attention (VA) span and reading differs across languages. Here we studied this relationship in Arabic, where the use of specific reading strategies depends on the amount of diacritics on words: reading vowelized and nonvowelized Arabic scripts favor sublexical and lexical strategies, respectively. We hypothesized that the size of the VA span and its association to reading would differ depending on individual "script preferences." We compared children who were more proficient in reading fully vowelized Arabic than nonvowelized Arabic (VOW) to children for whom the opposite was true (NOVOW). NOVOW children showed a crowding effect in the VA span task, whereas VOW children did not. Moreover, the crowding in the VA span task correlated with the reading performance in the NOVOW group only. These results are discussed in light of individual differences on the use of reading strategies in Arabic.


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If you would like to submit photos of class parties/reunions, graduations, etc. please e-mail them to email@example.com. Be sure to include the names of ALL persons in the photos, if you do not include names for everyone in the photo the photo will NOT be accepted. If any person in your photo has opted to NOT allow their photo to be used on the website, the photo(s) will not be approved. Inappropriate photos will NOT be accepted…..please keep them clean and family friendly.
There are currently no galleries!


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Team Profile:
Founded in October 2013, the team is represented by the best Indonesian Dota players who are already well known in the international gaming community. Rex Regum has grown to become one of the most respected Dota 2 teams in Southeast Asia. The team's mission is to become the best Dota team in the world by winning "The International", the largest Dota 2 tournament in the world, Which is held in Seattle, USA. Rex Regum believes that the key to their success is by combining the skills of the experienced players with those of up and coming young players.m.
Team achievement:
2016 South East Asia (SEA):1st Place Division 2 JDL League Season #10 (Moving to Division 1)
1st Place BTS SEA Series #4 Open Qualifier
5th-8th AGES Malaysia
National:1st Place ICE Dota 2 Tournament
1st Place Theprime Dota 2 Online Tournament 2016
1st Place Informatics Festival Dota 2 LAN Tournament
1st Place Lenovo Gaming League Jakarta Qualifier
2nd Place Lenovo Gaming League
1st Place ASEAN Games for E-Sports 2016 Indonesia Qualifier
1st Place Acer Predator Dota 2 Online Tournament Series 2
1st Place AES Summer Series Season 1
1st Place Fearless Dota 2 Online Tournament
2015:3rd Place Master Gaming Series 2015
3rd Place Dell Dota 2 Tournament
1st Place SEAC Dota 2 Tournament (SEA)
2nd Place IESL 2015 Dota 2 Tournament Jakarta
1st Place World of Gaming Dota 2 Tournament 2015
3rd Place FJB-E.G. Dota 2 Tournament Season 3
1st Place BINGO Binus Dota 2 Tournament 2015
1st Place S-Prime Dota 2 Tournament 2015
7th-8th Place MPGL Season 7 (SEA)
1st Place Informatics Celebration Dota 2 Tournament 2015
1st Place MPGL Season 7 Indonesia Qualifier
3rd Place GGS Dota 2 Tournament 2015
2nd Place GEST 2015 Indonesia Qualifier
3rd Place Live Gaming Dota 2 Tournament 2015
1st Place Eternal League Dota 2 Tournament Season 1
1st Place FJB-E.G. Dota 2 Tournament Season 1


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Olive Serviced Apartments is renowned in providing service apartments in Delhi Area and committed to giving 100% customer satisfaction. We are an award-winning hospitality agency offering serviced apartments at all prime locations of Delhi NCR. Each of our service apartments in Delhi NCR and Gurgaon has won prizes: The provider of apartments with the most promising services (2013-14) and Best Guest Houses (2015-16), as well as recognized from Booking.com and TripAdvisor as a leader of the industry.
Each apartment is aimed at all those discerning travelers seeking privacy, personalized service and a safe haven in the region of the national capital. All the service apartments for rent in Delhi are focused on quality services, beautifully decorated and furnished, represent luxury and modern comforts to make your stay productive and peaceful.
Olives offer vacation rental homes and holiday homes in Delhi across multiple locations such as Defence Colony, Green Park, Saket, & Greater Kailash, South Delhi, City-Centre Delhi, and Panchsheel Enclave. We believe to offer best facilities like fully furnished spacious apartments, 1/2/3/4 BHK for the short-term & long-term stay with our luxuries amenities friendly staff, homely environment, safe housekeeping, 24*7 security, and power backup serviced apartments in Delhi.
Our Serviced Apartments in Delhi are ideal for Tourists, Corporates, and Families traveling together. We have different types of serviced apartments available: Classic rooms for single budget travelers, Double occupancy Winsome rooms for couples and 1 BHK Apartment and Executive suites for the luxury-seeking.
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If you are looking for best-serviced apartments in Delhi, feel free to call @+91-7290029000 or Email us at - firstname.lastname@example.com.
Copyright © 2020 Olive Serviced Apartments. All rights reserved.


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Plant-based company leCupboard plans to install the first of 30 food vending machines in strategic locations throughout San Francisco over the next three months. The goal is that these interactive machines will help people fight disease with food.
"The goal is to reconnect preventative healthcare with food," said founder Lamiaâ Bounahmidi. "Everyone is going around talking about food as medicine, but there is more to it than going organic or eating vegetables."
The vending machines will be stocked with options such as chia pudding breakfast jars, lentil salads with cashew cheese and pumpkin seeds, and zucchini noodles with walnut pesto, for approximately $8 per meal.
Bounahmidi hopes to spread plant-based vending machines across the country with 200 machines nationwide in the next year.


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Sophie Turner is certainly celebrating her career high! With Game of Thrones and Dark Pheonix in her kitty, she's more than just a pretty face in the industry now.
Taking some time out, the actress turned muse for Vogue China's recent issue and we can't deal with this beaut!
Recommended Read: Irina Shayk, Gigi Hadid, Bella Hadid strut it out for Versace's S/S 2020 menswear showcase and we're digging this surge of gender fluidity!
Styled by Alex White, this diva stunned in Louis Vuitton ensembles on the cover.
Straight hair, a spiced pout and sunkissed cheeks complimented her look.
Catch some more looks from the cover shoot here:
(SOURCE: Instagram)


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7 Great Networking Events in Indianapolis for Every Stage of Your Career
Looking to expand your professional network in Indianapolis? Check out these seven networking events to connect with others in your industry, meet new colleagues — or maybe even your next boss.
Attending a bunch of networking events can seem daunting or even like a waste of time if you're not in sales. But when you do it right, networking can be a great way to develop your career, expand your network of connections and learn more about your industry and the local economy.
The trick is to find groups that have goals you can connect with and members you can relate to. Whether you're an experienced networker or a novice, there is an Indianapolis networking group to help boost you your career — and several that will improve your social life, too. (Click here to tweet this list.)
1. Network After Work Indianapolis
There's no doubt about it — Network After Work puts on some of the largest, best-attended networking events in the city, regularly attracting over 100 professionals. With a crowd that size, the get-togethers are more of a "mixer" than a "meeting," but color-coded name tags help attendees identify other participants by industry for easier introductions. As attendee Harlow Stevens puts it, the events have "Great turnout, [with] lots of interesting people there."
If you're looking for casual connections in a convivial atmosphere, Network After Work is the place to go. Admission is $12 in advance, $15 at the door with pre-registration or $20 at the door without pre-registration. The event lasts from 6-9 p.m. with light appetizers and a free cocktail from 6-7 p.m.
2. Young Professionals of Central Indiana
Networking is about much more than just making a sale. Young Professionals of Central Indiana is an organization devoted to helping the next generation of professionals develop all aspects of their lives, offering leadership and mentoring opportunities to community engagement and career networking events.
In addition to typical educational and business networking events, YPCI offers the opportunity to connect with other members through golf outings, casino nights and group-wide parties. The cost of membership in Young Professionals of Central Indiana is only $50 annually, and members receive discounted or free attendance to meetings and programs hosted by the group.
3. Network of Women in Business
For more than 35 years, the Network of Women in Business has provided opportunities for professional women in Indianapolis to develop their careers and expand their networks. NOWIB offers several meetings each month, including Southside, Northside, and Downtown locations, as well as Quarterly Power Lunches and occasional meetings focused on a particular industry or niche. Roundpeg chapter president Lorraine Ball says that she walks away from meetings feeling "energized, motivated, and inspired!"
Annual membership in NOWIB is $150, with a discounted yearly rate of $55 for full-time students. The group is run entirely by volunteers, and the Network encourages all participants to get involved and help shape the direction and character of the meetings. Guests are welcome to attend up to two meetings before determining if a membership is right for them.
4. Rainmakers
For Indianapolis professionals who are looking for a more in-depth, intensive networking opportunity, Rainmakers may be just the right fit. The Rainmakers commitment is to offer more monthly meetings than other groups, more customization to their members' needs, more training, and more connections. The group offers 10 networking and three training events each month, including groups focusing on young professionals, boomers and beyond, the technology industry and more.
Membership in Rainmakers runs $699 annually and gives members access to all monthly meetings, online groups, membership database and networking support from the Rainmakers staff. In addition to in-person networking, Rainmakers members also have access to private groups on Facebook and LinkedIn to continue making connections and passing referrals throughout the month.
5. Fast-Track Professional Networking
Prefer a networking opportunity that's a bit more fast-paced and low-commitment? Linking Indiana's speed networking events are a great way to make a lot of new connections in a highly structured environment. After registration, appetizers, and open networking, participants are seated in groups and asked to answer three questions in three minutes: Who are you? What do you do? How can you help others, and what kind of help are you looking for yourself?
A big stack of business cards is a must-have for anyone looking to attend these events, as connections are made quickly and often! Registration for Fast-Track Professional Networking is required at a cost of $20 in advance or $25 at the door.
6. Indy Rainbow Chamber
The Indianapolis Rainbow Chamber was founded to facilitate connections between professionals in the Gay, Lesbian, Bisexual, Transgendered, and Allied (GLBTA) community. Monthly After Hours networking events are held from 5:30-7 p.m on the first Wednesday of each month.
These free events are a great opportunity for Indianapolis professionals to build their network in a relaxed and supportive environment. Interested businesses and professionals can also join the chamber at an annual rate of $50 to $750, depending on company size and membership type.
7. TechPoint
More than a networking group, TechPoint is the clearinghouse and support structure for all things tech in Indianapolis and beyond. Each month the group hosts two events that are free and open to the public: the "New Economy, New Rules" breakfast series and the "Tech Thursday" evening networking meetings.
These events offer a heavy educational component to attendees who range from experienced industry veterans to students and technology industry newcomers. In addition, TechPoint holds awards ceremonies, hackathons, entrepreneur bootcamps and more. Technology industry professionals and hopefuls can find valuable information on the group's website, including job openings, internship programs and local industry trends.
Shelby Blanchard Stogner is a freelance copywriter and blogger who helps businesses get the word out about the great work they're doing. Visit her on the web at www.sbscopywriting.com for marketing tips and advice on how your business can benefit from zombies.
One Comment
Thanks for including TechPoint in this article! As the voice and catalyst for growing Indiana's tech ecosystem, we strive to offer a variety of educational and networking events throughout the year. We're eager to be a solid resource and conduit for tech talent to connect with the industry …
Comments are closed.


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Peter Quillin aka Kid Chocolate came to the Everlast studio on Tuesday to tape the third episode of TALKBOX, the podcast I host.
I had a good time chatting with the NY based boxer, and we touched on a wide range of subjects.
The 32-1-1 hitter tasted loss for the first time in December, and he shared how that affected him. Quillin told TALKBOX Nation that he is looking forward to fighting again before the end of the year, and yes, he's looking to move to super middleweight. Early fall, probably, he said.
We chatted about this age, which allows critics to shout loud and proud about all the things they hate about you. He admits that scorn surprised him.
More good stuff from Quillin…he told us he had a nervous breakdown before meeting Gabe Rosado in 2013; how he sparred Gennady Golovkin and Julio Cesar Chavez Jr the same day; that he called up a guy who was threatening him on Instagram and asked him why he was being like that.
There's a bunch more solid stuff from the fighter, including Quillin busting on my big bald head.
Please do check out the third ep, this material with Quillin, and feel free to listen to ep 1 with Rosie Perez and the second, an homage to Muhammad Ali.


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The long-time rumblings about age discrimination at IBM have finally produced a lawsuit. A 60-year-old Texas man alleges in a suit filed May 25 that he was improperly laid off amid the company's push to hire millennials.
Jonathan Langley, a former salesman in IBM's Hybrid Cloud unit, alleges the company sent him packing after a 24-year career that consistently "met or exceeded" the company's performance expectations. He also claims the company lied to investigators from the U.S. Equal Employment Opportunity Commission about the reasons for his dismissal.
The reality is if he had "been younger, and especially if he had been a Millennial, IBM would not have fired him," the federal lawsuit says.
Langley's Austin-based legal team filed the case just before the Memorial Day holiday, and in it highlighted a recent ProPublica/Mother Jones report that alleges the company is systematically pushing out its older workers, tilting its in-house evaluation and layoff process even against high performers. According to the report, IBM had "ousted an estimated 20,000 U.S. employees ages 40 and over since 2014, about 60 percent of its American job cuts during those years."
Steve Groetzinger, a Triangle resident and former salesman for the company's Security Division, says he was one of those targeted. Groetzinger, now 66, was working on a sales proposal to North Carolina's state government when he was laid off in 2016.
"When I looked around at all the people who've been laid off recently, there was a pattern," Groetzinger said. "Everybody was over 50."
Groetzinger isn't inclined to join the litigation — "I'm over it, I'm retired and I'm doing fine," he said — but he points out that other layoff victims aren't as lucky.
Some are "people a little younger than me who still had houses to pay off or kids in college," he said. "To them, this is really bad."
IBM, which employs thousands at its corporate campus in Research Triangle Park, has gone through multiple rounds of layoffs in recent years, including one just before the Memorial Day holiday that targeted workers in its Watson Health project.
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The situation has led to complaints to the EEOC, which appears to be taking an interest the matter. Pro Publica reported recently that the U.S. Equal Employment Opportunity Commission has launched a nationwide probe of age bias at IBM. It cited as its sources ex-employees who had spoken with investigators and people familiar with the agency's actions, including a former general counsel for the EEOC. The EEOC does not comment on on-going investigations.
Litigation is the next frontier, but lawsuits are complicated by severance agreements that call for the use of arbitration to resolve age-discrimination claims.
Langley is suing IBM on his own, but the issue is serious enough that it could "potentially" spawn a class-action lawsuit against the company, said David Lopez, a former EEOC general counsel who's now with a San Francisco law firm, Outten & Golden, that specializes in employment law.
Nor is IBM the only tech-industry player that's under fire. Lopez and his firm are involved in a lawsuit that accuses a number of companies, Amazon among them, of using age-restricted employment ads on Facebook to exclude older workers.
"When you start peeling the onion, you start to see that age discrimination in the hiring process is pervasive," Lopez said.
Lopez is also representing a former IBM program manager from Georgia, Coretta Roddey, who suspects her "over 40" age has something to do with her inability to return to the company after stints elsewhere in the private sector.
Roddey said she left IBM on good terms and was deemed re-hireable. But subsequent interviews or recruiting contacts, including one with an IBM human-resources manager based in the Research Triangle Park, never turned into an offer. She's filed an EEOC complaint.


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Sunday, November 14, 2010
when you think back to the times when you were in high school and each person had "dreams" of things they wanted to do. it is clear today that most people that i know from home are NOT doing anything that they wanted to do. i am really curious as to why alot of people changed their career paths. i know alot of people that i know who wanted to be doctors are now nurses or work lower on the food chain in health care.
i am so determined to become a doctor. i have serously evaluated my life and tried to consider possible doing something else with my life...but i realize there is absolutly nothing more i want to do in this world than to be a doctor and change peoples lives in a way that most people cant. i just think i was meant to do that. i mean with all of the oppertunities that i have been given and the influences i have had from the medical doctors i have met i just cant help but want to try harder and harder to obtain that goal. i have time i just have to do it!
but it is sad to look at the people that once had so many goals and see them settle. i mean i am sure they are not just settling to settle. they are doing it for thier family...to create a life for their kids/husbamd. yea another crazy not...taking not of all the people that are marrried/have kids/or about to get married. i have one friend that is divorced...LoL so i guess its not too bad to just know one person that it did not work out for. but still...it is just crazy to see how over the past 7 years how drastically peoples lives have molded and changed. i look forwerd to the next 7 and cant wait to see where i am 7 years from now :) hopefully i will be almost done with medical school...LoL


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Please be advised that due to the COVID 19 situation and restrictions, Taffy's is closed until further notice.
Where else in the South West can you enjoy watching Salt Water Taffy, that American seaside sweet sensation, being pulled and wrapped the old-fashioned way?
Strawberry, Banana, Passionfruit and many more flavours of taffy are cooked in a big copper kettle, cooled, and placed on a specially made pulling machine. It's pulled until it's soft and glossy, then goes into our bright red 1920's taffy wrapping machine, where it's cut into bite size pieces and wrapped in wax paper. From there. it's a short trip into your mouth!
Taffys is located on the Marlston Waterfront, just below the Barbados Restaurant and next to Aristos Waterfront Cafe.
We use traditional family recipes that have been perfected over 60 years of candy making, and are now available to you from Taffy's kitchen.
Aunt Dorothy's Peanut Brittle is simply the best you'll ever taste (we didn't make that up – our customers told us so!)
Mum's Fudge is marshmallow-based and melts in your mouth. A Christmas tradition for over 50 years is now your to try at Taffys.
Buckeyes, soft centred chocolate-dipped delights, are made in heaps of flavours like peanut butter, peppermint, and rum and raisin.
Taffys' also make rich caramels, white chocolate covered pretzels (surprisingly good!), chocolate drenched almonds, and many other tempting confections.
Bring the whole family to experience Taffys sweets made by hand, right before your eyes, savour the delicious smells and indulge in candies, chocolates, ice cream and thick shakes!


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Much delayed, but I've finally tackled the second album by Welsh critics favourite (or at least she was when she did her first album) Marina & The Diamonds. She's a solo artist despite the name, and had a decent breakthrough with debut album "The Family Jewels", including the amazing "I am not a robot".
The comeback felt a little disjointed, with inital buzz around single "Radioactive" not really crystallising into sales, and after a re-think the album was launched earlier this year without the song. Despite comfortable out-peaking her debut album on home turf with a rather shock No.1, the album didn't have any longevity and as I write this possibly hasn't sold even half of "The Family Jewels" modest total.
Still, when have poor record sales determined the quality of an album? Will this be a hidden gem? (ugh, sorry she's just asking for jewel-based puns isn't she?)
09 Bubblegum Bitch – Opening with a punch uptempo pop track now, and returning to the cute bimbo style of some of the sweeter moments of "The Family Jewels" like "Oh No!". That chorus is pretty great, very catchy and instant. The tempo is straightforward and pacey, I love it. It reminds me a bit of Toni Basil actually, which is no bad thing (in small doses). Why didn't she use this as a single, considering all the dead ducks she's released so far?
08 Primadonna – About the only hit she managed to wring out of this campaign, and harking back to her earlier sound a bit. Her voice is sickly sweet for the organ-backed intro, but a big squelchy electro beat kicks in and we are on even more familiar sound for her. It's sort of Gwen Stefani-esque in places, which for me at least is a good thing. I like the two different sounds to this song, which keeps going back and forth between cutesy and ballsy. It's not got a great hook in the way her "Jewels" singles did, but not bad.
07 Lies – Downtempo now, and a bit of a breakup song. Poor Marina's been cheated on, and is attempting a rather toothless chewing-out of her lover. Sure he cheated but she doesn't want to lose him. It's got a note of desperation in there, which is starting to resonate a little bit the more I listen to it. I like the bridges, even if the chorus is a bit of a drag. Decent.
08 Homewrecker – The spoken intro is a bit clunky and pseudo-intellectual, can't say I'm too keen, but luckily she's right into the meat of the song before long. Femme Fatale time now, as Marina assumes the role of the titular slut. Actually the chorus and tempo is about all this one's got going for it, but these songs are so short that's not much of a handicap. I just don't like those talky bits.
09 Starring Role – A slowie now, and one of the standout tracks for me. Her voice does get on my nerves a bit when she's always in that upper register, but it fits this tune nicely. Not sure what she's getting at with this one, she wants to be the centre of attention but it seems to be wrecking her relationship. It's got a bit of a melancholy note to it, like "let's just get drunk, forget we don't get on". But then she drops bitch-bombs like "send my best regards from hell". Maybe the guy's playing around with another woman and she's not having it? A bit muddled but I really like it.
08 State of dreaming – In some ways this album reminds me of Nicola Roberts' solo debut, there are just musical moments that have that same sort of atmosphere. A few moments in this song are like that, though this is a bit of an indulgent ballad. It sounds lovely, but I'm grateful when the tempo suddenly picks up into an almost Coldplay-sounding bittersweet but big track. That's a weird comparison, but it's only meant in a good way. It really develops nicely, except that rather shrill middle-eight.
07 Power & Control – Maybe this is the point where her muddled-sound and samey vocal tone get a bit much for me. I mean I can see why this was pushed as a single (unsuccessfully), the chorus is memorably simple, and I do like the general feel of the production but there's just something a bit missing here.
07 Living Dead – No I was wrong, THIS is the point I'm getting a bit bored. Those opening lyrics are just nonsense, and just gets on my nerves. But despite myself, I love that chorus. Living de-de-de-dead, whatever it's about, it's just a good hook. Just the verses don't support this song well enough.
06 Teen Idle – No THIS is where I get bored… sigh, why do all the songs have some big saving grace, it's making a mockery of my scoring system. A bit of a flimsy first minute, just reeling off lists of stuff she wants to be. Didn't we have this already? I feel that "Oh No" covered that whole "Oh I'm such a complicated contradiction of a person", but that at least had some punch to it (literally in some places, I remember some boxing gloves being involved).
05 Valley of the dolls – More maudlin wailing, is she trying to be Florence? I can't say this is doing much for me at all, though I do like that "in the Valley" refrain. But that's a very slim portion of the song, and the rest is just a nondescript midtempo. Why couldn't we have had more songs like Bubblegum Bitch?
07 Hypocrates – A similar tempo to "Valley of the Dolls", but things are a little more upbeat and optimistic. It's nice enough to listen to – in fairness this album as a whole doesn't really drag – and it even has a reasonably likeable chorus. It's just a bit nothingy, with a few nice moments. Sort of like the bulk of female singer-songwriters in the mid-00s, I vaguely like it, but I'd struggle to recall it.
08 Fear and Loathing – A dramatic finish to a pretty brief album (perhaps it's a good thing for the songs not to outstay their welcome). Finally she gets one of her atmosphere pieces, even if a 6-minute epic wasn't quite necessary. Just builds well, and her voice doesn't get on my nerves too much. The lyrics are a bit clunky, she was determined to rhyme as many things as she could, wasn't she? Hangs together well and doesn't get too drawn out.
Well it's not a perfect album, but it's certainly something I can listen to from start to finish without getting too tempted to skip tracks. The less desirable tracks are almost over by the time they register on my annoying radar, and the result is a pretty tight album. She might be preoccupied with having a persona to really let loose with some great pop songs, but maybe the commerically dry period she's found herself in (with a new potential for international success if she plays her cards right) could yet be turned around.
It looks like there's some sort of re-issue on the cards, and there's no guarantee it'll work but I hope she at least gets a chance to go for broke with another album.
Keepers for the iPod: Bubblegum Bitch, Starring Role, State of Dreaming