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Transgenic plants producing peroxisomal polyhydroxy- alkanoate (PHA) from intermediates of fatty acid degradation were used to study carbon flow through the β-oxidation cycle. Growth of transgenic plants in media containing fatty acids conjugated to Tween detergents resulted in an increased accumulation of PHA and incorporation into the polyester of monomers derived from the β-oxidation of these fatty acids. Tween–laurate was a stronger inducer of β-oxidation, as measured by acyl-CoA oxidase activity, and a more potent modulator of PHA quantity and monomer composition than Tween–oleate. Plants co-expressing a peroxisomal PHA synthase with a capryl-acyl carrier protein thioesterase from Cuphea lanceolata produced eightfold more PHA compared to plants expressing only the PHA synthase. PHA produced in double transgenic plants contained mainly saturated monomers ranging from 6 to 10 carbons, indicating an enhanced flow of capric acid towards β-oxidation. Together, these results support the hypothesis that plant cells have mechanisms which sense levels of free or esterified unusual fatty acids, resulting in changes in the activity of the β-oxidation cycle as well as removal and degradation of these unusual fatty acids through β-oxidation. Such enhanced flow of fatty acids through β-oxidation can be utilized to modulate the amount and composition of PHA produced in transgenic plants. Furthermore, synthesis of PHAs in plants can be used as a new tool to study the quality and relative quantity of the carbon flow through β-oxidation as well as to analyse the degradation pathway of unusual fatty acids.
Plant peroxisomes are involved in a variety of metabolic functions including photorespiration, detoxification of reactive oxygen species and the catabolism of fatty acids and amino acids ( Olsen 1998). The β-oxidation and glyoxylate cycles participate in the breakdown of fatty acids to acetyl-coenzyme A (CoA) and its conversion to succinate, respectively. During germination of seeds storing triacylglycerides, the enzymes of both the β-oxidation and glyoxylate cycles are abundant in peroxisomes. Once photosynthesis is established, the activity of these two cycles greatly diminishes, while the enzymes involved in photorespiration become abundant. Reactivation of β-oxidation and glyoxylate cycles occurs during senescence. The intracellular concentration of hexose sugars or the flux of hexose sugars into glycolysis may provide important signals giving rise to changes in expression of genes involved in the glyoxylate cycle ( Graham et al. 1994 ; Ismail et al. 1997 ). It is not know whether the same signals could also be involved in regulation of the β-oxidation cycle. Although the enzymes of the β-oxidation and glyoxylate cycles are normally present at very low levels in photosynthetic leaves and developing seeds, analysis of transgenic Brassica napus expressing the California bay lauroyl-acyl carrier protein (ACP) thioesterase revealed an increase of isocitrate lyase (ICL) activity in leaves and of both ICL and acyl-CoA oxidase (ACOX) activities in developing seeds ( Eccleston & Ohlrogge 1998; Eccleston et al. 1996 ). Acyl-ACP thioesterases are plastidial enzymes catalysing the hydrolysis of acyl-ACPs, resulting in the termination of chain elongation by the fatty acid synthase complex. Transgenic plants expressing the lauroyl-ACP thioesterase were shown to accumulate lauric acid in seed triacylglycerides ( Voelker et al. 1992 ). In contrast, although the level of lauric acid synthesized in chloroplasts isolated from these transgenic plants is relatively high, no lauric acid is detected in leaves from the same plants ( Eccleston et al. 1996 ). These results suggested that the lauric acid synthesized in leaves is rapidly degraded through the peroxisomal β-oxidation cycle, a hypothesis supported by the increase in isocitrate lyase activity ( Eccleston et al. 1996 ). It is not known what regulates activation of the enzymes of the β-oxidation and glyoxylate cycles in this system, but the level of fatty acid intermediates could be such a factor.
Over the past decade, much attention has been focused on understanding the mechanisms implicated in the control of carbon fluxes to particular metabolic pathways. In many cases, this knowledge has practical benefits, such as increasing levels of starch, lipids or amino acids in storage tissues. Control over the carbon flux to the peroxisomal β-oxidation pathway has gained interest due to its potential impact on the accumulation of unusual lipids in transgenic oil crops ( Eccleston & Ohlrogge 1998), as well as on polyhydroxyalkanoate (PHA) production in plants ( Poirier 1999).
PHAs are polyesters of hydroxyacids naturally synthesized by a wide variety of bacteria ( Poirier et al. 1995 ; Steinbüchel 1991). PHAs have attracted considerable interest because of their plastic and elastomeric properties, as well as biodegradability, making them an interesting source of renewable and environmentally friendly polymers ( Poirier et al. 1995 ; Steinbüchel 1991). Synthesis of PHAs in agricultural crops is seen as an alternative to bacterial fermentation for the production of PHAs on a large scale and at low cost ( Poirier & Nawrath 1998; Poirier et al. 1992 ; Poirier et al. 1995 ). Expression of the Ralstonia eutropha (formerly Alcaligenes eutrophus) polyhydroxybutyrate (PHB) biosynthetic enzymes in the plastids of transgenic Arabidopsis thaliana led to the accumulation of PHB up to 14% of the leaf dry weight ( Nawrath et al. 1994 ). PHB is a plastic with poor physical properties, being relatively stiff and brittle. In contrast, medium-chain-length PHAs (MCL-PHAs) are co-polymers having physical properties ranging from soft plastics to elastomers, rubbers and glues ( De Koning 1995). MCL-PHAs are composed of 3-hydroxyacid monomers ranging from 6–16 carbons in length, and are synthesized in a number of pseudomonads from 3-hydroxyacyl-CoA intermediates generated by the β-oxidation of alkanoic acids ( Poirier et al. 1995 ; Steinbüchel 1991). It has recently been demonstrated that targeting of a PHA synthase from Pseudomonas aeruginosa into the peroxisomes of A. thaliana led to the synthesis of MCL-PHAs containing saturated and unsaturated monomers ( Mittendorf et al. 1998a ; Mittendorf et al. 1998b ). In these transgenic plants, PHA inclusions accumulated primarily in the peroxisomes and maximal synthesis was linked to the β-oxidation of reserve fatty acids in germinating seedlings.
In the present study, transgenic plants accumulating peroxisomal MCL-PHA were used as a tool to study factors influencing the activity of the β-oxidation cycle as well as the relative quantity and the nature of the fatty acids that can be channelled to β-oxidation and PHA synthesis.
Modulation of PHA synthesis by Tween fatty acid esters
To assess whether the flow of fatty acids to the peroxisomal β-oxidation and PHA biosynthetic pathways could be increased in plants, seedlings from transgenic A. thaliana line 3.3, which constitutively expressed the PhaC1 synthase from P. aeruginosa in peroxisomes ( Mittendorf et al. 1998a ; Mittendorf et al. 1998b ), were grown in liquid cultures supplied with various fatty acid conjugates. Tween-80 and Tween-20 are detergents of polyoxyethylenesorbitan esterified primarily to oleic acid (C18:1) and lauric acid (C12:0), respectively. It has been shown previously that plant cells can incorporate fatty acids present in Tween into their membrane lipids ( Terzaghi 1986a; Terzaghi 1986b; Terzaghi 1989). While the addition of 5% Tween-80 (equivalent to 28 m m oleic acid) resulted only in a marginal increase in PHA accumulation, addition of 5% Tween-20 (equivalent to 20 m m lauric acid) resulted in an approximate eightfold increase in PHA ( Table 1). Tween-80 feeding resulted in an increased proportion of all PHA monomers derived from the β-oxidation of oleic acid, namely 3-hydroxyhexadecenoic acid (H16:1), 3-hydroxytetradecenoic acid (H14:1), 3-hydroxydodecanoic acid (H12), 3-hydroxydecanoic acid (H10:0), 3-hydroxyoctanoic acid (H8) and 3-hydroxyhexanoic acid (H6) ( Table 1 and Fig. 1). PHA isolated from plants grown in media supplemented with Tween-20 showed significant increase in the proportion of all saturated 3-hydroxyacid monomers ranging from 6–12 carbons, which are primarily derived from the β-oxidation of lauric acid ( Table 1). Similar results were obtained for plant cultures fed with 2.5% Tween-80 and Tween-20, while addition of Tween backbone (Tween without fatty acids) had no significant effects on either PHA quantity or monomer composition compared to cultures grown in the absence of Tween (data not shown).
Table 1. Influence of Tween on the quantity and monomer composition of PHA produced in transgenic plants
a3-Hydroxyacid monomers are denoted by the prefix H. The monomer H10:1 and odd-chain monomers were not quantified.
Seedlings from phaC1-transformed line 3.3 were grown axenically in liquid medium containing half-strength Murashige and Skoog (MS) salts and 2% sucrose for the first 7 days, and in the same medium supplemented with 5% (w/v) Tween detergent for an additional 7 days.
PHAs produced from cultures supplied with Tween detergents were also analysed by 1H-NMR. One-dimensional 1H-NMR does not provide a level of resolution sufficient for the precise assignment of all peaks to particular carbon groups in PHA containing over 15 different saturated and unsaturated monomers. Nevertheless, comparative analysis of bacterial PHA containing saturated and unsaturated H6 and H8 monomers with linolenic acid and plant MCL-PHA clearly confirmed that PHA isolated from plant cultures supplied with Tween-80 contained unsaturated monomers and that their proportion greatly decreased in PHA isolated from cultures supplied with Tween-20 ( Fig. 2). These results indicate that addition of an external source of fatty acids in the form of a Tween conjugate can lead to an increased flow of fatty acids towards peroxisomal β-oxidation. It furthermore indicates that the amount of PHA synthase expressed in these transgenics was not limiting the synthesis of PHA in plants relying on the endogenous fatty acid pool, but rather that the supply of fatty acids to β-oxidation was a key factor.
Peroxisomal β-oxidation of unusual fatty acids
Plants can synthesize a broad range of unusual fatty acids which are mainly accumulated in their seed oils ( Van de Loo et al. 1993 ). Although plants producing unusual fatty acids, such as hydroxylated or epoxidized fatty acids, must be able to degrade them during germination, little knowledge is available on these catabolic pathways. Furthermore, there are examples of transgenic plants which accumulate in their seed triacylglycerides containing unusual fatty acids that they do not normally make, without apparent negative impact on seed germination, again indicating that appropriate degradation of unusual fatty acids occurs in these plants ( Broun & Somerville 1997; Lee et al. 1998 ).
To investigate the range of fatty acids which could be degraded by β-oxidation and analyse whether their degradation intermediates could be included into MCL-PHAs, unusual fatty acids were supplied exogenously to transgenic plants growing in liquid media containing 0.2% sucrose and 5% Tween-80. Growth of plants in media containing 0.2% sucrose generally lead to a higher level of PHA per g dry weight compared to plants growing in media containing 2% sucrose ( Table 1 versus Table 2). This is probably due to the higher shoot to root ratio of plants grown in low sucrose. While addition of 0.35 m m octanoic acid resulted only in minor changes in the composition of PHA, with a relatively small increase in the proportion of H8 monomer, addition of 1.8 m m tridecanoic acid resulted in a large increase in the proportion of H13 and H11 monomers, and a more modest increase in H9 and H7 monomers ( Table 2). Furthermore, addition of 0.47 m m of tridecenoic acid (C13:1 Δ12) and 0.87 m m 8-methyl nonanoic acid resulted in the synthesis of PHA containing additional novel monomers. For plants fed with C13:1 Δ12, the PHA contained 10, 7, 8 and 4 mol% of H13:1 Δ12, H11:1 Δ10, H9:1 Δ8 and H7:1 Δ6 monomers, respectively. Plants fed with 8-methyl nonanoic acid produced a PHA containing 33, 19 and 7 mol% 8-methyl H9, 6-methyl H7 and 4-methyl H5 monomers, respectively. In plant PHA isolated from cultures fed with C13:1 Δ12, the presence of monomers with terminal unsaturated bonds was further confirmed by 1H-NMR ( Fig. 2e). In all cases, changes in the plant PHA monomer composition following addition of unusual fatty acids can be explained by the incorporation of monomers derived from the 3-hydroxyacyl-CoA intermediates of the β-oxidation of these unusual fatty acids.
Table 2. . Influence of exogenous unusual fatty acids on PHA synthesis in plants
Seeds from phaC1-transformed line 3.3 were grown axenically in liquid medium containing half-strength MS salts and 0.2% sucrose for the first 7 days, and in the same medium supplemented with 5% (w/v) Tween-80 and free fatty acids for an additional 7 days. Monomers present in trace amounts (H10:1 and H16:1) were not quantified. n = 3–4; data are means ± SD.
Of the various enzymes involved in fatty acid β-oxidation, ACOX is thought to be a good indicator of the level of β-oxidation since it appears to be the main step controlling the flux through β-oxidation ( Aoyama et al. 1994 ; Holtman et al. 1994 ). The levels of both medium-chain and long-chain ACOX were measured in plants fed with 2.5% Tween-80, Tween-20 and Tween backbone ( Fig. 3). Addition of Tween-20 to the growth media increased medium-chain and long-chain ACOX activities in transgenic plants by approximately fourfold. In contrast, no significant changes in ACOX activity were observed with the addition of either Tween-80 or Tween backbone.
Modulating the monomer composition of MCL-PHAs using endogenous fatty acids
The physical properties of PHAs are dependent on their monomer compositions. MCL-PHA synthesized in transgenic A. thaliana expressing the peroxisomal PhaC1 synthase contains a high proportion of saturated and unsaturated long-chain monomers (40–50 mol% H12 and higher, 20–30 mol% polyunsaturated monomers), making the polymer soft and sticky. Better elastic properties and a higher melting point can be achieved by increasing the proportion of shorter and more saturated monomers. In a first strategy aimed at modulating the proportion of different fatty acids going to β-oxidation, and therefore influencing the proportion of various monomers in MCL-PHAs, we have expressed the peroxisomal PhaC1 synthase in various A. thaliana mutants deficient in fatty acid desaturation. The fad2-1 mutant is deficient in the extra-plastidial 18:1 desaturase, resulting in an increase in mono-unsaturated fatty acids and a decrease in di-unsaturated and tri-unsaturated fatty acids present in leaf, root and seed lipids ( Miquel & Browse 1992). The fad3/fad7-1/fad8 triple mutant has a large reduction in the 18:2 and 16:2 desaturases, resulting in a more than 10-fold decrease in tri-unsaturated fatty acids and a fivefold increase in di-unsaturated fatty acids ( McConn & Browse 1996). Transgenic plants expressing the peroxisomal PhaC1 synthase in the wild-type background were crossed to the fad2-1 and fad3/fad7-1/fad8 triple mutants, and F2 plants homozygous for the phaC1 gene in the different mutant backgrounds were selected.
The quantity and composition of the PHA synthesized in these various transgenic lines grown for 14 days in liquid media is shown in Table 3. Transgenic plants in the fad2-1 background produced a PHA with an increased proportion of monomers derived from mono-unsaturated fatty acids (H16:1, H14:1) or both mono-unsaturated and saturated fatty acids (H12, H10, H6), and a decrease in monomers derived from tri-unsaturated fatty acids (H14:3, H12:2, H8:1) and di-unsaturated fatty acids (H16:2, H14:2, H12:1). Transgenic plants in the fad3/fad7-1/fad8 background produced a PHA with a large decrease in monomers derived from tri-unsaturated fatty acids (H16:3, H14:3, H12:2 and H8:1) and an increase in monomers derived from di-unsaturated fatty acids (H16:2, H14:2, H12:1), as well as an increase in H8 derived from di-unsaturated, mono-unsaturated or saturated fatty acids. The changes in the PHA monomer composition produced in the various mutants reflected well the relative changes in abundance of fatty acids which are used as the source of substrates for PHA synthesis ( Fig. 1). The only exceptions are the relatively stable level of the H16:3 and H8 monomers in transgenic plants in the fad2-1 background. The amount of MCL PHA produced in the various lines was between 0.54 and 0.73 mg g–1 dry weight for 14-day-old plants, indicating that although the nature of the fatty acid going to β-oxidation was changed, the quantity remained relatively stable.
Table 3. . Composition of PHA produced in mutants affected in fatty acid desaturases
The monomer 10:1 and odd-chain monomers were not quantified.
Seedlings from phaC1-transformed line 3.3 (Columbia wild-type background) and lines expressing the same transgene in the fad2-1 and fad3/fad7-1/fad8 mutant backgrounds were grown axenically in liquid medium containing half-strength MS salts and 1% sucrose for 14 days.
c PHA content (mg g –1 dry weight) n = 3; data are means ± SD.
0.68 ± 0.06
1.3 ± 0.1
19 ± 1
22 ± 2
5.3 ± 0.2
4.7 ± 0.2
3.4 ± 0.2
1.4 ± 0.1
4.8 ± 0.3
3.0 ± 0.1
6.3 ± 0.3
15 ± 1
3.5 ± 0.2
0.3 ± 0.1
3.6 ± 0.1
7.4 ± 0.3
0.54 ± 0.06
2.3 ± 0.2
17 ± 1
15 ± 1
12 ± 1
11 ± 1
0.6 ± 0.1
0.79 ± 0.06
4.0 ± 0.1
15 ± 1
1.3 ± 0.1
10 ± 1
2.5 ± 0.1
0.7 ± 0.2
0.64 ± 0.06
8.4 ± 0.2
0.73 ± 0.03
1.4 ± 0.1
44 ± 1
5.5 ± 0.1
4.1 ± 0.1
11 ± 1
4.3 ± 0.1
2.6 ± 0.3
14 ± 1
0.11 ± 0.06
3.7 ± 0.2
0.3 ± 0.1
8.9 ± 0.1
Increased flow of carbon towards PHA in transgenic plants expressing an acyl-ACP thioesterase
Since experiments in liquid cultures indicated that the supply of fatty acids to the β-oxidation pathway and PHA synthesis could be increased by the exogenous addition of unusual fatty acids, such as lauric acid, we sought to determine whether a similar increase could be generated endogenously through genetic engineering.
The FatB3 gene encoding a medium-chain acyl-ACP thioesterase has recently been cloned from Cuphea lanceolata ( Martini et al. 1999 ; Töpfer et al. 1995 ). Expression of the full-length FatB3 cDNA in A. thaliana under the control of the B. napus napin promoter led to the accumulation of triacylglycerides in seeds containing 18 mol% capric acid, 3.5 mol% lauric acid and 3.4 mol% myristic acid (Y. Poirier, unpublished results), confirming that the FatB3 thioesterase from C. lanceolata has highest specificity for capryl-ACP ( Martini et al. 1999 ).
The FatB3 gene under the control of a truncated CaMV 35S promoter was transformed into transgenic A. thaliana line 3.3 expressing the PhaC1 synthase in the peroxisomes. Two independent transgenic lines, TP2.4 and TP2.11, homozygous for both the FatB3 thioesterase and PhaC1 genes, were analysed for fatty acid and PHA compositions. Seeds from the double transgenic line TP2.4 accumulated lipids containing 2.2 mol% capric acid, while seeds of line TP2.11 accumulated lipids containing 2.5 mol% capric acid. Leaves from both double transgenic lines did not show the presence of medium-chain length fatty acids (detection limit < 0.05 mol%), confirming that like B. napus and A. thaliana plants expressing a lauroyl-ACP thioesterase ( Eccleston et al. 1996 ; Hooks et al. 1999 ), A. thaliana transformed with a capryl-ACP thioesterase can accumulate medium-chain fatty acids in seed lipids but not in leaf lipids. Growth of the FatB3/phaC1 double homozygous transgenic plant lines was slower than either plants homozygous for only the phaC1 gene or heterozygous for both phaC1 and FatB3. The fresh weight of 26-day-old soil-grown FatB3/phaC1 double homozygous transgenic plants was approximately 30% of wild-type or phaC1-transformed plants. Reduction in growth has also been observed for some plants expressing only the CaMV35S–FatB3 gene construct, although the number of plants analysed were too small to clearly establish a correlation between level of thioesterase expression and reduction in plant growth.
PHA was extracted from 10-day-old double homozygous seedlings grown on agar plates and from leaves of 40-day-old soil-grown plants homozygous for the phaC1 gene and either homozygous or heterozygous (in a 1:2 ratio) for the FatB3 thioesterase. The data for the quantity and monomer composition of PHA are summarized in Table 4. Care should be taken in comparing the values of PHA in transgenic line PhaC1 3.3 grown for 10 days on plates containing 2% sucrose ( Table 4) with plants grown in a similar liquid media for 14 days ( Tables 1 and 3). Because most of the reserve lipids are in the cotyledons, this tissue is expected to have a larger amount of PHA compared to roots and leaves. Plants grown on agar plates for 10 days have large cotyledons, emerging primary leaves and a small root sytstem. In contrast, plants grown in liquid media for 14 days have a large root system and well developed primary leaves. Thus, the amount of PHA in plants grown on plates for 10 days (2.2 ± 0.3 mg g–1 dry weight, n = 4; mean ± SD) is consistently higher than for plants grown in liquid for 14 days (0.7 ± 0.1 mg g–1 dry weight, n = 5). The same transgenic plants grown in soil for 45 days have a PHA content of 0.10 ± 0.03 mg g–1 dry weight (n = 5), reflecting the fact that as the plant expands during post-germinative photosynthetic growth, the rate of increase in dry weight of the plant outpaces the synthesis of PHA in the peroxisomes ( Mittendorf et al. 1998a ).
Table 4. . PHA synthesis in transgenic plants expressing the phaC1 and FatB3 genes
Monomers 10:1 and 16:1 and odd-chain monomers were not quantified.
b Plants were grown under continuous illumination either for 10 days on solid medium (MS, 1% sucrose and 20 μg ml –1 hygromycin for TP lines or 50 μg ml–1 kanamycin for PHAC3.3 line) or for 40 days in soil.
c PHA content (mg g –1 dry weight).
Homozygous for both phaC1 and FatB3 genes.
Homozygous for phaC1 and either homozygous or heterozygous (in a 1:2 ratio) for FatB3.
Analysis demonstrated that 10- and 40-day-old double transgenic plants produced approximately three and eight times more PHA, respectively, in their vegetative tissues compared to plants expressing only the PhaC1 synthase ( Table 4). Furthermore, double transgenic plants contained a higher amount of saturated short-chain monomers, with H10 and H8 monomers reaching 74 mol% of the polymer in 40-day-old double transgenics, as compared to 18 mol% in plants expressing only the PhaC1 synthase.
The level of ACOX activity was measured in both TP2.4 and TP2.11 lines. A significant increase in both medium-chain and long-chain ACOX activity was measured in the double transgenic lines compared to the parental line expressing only the PhaC1 synthase ( Fig. 4).
It has been shown previously that exogenous Tween–fatty acid conjugates are imported into soybean cells where they are hydrolysed to give free fatty acids, which are in turn incorporated preferentially into extra-plastidial glycerolipids ( Terzaghi 1986a; Terzaghi 1986b). Shintani & Ohlrogge (1995) have also shown that addition of Tween–fatty acid esters to tobacco suspension cells caused a reduction in acetate incorporation into fatty acids which could be best explained by a biochemical or post-translational modification of the acetyl-CoA carboxylase. The effects of Tween on PHA accumulation in transgenic plants expressing a PHA synthase in the peroxisome clearly indicate that in addition of being included in membrane lipids, external fatty acids are also directed towards the peroxisomal β-oxidation cycle. Addition of Tween-80 or Tween-20 modified the PHA monomer composition by increasing specifically the proportion of 3-hydroxyacid monomers derived from the β-oxidation of the major fatty acid present in the conjugate.
It is striking that addition of Tween-20, containing primarily lauric acid, was more effective at increasing the amount of PHA than addition of Tween-80, which contains primarily oleic acid ( Table 1). Furthermore, larger shifts in PHA monomer composition were achieved with Tween-20 compared to Tween-80 ( Table 1). It is unlikely that differences in the affinity of the PHA synthase for the various 3-hydroxyacyl-CoA intermediates generated by the β-oxidation of oleic and lauric acids could account for this large difference since four out of six monomers generated by the degradation of these fatty acids are identical (i.e. H12, H10, H8 and H6) ( Fig. 1). These results thus indicate that the proportion of external fatty acids channelled towards peroxisomal β-oxidation is larger for Tween–laurate compared to Tween–oleate. This may be due to preferential penetration of Tween–laurate into the plant cells or to a higher rate of fatty acid hydrolysis from the Tween backbone. Alternatively, it is possible that this difference may also be due to an intrinsic metabolic response that specifically targets unusual fatty acids, such as lauric acid, towards β-oxidation, in comparison to usual fatty acids, such as oleic acid, which may be incorporated into membrane lipids (see below).
Addition of 2.5% Tween-20 to plant cells resulted in a large increase in ACOX activity, while addition of an equivalent amount of Tween-80 or Tween backbone had no significant effects on ACOX activity. This indicates that the increase in ACOX activity is not due to a non-specific toxic effect of detergents on cells, but rather is a response to the presence of fatty acids in the media, with plant cells being more responsive to unusual fatty acids compared to fatty acids normally present in membrane lipids. A significant increase in both medium- and long-chain ACOX activity has also been observed in shoots of transgenic A thaliana expressing constitutively the C. lanceolata capryl-ACP thioesterase ( Fig. 4). Analysis of transgenic B. napus expressing the lauroyl-ACP thioesterase revealed an increased activity of only the medium-chain ACOX, suggesting a correlation between the length of the synthesized fatty acid and the specific induction of a particular ACOX ( Eccleston & Ohlrogge 1998). Our results show no such direct correlation since FatB3 has high activity for C10 acyl-ACP while an increase in long-chain ACOX is evident in both transgenic lines tested. In plants fed with Tween-20, both medium- and long-chain ACOX were also increased. In contrast to rapeseed, constitutive expression of the California bay lauroyl-ACP thioesterase in A. thaliana was recently shown not to lead to increased activities of the various enzymes involved in β-oxidation, including ACOX ( Hook et al. 1999 ). These data, combined with our results on PHA synthesis in peroxisomes (see below), indicate that ACOX activity may not be a reliable indicator of the relative flow of fatty acids through β-oxidation, but that peroxisomal PHA may fulfil such a role.
Mechanisms are thought to exist in plants to exclude the accumulation of unusual fatty acids in membrane lipids ( Battey & Ohlrogge 1989; Stymne 1993). This concept is supported by the absence of medium-chain and hydroxylated fatty acids in membrane lipids from the vegetative tissues of transgenic plants expressing a medium-chain thioesterase and a hydroxylase, respectively ( Broun et al. 1998 ; Eccleston et al. 1996 ; Hooks et al. 1999 ; Van de Loo et al. 1995 ). Co-expression in transgenic A. thaliana of C. lanceolata FatB3 thioesterase in the plastid and PhaC1 synthase in the peroxisome resulted in an eightfold increase in PHA accumulating in 40-day-old shoots. Furthermore, the PHA monomer composition was strongly shifted towards H8 and H10 saturated monomers, reflecting the higher specificity of the FatB3 thioesterase towards capryl-ACP. Clearly, medium-chain fatty acids are being synthesized in the shoots of transgenic plants expressing FatB3 but are degraded by the β-oxidation pathway before their accumulation to detectable levels in membrane lipids. These results support the hypothesis that plant cells must have mechanisms which sense levels of free or esterified unusual fatty acids, resulting in changes in the activity of the β-oxidation cycle as well as in the removal and degradation of these unusual fatty acids.
Nearly 100 different hydroxyacid monomers have been shown to be included into bacterial PHAs, a large proportion of which are derived from the β-oxidation of unusual lipids or alkanoic acids added to the bacterial growth medium ( Steinbüchel & Valentin 1995). Feeding plant cells with a number of unusual fatty acids has shown that the plant β-oxidation pathway is also capable of generating a number of unusual substrates which can be included in MCL-PHAs, even from lipids which are not normally synthesized in significant quantities by the plant ( Table 2). For the different unusual fatty acids tested, all novel monomers (≥ 6 carbons) derived from degradation through peroxisomal β-oxidation were detected in PHA. These results indicate that analysis of the monomer composition of plant MCL-PHAs can be used as a tool to study the pathway of degradation of unusual fatty acids. For example, it is largely unknown how unusual fatty acids, such as hydroxylated or epoxydized fatty acids, are degraded in plants ( Gerhart 1993). Analysis of the monomer composition of plant PHA isolated from cells fed with unusual fatty acids could thus provide valuable information on the biochemistry of their degradation.
The physical properties of PHAs are strongly dependent on their monomer composition. Thus, while PHB homopolymer is a stiff and brittle plastic, poly(hydroxyvalerate–hydroxybutyrate) co-polymer is more flexible, while MCL-PHAs are typically soft elastomers. Gaining control over the monomer composition through modification of plant metabolism is therefore important for the success of producing PHAs in plants. In this study, we have used two strategies to modulate the quality of monomers channelled to PHA synthesis. In the first strategy, the PHA synthase was expressed in A. thaliana mutants deficient in the desaturation of lipids. PHA synthesized in these plants showed significant changes in monomer composition, such as reduction in tri-unsaturated and di-unsaturated 3-hydroxyacids ( Table 3). The changes in PHA monomer composition reflected well the relative proportion of various fatty acids present in the plant cell, with the exception of the stable level of H16:3 and H8 monomers in the fad2-1 mutant background. The reason for these discrepancies is unknown but could reflect changes in the availability of various 3-hydroxyacyl-CoAs to the PHA synthase due, for example, to changes in activities of different enzymes of the β-oxidation cycle ( Hooks et al. 1996 ). It is notable that the amount of PHA synthesized in the fad3/fad7-1/fad8 mutant background is not increased compared to wild-type ( Table 3). This indicates that large changes in the proportion of the usual fatty acids present in membrane lipids are not sufficient to increase the flow of fatty acids towards β-oxidation, reinforcing the concept that unusual fatty acids have a greater impact on this flow then usual fatty acids.
In the second strategy, both the quality and quantity of PHA synthesized were modulated. Shoot tissues of transgenic plants expressing the PhaC1 synthase in the peroxisomes accumulated a relatively low amount of PHA (0.1 mg g–1 dry weight in 40-day-old leaves) containing a high proportion of long-chain (59 mol% ≥ 12 carbons) or unsaturated (68 mol%) monomers, making the polymer a sticky soft material with low crystallinity and melting point. Co-expression of FatB3 thioesterase in the plastid and PhaC1 synthase in the peroxisome resulted in an eightfold increase in PHA accumulating in 40-day-old shoots, with the monomer composition being strongly shifted towards saturated short-chain monomers (81 mol% H6, H8 and H10), bringing the properties of the polymer towards an elastomer ( Table 4).
Unusual fatty acids containing functional groups, such as hydroxy, epoxy, keto, furanoid and cyclic groups, are attractive in the context of functionalizing PHAs as they represent a source of monomers with reactive side groups which can be used to change the properties of PHAs after extraction ( De Koning et al. 1994 ). It is expected that, as for the synthesis of medium-chain fatty acids in leaves, the synthesis in vegetative tissues of fatty acids with unusual side groups will lead to an increase in their degradation by the peroxisomal β-oxidation cycle. Thus, from this work, a working model emerges whereby the quantity and quality of the PHA synthesized in the vegetative tissues of plants could be modulated through the co-expression of enzymes responsible for the synthesis of unusual fatty acids in conjunction with a peroxisomal PHA synthase capable of using the β-oxidation intermediates from these unusual fatty acids to synthesize novel PHAs.
Plant material and growth conditions
Transgenic A thaliana line 3.3 expressing the P. aeruginosa PhaC1 synthase in the peroxisomes was transformed with the FatB3 gene by the vacuum infiltration method ( Bechtold et al. 1993 ). Transformants were selected on media containing Murashige and Skoog (MS) salts, 1% sucrose, 0.7% agar and 20 μg ml–1 hygromycin. Hygromycin-resistant plants were subsequently transferred to soil and grown under continuous fluorescent light at 19°C. For feeding experiments, axenic plants were grown under constant agitation (100 rev min–1) for 14 days in liquid media containing half-strength MS salts, 0.2–2% sucrose and Tween detergents or Tween backbone, either alone or in combination with free fatty acids. The fad2-1 mutant was obtained from the Nottingham Arabidopsis Biological Resource Center, while the fad3/fad7-1/fad8 triple mutant was provided by John Browse (Washington State University).
Production of Tween backbone
Tween from which fatty acids have been removed (Tween backbone) was prepared according to Terzaghi (1986a) with slight modifications. Ten g of Tween-80 was mixed with 30 ml of aqueous 1 m KOH and refluxed at 85°C for 20 h. The reaction mixture was neutralized with HCl and dried by lyophilization. The mixture was redissolved in diethyl ether and sufficient methanol to solubilize the Tween backbone. Following filtration through glass wool, the Tween solution was applied to a column packed with 100 g of 60–200 μm mesh silica gel 60 (Merck, Darmstadt, Germany) in diethyl ether. Free fatty acids were eluted with four column volumes of diethyl ether, then Tween backbone was eluted with four volumes of methanol. The dried Tween backbone was diluted with four volumes of water and extracted several times with hexane. Tween backbone contained less then 0.1% fatty acids.
The FatB3 gene from C. lanceolata was initially subcloned into the pART7 vector ( Gleave 1992) modified to contain only the first 200 bp of the CaMV 35S promoter, and finally inserted in the binary vector pBJ49 (B. Janssen, unpublished results). The binary Ti plasmid was introduced in the Agrobacterium tumefaciens strain GV3101 ( Koncz & Schell 1986) by electroporation.
ACOX activity measurements
Plant tissue (50 mg) was homogenized in 50 μl of cold extraction buffer containing 150 m m Tris–HCl (pH 7.5), 10 m m KCl, 10% (v/v) glycerol, 10 μm flavin adenine dinucleotide, 1 m mβ-mercaptoethanol, 1 m m EDTA, 0.1 m m PMSF, 0.01% Triton X-100 and 1% polyvinylpyrrolidone. The extracts were clarified by centrifugation at 14 000 g for 10 min at 4°C, and 20 μl of the supernatants were used per 1 ml of reaction mixture. ACOX activity was measured essentially as described by Hooks et al. (1996) using decanoyl-CoA and palmitoyl-CoA as substrates. Hydrogen peroxide production was followed at 500 nm in a UNICAM UV4 spectrometer.
PHA extraction and analysis
Extraction of PHA from plant material and analysis by gas chromatography and mass spectrometry (GC–MS) was performed as previously described ( Mittendorf et al. 1998a ; Mittendorf et al. 1998b ). Fresh or dried frozen plant material was ground in a mortar and lyophilized. The powder was extracted with methanol in a Soxhlet apparatus for 24 h followed by PHA extraction with chloroform for 24 h. The PHA-containing chloroform was concentrated using a Rotovapor and either filtered over glass wool or extracted once with water to remove residual solid particles. PHA was precipitated by the addition of 10 vol cold methanol and subsequently washed by two cycles of chloroform solubilization and methanol precipitation. PHA dissolved in chloroform was trans-esterified by acid methanolysis and analysed by GC–MS using a Hewlett-Packard 5890 gas chromatograph (HP-5MS column) coupled to a Hewlett-Packard 5972 mass spectrometer. In some experiments, a simplified procedure was used in which plant tissues were extensively extracted with methanol at 65°C, the residual material was trans-esterified by acid methanolysis and the PHA content was analysed by GC–MS utilizing the ion selective mode. Identification of monomers present in plant PHA was facilitated by the use of commercial standards and purified bacterial PHAs. 3-Hydroxyoctanoic acid, 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid and 3-hydroxytetradecanoic acid were purchased from Sigma (St Louis) while 3-hydroxyhexanoic acid was obtained from D. Seebach (ETH, Zürich). MCL-PHA purified from P. putida grown on linoleic acid and in which the monomer composition was determined by GC–MS as well as NMR was obtained from G. Eggink (ATO-DLO, Wageningen). PHA containing 3-hydroxyoctenoic acid (H8:1 Δ7) and 3-hydroxyhexenoic acid (H6:1 Δ5) was obtained from B. Witholt (ETH, Zürich), and PHA containing 8-methyl-3-hydroxynonanoic acid and 6-methyl-3-hydroxyheptanoic acid was a gift from B. Hazer (Marmara Research Centre, Turkey). 1H-NMR spectroscopy was performed on a Bruker AC-P200 spectrometer at 200 MHz using deuteriochloroform as solvent and tetramethylsilane as internal standard with a sample concentration of ≤ 10 mg ml–1.
We thank Giovanni Ventre and Stephanie Stolz for their technical assistance. We are grateful to Baki Hazer, Robert Lenz, Gerrit Eggink, Michele Kellerhals and Bernard Witholt for providing MCL-PHA samples, and Bart Janssen for the pBJ49 vector. We are also grateful to John Browse for providing the fad3/fad7-1/fad8 mutant line. We thank Edward Farmer and Christiane Nawrath for critical reading of the manuscript. This work was supported, in part, by grants from the Ciba-Geigy Jubiläums Stiftung, the Herbette Foundation and Monsanto. V.B. and L.A. were recipients of fellowships from Colciencias of Colombia, and the Georgine Claraz Foundation, respectively.
EMBL accession number AJ131740(Cuphea lanceolata FatB3 gene).

domain: chemistry


text:
BBC One has announced a new production deal with Agatha Christie Productions Ltd.
Seven new Agatha Christie adaptations will be produced over the next four years.
The first novel to be adapted is Ordeal By Innocence, with other titles confirmed so far including Death Comes As The End, a murder mystery set in Ancient Egypt, and The ABC Murders, a race against time to stop a serial killer who is operating across 1930s Britain.
Ordeal By Innocence will be written by Sarah Phelps (And Then There Were None).
Christie’s The Witness For The Prosecution, also adapted by Phelps, is currently filming and stars Toby Jones, Andrea Riseborough, Kim Cattrall and David Haig.
Charlotte Moore, Director, BBC Content, commented: “These new commissions continue BBC One’s special relationship as the home of Agatha Christie in the UK. Our combined creative ambition to reinvent Christie’s novels for a modern audience promises to bring event television of the highest quality to a new generation enjoyed by fans old and new.”
Hilary Strong, Chief Executive Officer, Agatha Christie Limited added: “And Then There Were None was a highlight of the 2015 BBC One Christmas schedule, and we are truly delighted to be building on the success of that show, first with The Witness For The Prosecution, and then with adaptations of seven more iconic Agatha Christie titles. What Sarah Phelps brought to And Then There Were None was a new way of interpreting Christie for a modern audience, and Agatha Christie Ltd is thrilled to be bringing this psychologically rich, visceral and contemporary sensibility to more classic Christie titles for a new generation of fans.”
Are you looking forward to the new Agatha Christie adaptations? Let us know below…

domain: artistic


Brett Hudson: How is technology impacting social and economic divisions in cities?
Ubiquitous information and communication technology (ICT) holds overwhelmingly positive promise to bridge social and economic divides within cities.
The rise of ICTs has diminished the role that long-standing institutions play in many parts of society. Institutions have traditionally provided a structured and cost-effective way for individuals to meet their needs and goals.
As ICTs became pervasive, they inspired a “do-it-yourself” (DIY) culture that encourages people to tap into their own innate potential and immediate networks rather than relying on institutions to accomplish those same goals. DIY culture is a natural result of ubiquitous ICTs; these technologies have dramatically lowered the cost for individuals to accomplish far more of their respective goals using far less time and resources, which rewards the individual with an unprecedented power that can exist entirely outside of any institution.
Many traditional institutional structures simply cannot compete with the DIY culture, which forces them to adapt, become irrelevant or simply be supplanted through disruptive innovation. Consequently, the modern economy’s DIY culture is pulling power away from embedded institutions and delegating it to individuals—particularly networks of individuals—leading to an inevitable transition from an institution-based society to a network-based society.
Why is this transition so important?
A network-based economy provides a fundamentally new power structure that rests outside of the traditional institutional structure. Tapping into the DIY culture has become increasingly commonplace, even essential, for forward-thinking business models. In recent years, ICTs have dramatically expanded the potential of network-based tools like crowdsourcing, crowdfunding and crowdspeaking (crowd-anything, really). The pervasiveness of these models indicates the potential power that an individual can exercise when tapping into a broad network versus going through a confined institutional structure. Why raise capital through institutional finance to seed your idea when you can engage an entire network of individuals with a greater likelihood of success?
This transition to a network-based economy also creates a challenge. While private sector institutions are built to adapt or be innovatively disrupted, many public sector institutions are not. This obstinate institutional structure could indicate why the public is becoming increasingly disaffected with government, particularly among Millennials that tend to be entrenched in DIY culture. Government is slow to adapt to technological changes and fundamental shifts in society, but given its nature, cannot be innovatively disrupted or supplanted with more effective ideas. When government loses its ability to solve problems, the public becomes disengaged, angry or both.
There is hope.
While the rise of ICTs are making it difficult for governments’ institutional structures to stay relevant in the DIY economy, embracing technology and DIY culture at the local level of government may also be its savior. The tech-driven DIY culture is forcing private sector institutions to identify business models that cede or share power with individuals. City governments can do the same to re-engage and unleash a latent creative potential in their citizenry.
For example, new network-based civic organizations are coming to fruition, like civic crowdfunding platforms neighbor.ly and citizinvestor, which could expand the role in which citizens play in their city’s financial decisions. As more of these kinds of platforms come online, it will continue to shift the influence away from the institution and into the hands of the individual, which expands the influence that she can exert over her community.
A transition away from institutional-based governance into network-based governance holds promise to overcome economic and social divisions within our communities. When cities embrace ICT and DIY culture, it opens up the potential to engage and empower new voices that have not participated in government’s traditional institutional structure. A shift in the power structure can equip underserved communities, which have not traditionally had the voice, power or resources to influence the institutional structure of government, with the necessary tools to create change. Over time, citizens that are closest to their community’s challenges could ideally overcome persistent social and economic structural problems that traditional institutions of government simply could not solve.
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Spotlighting innovations in urban sustainability and connected technology
Dedicated anti-trafficking actors across the nation are trying to build better systems in big jurisdictions like New York, San Francisco, and Los Angeles, and in smaller but scrappy jurisdictions like Waco, Texas and Boaz, Alabama. They all share the same need, for stronger interconnectedness as an anti-trafficking field, and more collaboration.
The Forging Freedom Portal is a one-stop shop where a police officer planning a victim-centered operation can connect with their law enforcement counterparts, and the right service providers ahead of time, collaborating to make sure they’re planning for the language skills, social services, and legal support that victims may need. The portal is a place where the people who care most about ending human trafficking, who are doing the hard work every day on the ground, can learn from each other and share best practices to raise the collective standard of this work.
Maximizing both the mobility and safety of road users at urban and suburban intersections is of utmost importance to city leaders and citizens today. Trends such as micromobility, connected and automated vehicles, and an explosion of available data, coupled with increasing numbers of bikes and pedestrians on our streets, result in both challenges and opportunities.
The increasing ability to provide intersection connectivity, edge computing and cloud storage, along with growing tool sets, such as Signal Performance Measures (SPM) and advanced video detection, provide new and exciting opportunities to traffic engineers. Possible combinations of Vision Zero intersection solutions, Near-Miss analyses, and the ability to make real-time operating decisions at our intersections can be overwhelming. Still, they must be embraced to ensure public officials are accountable to the traveling public.
I caught up with Joe Bergera – CEO of Iteris – recently and we discussed a cloud-first strategy for cities and the benefits, particularly during the pandemic. Organizations, cities and companies that have replicated some of their business processes in the cloud have navigated the pandemic quite well. We discuss why that is, and what can be done to help other cities during this time.


Are you struggling to find the best deal on Tractor Attachments ? Do you find yourself selecting between various choices when it’s time to purchase Tractor Attachments and which model is best suited to your needs? Do you feel the same? Don’t worry if it’s you. The right Tractor Attachments to suit your needs can be challenging for many people. You’re likely to be keen on Tractor Attachments as you’re here. You must find reliable sources that offer credible alternatives before you take any decisions about what product will be the best fit for your needs. The internet is a fantastic resource for finding information about products.
You can look through a variety of different sources, including buying guides or rating websites that provide the most highly rated items in particular categories; or word-of-mouth reviews from family and friends members who have purchased similar items before. You can then write about your experiences in forums where other people are sharing their opinions among themselves, while watching YouTube videos which provide real-time feedback regarding what works well with various types of equipment as well as help YOU make the right choices! It’s just not always that simple. To ensure that your search is as simple and effortless as is possible We have put together an extensive list of the top Tractor Attachments on the market. You may be asking why we came up with this list. What’s the method to come up with this list?
We have checked over 67428 reviews of the Tractor Attachments listed below to make the best list.
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Required A Quick Selection? Only Observe This Top 10 Best Tractor Attachments Listing of 2022
BestEquip Pallet Forks 1500lbs Capacity, Tractor Forks 43 inch Length, Clamp on Bucket Forks w/Adjustable Stabilizer Bar, Fork Loader Attachment for Skid Steer, Tractor Forks for Agriculture and Farm
- 【1500 LBS LOADING CAPACITY】-Working Capacity: 1500 lbs. These heavy-duty pallet forks can carry loads of up to 1500 lbs, so you can carry a wide variety of items. Attention: Please choose the right pallet fork according to your tractor's bearing capacity and place the loads evenly at the back position during use.
- 【HIGH COMPATIBILITY】-Overall Length: 43''. Forks Length: 30''. Fork Width: 2.75''. Fork Height: 1.95''. Bucket lip gap: 2.95". The 5/18'' screw rod is for added strength. This tractor bucket fork is compatible with almost all loader bucket skid steer tractors. Perfect for moving materials, logs, lumber, firewood, hay bales, brush, and broken fences.
- 【EASY TO ASSEMBLE & USE】- Simply Slide our forks inside and rotate the screw rod to clamp it down to lock tightly. Please make sure that the weight of the material you want to move plus pallet forks weight less than the capacity of the skid steer to ensure safety. (e.g., the skid steer has a 2,500 lbs lift capacity and the pallet forks weigh 300 lbs, the actual amount of the load could not exceed 2,200 lbs)
- 【17"-31.5" ADJUSTABLE WIDTH】- By using an adjustable stabilizer bar to prevent your forks from spreading apart conveniently, you can get your desired distance when lifting debris. The stabilizer bar can be adjusted from 17" to 31.5".
- 【SECURITY & DURABLE STRUCTURE】-Reliable welded steel adopted by our clamp-on bucket forks is rust-resistant. Thick and rectangular tube walls add stability. Also, chain holes and protection slots allow you to keep your loads right on the forks where they should be, ensuring security during work.
IMAYCC Enhanced Tractor Bucket Hooks 3/8" (2 Pack),Heavy Duty Tow Hook Grab Hooks for Tractor Bucket,G70 Forged Steel Bolt Hook Suitable for Rv,Utv,Tractor Bucket Accessories Max 15,000 lbs, Black
- 【Enhanced Utility of Tractor Buckets】The Grab hook Mounted on Bucket, and Use with Chain for Logging,lifting,dragging,moving Etc,also Used As Tow Hooks,is a Good helper for Tractor.
- 【Heavy Duty & Reliable】 Built with G70 Forged Steel 3/8" Grab Hook,Come with Thickened Back Plate, Solid Base, Strong Hooks, High-strength 10.9-Grade bolts,which ensure its Break Strength Reaches Up To 15000Lbs.
- 【Long Service Life】This Bolt-On Bucket Hooks adopt galvanizing process, preferably powder for spraying treatment offer strong surface adhesion and Anti-Rust;Perfect Welding ensure strong bearing capacity and prevent breakage.
- 【Easy to Install】This Bucket Hooks set Contains 2 Bolt On Grab Hooks 3/8", 2 Steel Plates 4.25 x 3.26 Inch, Grade 10.9 Bolts X4 , Grade 10.9 Nuts X4 , you just need to punch the holes and then fix the tractor hooks to the tractor bucket,Install in Minutes easily.
- 【What You Get】 2 pack Tractor Bucket Hooks and 1 year worry-free after-sale service,feel free to contact with us for any issue with the product ,we will reply you within 24 hours.
ENIXWILL Clamp On Bucket Tractor Hitch 2" Receiver Fit for Deere Bobcat Loader Front Bucket Clamp
$65.99 in stock
- 【Universal】2" receiver clamp on bucket hitch. Great for around-the-yard work requiring trailer hitches.such as boat, RV, trailer, camping accessories, etc.
- 【Design】Two side baffles are designed to prevent sliding left and right during transportation.
- 【Easy Installation】Slide this clamp on bucket hitch receiver to the front of your tractor and turn the handle to secure the clamp.
- 【Material】Made of heavy-duty steel, providing high strength load capacity, coated with red powder to prevent rust and corrison.
- 【Easy to Use】It is used for a quick change device at the front of the bucket tractor. It can quickly clamps on the bucket of your tractor in seconds.
Keeswin Lawn Tractor Leaf Bag Grass Catcher Bag, 78.7 x 51.1 Inch Large Capacity for Fast Leaf Collection, Heavy Duty Material Fits for Most Two Bag Material Collection Systems for Ride-in Lawn Mowers
- Sturdy Material: The tractor leaf bag is made of 420D Oxford fabric, which can withstand the abrasion of the lawn and avoid tearing due to surface resistance. The size is 78.7 * 51.1 inches, which is slightly smaller than others, avoiding being too heavy to lift when it is full.
- End Zipper Design: The grass catcher bag has a quick cleaning zipper at the end. When the bag is full, you can open the zipper, easily pour it out or open the engine to blow out the contents, and finally pull it back and lock the zipper to continue collecting!
- Mesh Vent Design: The mesh vent design allows the airflow of the lawn mower to escape and leave leaves, prevent the bag from falling off due to excessive air, and increase the air permeability of the bag.
- Easy to Install :Drawstring design makes the installation more reliable. Set the bag into the tractor collection system and tighten the drawstring cord. After leaf collection, just need to open the zip at the end ,easily pour the leaves out.
- Attention: This leaf bag is suitable for most ride-on lawn tractors and zero-turn mowers. Please make sure that the bag will not come into contact with the hot exhaust. Material collection system/grass catcher is NOT included, bags only.
Titan Attachments 48â€ Light-Duty Single Cylinder Root Grapple Rake fits John Deere Tractors, Economy Series
- HOOK AND PIN CONNECTION: This grapple bucket fits John Deere tractors equipped with the hook and pin carrier. Top pin set fits H120, D120, 210, 200x, 200cx, 300cx, 300x, 305, 400x, 400cx, 410, 419, 420, 430, 440, and 460 series loaders. The bottom pin set fits 500, 510, 520, 521, 540, 541, and 542 series Ag loaders.
- SINGLE 3,000 PSI CYLINDER: To help your tractor operate smoothly, this attachment utilizes a single 3,000 PSI cylinder. Itsâ€ convenient cylinder assists in providing powerful lifting capability, so you can move and transport rocks, logs, brush, debris, and other heavy objects. Clear out your yard using this strong root grapple rake.
- GREAT FOR LANDSCAPING: If you are looking to beautify your yard, consider this handy Root Grapple Rake Attachment. It is ideal for picking up and moving rocks, logs, brush, debris, and other odd-shaped material. This attachment also features an extra-wide opening that makes pushing and gripping large loads easy.
- DIMENSIONS: The 48-inch Root Grapple Rake Attachment features an extra-wide rake opening style height of 45-inches. The structure is built with a 0.375-inch side and 0.375-inch tine thickness. The implement weighs 325-pounds.
- NOTE: When you order this grapple rake, you will need to provide your phone number so the trucking and shipping company can schedule delivery.
hikotor Heavy Duty 3 Point 2 Inch Receiver Drawbar Hitch Adapter Subcompact Trailer and Farm Equipment Replacement for BX Kubota John Deere LM25H WLM Tractor NorTrac Kioti Yanmar Cat
- HEAVY DUTY CONSTRUCTION - The hitch adapter is crafted of stainless steel, enhanced strenth and durability; Fully finished with black powder coating to resist rust and corrode, which affords to be left outside in the elements for long periods of time
- PREMIUM DETAILS - Overall Width 28" (from end of pin) and height 19" (bottom pin to top pin); Triangle design is for long-lasting using and relatively light weight, which is easy to hook up and lock to the tractor or trailer, saving your time and energy
- EXCELLENT PERFORMANCE - The tractor tow bar implement helps change device at the rear of the tractor for easy and quick assembly and disassembly and move trailers around on the property, widely used in heavy duty work of industrial and construction business
- COMPLETE KITS - Equipped with lifting pins that are 7/8 inches in diameter to fit all Category 1 tractors Standard 2" trailer receiver; Unique bottom pin makes it easy to use with smaller model tractors as well as larger tractors
- WIDE COMPATIBILITIES - The hitch drawbar adapter acts as a stabilizer, perfect for moving around Compact Sub-Compact Tractors BX Kubota, John Deere, LM25H WLM Tractor, NorTrac, Kioti, Yanmar, Cat and other cargo carriers
Titan Attachments Medium-Duty 46" Clamp-on Pallet Forks Stabilizer Bar Rated 4,000 LB Loader or Skid Steer
- TRANSPORT GOODS WITH EASE: Skip the heavy lifting and let your tractor do the work! Our pallet forks make it easier to move and transport bulky items. These pallet forks allow you to haul logs, lumber, and firewood. You can also move fence posts. Use these pallet forks to clear debris and brush as well as move hay bales. The versatility of our pallet forks is endless. Order today.
- ADJUSTABLE SPREADER BAR: It can be irritating when your forks spread too far apart because it makes picking up bales or logs more difficult. With our adjustable stabilizer bar, this issue will not be a problem. By turning the width adjustment knob, you can set the forks 19.5 to 36-inches apart.
- COMPATIBLE WITH MOST LOADERS: These pallet forks feature a universal design compatible with most lawn tractors, loaders, and mowers. Whether you own a New Holland, Kubota, Ford, or another make, these pallet forks will easily clamp-on.
- SECURE LIFTING: These lightweight pallet forks use chain holes to ensure you will get a secure lift, even when dealing with heavy objects. Rollback protection slots will keep you and your machinery safe from harm. These forks have a combined weight capacity of 4,000-pounds so that you can carry a wide variety of goods.
- DIMENSIONS: The Medium-Duty Clamp-On Pallet Forks with Stabilizer Bar is crafted with a fork width of 4-inches and a height of 2-inches. They allow for a bucket lip gap of 2-inches and weigh a combined 113-pounds.
Titan Attachments Mini Skid Steer Root Grapple 42" 318 lb. Quick Tach
- The 42-inch Mini Skid Steer Root Grapple is the perfect attachment for picking up all types of debris including rocks, brush, and even logs. It is designed to fit mini skid steer loaders including Toro Dingo, Ditch Witch, Multi One, Ram Rod, Vermeer, Finn Eagle, Boxer, Thomas, and others with Toro style mounting. It will not fit Bobcat, Kanga Kid, or Prodigy.
- Cylinder rod cover protects hoses and cylinder from accidental damage that can occur during everyday use.
- A grapple jaw opening of 24" allows this attachment to accommodate for many different job applications.
- This root grapple is ideal for those cleaning up sites with trees, roots, rocks, brush, etc.
- Grapple Width: 32.5"
SKYMIRO Clamp on Tractor Trailer Ball Bucket 2'' Fit Hitch Mount Receiver Adapter Tractor Bucket Attachments Compatible with John Deere Front Loader Black
$59.99 in stock
- HEAVY DUTY & ANTI-RUST: Our clamp on bucket hitch is well made of heavy duty alloy steel painted with anti-rust black powder coating, ensuring its long service life.
- DOUBLE-BOLT DESIGN: This clamp on trailer hitch receiver holds dual-thread bolt which can properly tie in with the hexagon nut at the bottom side of the tubing. High quality bolt is strong and sturdy enough to tighten and stay put on the front loader ball bucket without slipping sideways.
- WIDE APPLICATION: Our clamp on trailer is widely applicable to all kinds of vehicel with standard 2 inch hitch receiver , including boat, RV, trailer, tractor, camper, Marine-Personal-Craft, etc.
- EASY INSTALLATION: This clamp on trailer receiver can securely fasten on the front bucket with ease, spinning on the handle screw. Slightly adjust the inner protubing to reach the edge of the bucket and it won't loose and wobble back and forth.
- PACKAGE CONTENTS: 1 x Clamp On Trailer Receiver Hitch 2-inch Tractor Bucket , 1 x screw bolt and hexagon nut , 1 x hitch pin and clip
Arnold 19A30017OEM 46-Inch Snow Blade Attachment, 46 Inch, Black
- COMPATIBLE SNOW PLOW ATTACHMENT: Fits all Troy-Bilt, Bolens, Yard Machines (13C2775S and 13AM775S), Huskee and MTD Gold riding Mowers, 2001 and newer
- ADDITIONAL COMPATIBILITY: Fits Cub Cadet XT-1 and XT-2 Enduro Series Tractors and Series 1000 and 1500 LT, GT and LTX lawn tractors
- QUICK-CHANGE ANGLE: Snow blade adjusts from straight ahead to 25 degrees to the left or to the right. Durable and relaible strength that is capable of clearing large snow filled areas in no time
- ADJUSTABLE SKID SHOES: Use in a variety of conditions and terrains, even in gravel driveways
- PROTECT YOUR TRACTOR: The spring-loaded blade return protects your tractor and the blade from any kind of damage
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- Longevity: The high quality of a product may be actually identified through its toughness and stability of the product. A excellent as well as reliable Tractor Attachments are going to serve you for several years to find.
- Supply:There are consistently brand-new items to substitute the old ones. Products reoccur. Besides the addition of some brand new functions, some needed adjustments have actually additionally probably been actually created. When the manufacturer zero longer assists a supposedly really good Tractor Attachments, what is actually the aspect of utilization it? Our goal is to show items that are up-to-date, and marketed through a minimum of one respectable homeowner, if certainly not several.
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james Coffee Table, 2017-08-08 05:19:49. Ottoman is additional furniture together with the principal sofa that are stylish and multipurpose. If you want the storage ottoman above but simply don’t think that it will offer enough storage area, have a look at this Cocktail storage ottoman bench. Last, the Home Square-Shaped Faux-Leather Storage Ottoman with Tray is a simple product which arrives at a wonderful price.
Joanne Gill Coffee Table, 2017-08-26 06:57:57. Go at your own pace and make certain to consider about the way the true table will fit in your room. It is going to surely give your living room a modern appearance and display your keen awareness of taste. If you’re planning on doing this you’ll need to make sure there is sufficient room on the surface of the table.
Kristina Mcguire Coffee Table, 2017-03-13 23:43:42. Coffee tables ought to be the exact height as the surrounding seating, with 18 inches being an excellent average (although it is going to ride on your furniture). That is why it is crucially important to check when buying a glass coffee table whether it’s been made with tempered glass. Why splurge unnecessarily on wooden fitments as soon as you can make an aesthetic coffee table similar to this one. Farm-style coffee table also frequently provide floor-level shelving, which is ideal for lining up baskets. The very first coffee table with wheels within this list is fantastic for your living room ideas in case you have a farmhouse.
james Coffee Table, 2017-03-20 17:37:07. Pool tables take up quite a little bit of space. Ergonomic tables are available in many standard sizes with dimensions that may be customized. It’s not too large and a great table to drink coffee on. When you are attempting to locate a brand-new sewing table certainly look at the amount of sewing you’d love to carry out and just how major the task will likely be. Numerous contemporary sewing tables arrive fitted with wheels, to make sure you are able to in order to maneuver your table in 1 room to another, that’s undoubtedly extremely headache-free if a person isn’t able to use a predetermined sewing space, your sewing tasks could possibly be mobile.
Joanne Gill Coffee Table, 2017-03-16 04:02:54. Just take the time to consider the types and sizes and styles of tables you desire. Not if you purchase a Tarzan table. These tables are extremely colourful and are extremely attractive for kids. Repurposed tables are awesome since you know that they were already employed for something and they’re reused or repurposed. This fine table resembles something a craftsman would utilize. The ideal coffee table should not merely complement your living space, it also ought to be functional and durable.
james Coffee Table, 2017-03-14 01:05:40. Stand up and visit the nearest mirror that you may find, or when you have a mirror that’s small you could hold in your hand, that would be even better. As you research the mirror, I hope you realize for the very first time that you’re looking at Divinity. When you check in that mirror, like I hope you’re now, I hope you understand that you’re not your name. It is not simple to tell just by taking a look at glass whether it is tempered. While searching for a table top made from glass, you might not understand what kind of glass you’re getting even when you ask. Wine and drink glasses can be purchased to grow your drink baskets.
Audrey Chase Coffee Table, 2017-03-16 22:53:42. If you intend to actually use the table, I strongly suggest taking the opportunity to apply a topcoat. Glass tables are offered in as astonishing range that there’s something special for everybody. A sleek metallic table with a glass top will offer you a contemporary feel, though a rustic oak table will provide you with a casual country feel.
Beulah Griffith Coffee Table, 2017-03-12 05:15:46. How you decorate your table is all up to you, but, no matter what you do, you must plan. Before you choose a table to fit your requirements, ensure it’s child-friendly. Round tables are perfect for homes with kids and pets, since you don’t need to fret about sharp corners hurting anybody. Some round tables are usually designed to revive the attractiveness of your property. Artistically crafted tables can be ideally utilized in rooms meant especially for entertaining intent. Most tables are going to have center support already. Since convertible cocktail tables are often dual-purpose, it means you may make use of over 1 function.
Kristina Mcguire Coffee Table, 2017-03-15 17:18:19. Coffee tables are created of different varieties of materials such as steel, brass or a mixture of both for a traditional appearance. A coffee table goes nicely with your sofa collection. Prior to getting to actually shortlisting a coffee table, you have to plan your financial plan. A coffee table is likewise an important bit of furniture. Coffee tables are among the most vital and practical parts of furniture, adorning the living room. Much like other forms of furniture, the very first matter to think about if buying a new coffee table is the size of table which you require. It’s also important to be certain your new coffee table is sturdy enough for your requirements, especially if you will likely be placing quite a few objects on it, like books, magazines, remote controls and ornaments.
james Coffee Table, 2017-03-16 13:07:58. Knowing what you would like to place on the table and the way you need to utilize it will help in selecting the ideal size for your accent table. Coffee tables can be created from a variety of woods, from pine through to walnut. For many homes today, they are indispensable furniture that is used to add a focal point in the living room or any part of the house for that matter. The ideal thing to do with wooden coffee tables is, naturally, to attempt to keep them from staining. A suitable square coffee table is ready to make a perfect environment of your guest hall and living lounge of your home or your workplace.
Audrey Chase Coffee Table, 2017-03-16 05:34:34. What functions your table must fulfill ought to be one of the major considerations when you’re selecting it. If you are really considering one of these coffee tables in your living room then allow me to assure you that you won’t be mistaken and you’ll still understand the value of such a piece for several years to come. A coffee table is a glamorous object of furniture that may beautifully uplift the whole appearance of the living space, and boost its functionality. For many homes today, coffee tables are indispensable furniture that’s utilized to bring a focus in the living room or any portion of the house for this issue. If you’re buying a square coffee table make sure to mark out the floor area which will be covered to be certain it will not overwhelm the room. You may also have a square coffee table! What’s more, you need to go for a superb quality wooden coffee table that would persist for a very long time and it’s essential not to get caught up in the present time and blow the money on a good looking unit that may not be sturdy and functional.
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About Marrickville Rental Properties
Experience the Vibrant Lifestyle in Marrickville
Marrickville is a dynamic and diverse suburb located in the Inner West of Sydney. With its unique blend of cultures, lively markets, and vibrant street art scene, Marrickville offers a truly eclectic living experience.
Explore Marrickville's Local Attractions
One of the highlights of Marrickville is its thriving food scene. From trendy cafes to multicultural eateries, you'll find a wide range of culinary delights just around the corner. The suburb is also home to several parks and recreational areas, providing ample opportunities for outdoor activities and relaxation.
For art enthusiasts, Marrickville is a treasure trove of creativity. You can wander through the streets and discover colorful murals and graffiti art, showcasing the suburb's vibrant artistic culture.
If you're a music lover, Marrickville has a vibrant live music scene with plenty of venues hosting local and international acts. Whether you enjoy rock, jazz, or indie music, you'll find something to suit your taste.
Additionally, Marrickville is well-connected to the rest of Sydney, making it convenient for commuters. The suburb is serviced by multiple train stations and bus routes, providing easy access to the city and surrounding areas.
With its unique charm and convenient location, Marrickville is a popular choice for those seeking a vibrant urban lifestyle.
TenantApp helps you find apartments for rent in Marrickville, townhouses for rent in Marrickville, and houses for rent in Marrickville. You can also visit Wikipedia for more information about the suburb.
FAQs about Marrickville, New South Wales
1. What are some popular attractions in Marrickville?
Some popular attractions in Marrickville include the Marrickville Organic Food and Farmers Markets, the Factory Theatre, and the Addison Road Community Centre.
2. Are there any parks in Marrickville?
Yes, Marrickville has several parks, including the Marrickville Park, Henson Park, and Enmore Park.
3. What is the public transportation like in Marrickville?
Marrickville is well-served by public transportation, with multiple train stations and bus routes connecting the suburb to the rest of Sydney.
4. Are there any schools in Marrickville?
Yes, Marrickville is home to several schools, including Marrickville Public School and Casimir Catholic College.

domain: geography


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Atrium, Canalejas Complex
Centro Canalejas Madrid
Miguel de Guzmán
|LED Linear™ Partner
|XOOLUX NANO IP67
Canalejas complex, historic elegance in the centre of Madrid
The Canalejas complex is the result of one of the most important urban redevelopments in Europe. The project involves the restoration of seven historic buildings that house the first Four Seasons Hotel in Spain, more than twenty new luxury flats and a large shopping centre on three floors. Atelier Anoche has breathed soul into the façade of this architectural marvel, now elegant and distinguished in the centre of Madrid.
A successful lighting proposal that highlights the details and individual characteristics of each building while creating a common and coherent reading of the whole. Prior to the proposal, a thorough revision of the catalogue was carried out to restore all the details and historical elements of the building. This delicate work not only furthered the understanding of the architecture, but also provided the necessary clues to determine how the individual façade openings, cornices, columns, vertical elements and details of the ensemble should be illuminated.
One of the priorities was to maintain the aesthetics and enhance the inner courtyard. The light from the XOOLUX™ NANO highlights the fittings and openings of a unique architecture clad in warmly lit plant textures, making this space a friendlier place.
A large stone wall rises at the end of the courtyard. This element forms the background that will help us understand the interior and exterior space. A light show with ADONIS and KALYPSO that invites us to experience a new and unique environment, spectacularly lit.


BA leads fight to 'direct' Nats to re-open British airspace
British Airways has joined forces with a number of other UK airlines to collect data from test flights that they hope will lead to the current ash cloud flight ban being lifted.
In a statement released this afternoon, the airline said that it hoped data 'provided by the industry' would enable the Civil Aviation Authority (CAA)
to 'direct National Air Traffic Services (NATS) to
reopen UK airspace and allow British Airways to operate flights into
and out of the UK'.
Back in the skies: A number of test flights have taken place today
BA has conducted several test flights including one on Sunday with chief executive Willie Walsh onboard and reported 'normal operational performance'.
Elsewhere, holiday carrier Thomson Airways said it had flown six aircraft from Iceland to the UK today.
The planes arrived in Prestwick and Manchester this morning and the airline has confirmed 'no technical difficulties were experienced during the flights' and that no damage had been inflicted on the aircraft during the flight.
Fraser Ellacott, Director of Engineering for Thomson Airways, said: 'The safety of our customers and crew is our number one priority at all times. We are sharing this information with our sister airlines in the Tui Travel PLC group and the wider aviation industry with the intention of assisting the authorities during their review of the flight restrictions currently in place.'
The UK flight ban, now running into its sixth day, is costing airlines dear. Budget carrier easyJet said yesterday it had already lost around £40million.A total of 4,500 easyJet flights have been cancelled over the past five days and around 200,000 easyJet passengers are thought to have been affected by the near blanket ban on flights since Thursday lunchtime.
Grounded: Maintenance men work in front of easyJet planes at Stansted Airport this week
Airline analysts at UBS calculate the entire cost to the industry at up to 140million euros (£123million) a day, with no insurance cover available to protect against this type of event.
EasyJet confirmed it was 'actively working with government' in a bid to secure some financial support. Most European airlines are lobbying the European Unions and governments for compensation.
An easyJet spokesman said: 'This is a natural disaster and it's the Government's responsibility to look after its citizens - that hasn't happened.'
'At the moment the cost lies completely with the airlines - we're paying for all of it - and we think this cannot just be the airlines' responsibility.'
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Individuals pick to consume smoothies for a range of reasons, and weight-loss is a typical one for a great factor. Ten Day Smoothie Diet
You can make them in numerous various ways utilizing a broad choice of ingredients. Diet smoothies also offer numerous health benefits. People enjoy to drink smoothies since they are practical, taste excellent, and are both easy and fast to make.
One specific benefit related to drinking smoothies is that you can get a lot of nutrients into your system in a highly effective way, particularly if you are utilizing the right dish.
The 21 Day Smoothie Diet
The 21-Day Smoothie Diet is a scrumptious, easy-to-follow, plan for losing weight and improving your general health. It works by supplying the body with exactly what it needs to be successful.
For instance, given that the diet emphasizes a balance of carbohydrates, protein, and fats in every treat or meal (known as the macronutrient balance), you must be able to handle food yearnings and cravings better throughout the day.
A diet’s macronutrient balance describes the ratio of calories stemmed from fat, carbohydrates, and protein. Different theories have been advanced to attend to the debate concerning what the ideal balance needs to be. The 21 Day Smoothie Diet, nevertheless, provides its own program around this principle so that you do not even require to think of it.
It is recommended to follow a tried and checked smoothie routine instead of mixing numerous ingredients yourself.
Drew, who is a professional health coach produced the dishes in the 21-Day Smoothie Diet. Drew has actually assisted hundreds of individuals lose weight, even those who thought they had actually attempted everything and were almost quiting. Drew aims to assist you make visible changes in simply 3 weeks by following his yummy, simple-to-use system.
If you are looking to make a modification in your life and would like to see a visible, healthy modification in your body, give the 21 Day Smoothie Diet a shot today.
Health Advantages of Smoothies Ten Day Smoothie Diet
People that prefer drinking smoothies prepared with only healthy ingredients such as fresh vegetables and fruits, take pleasure in the antioxidants, vitamins, and many other nutrients consisted of therein.
If you make it a point to include spinach in your smoothies, you can quickly and effectively get your day-to-day dose of iron. If you use a banana in your smoothies, you will get potassium, which is good for the heart, kidneys, and nervous system. You can take pleasure in the numerous health benefits from the antioxidants consisted of within if you add blueberries to the mix.
Diet smoothies can promote your weight-loss efforts by replacing the calories in your routine diet with nutrients that promote health.
Smoothies are quickly absorbed and sustain the body with energy, which means that you can wind up sensation fuller for longer after taking them. You will suffer from fewer yearnings throughout the day that may lead you to consume more calories than you really require if you take smoothies.
Smoothies can also offer the body with a great amount of fiber, but this will mostly depend on what you put in them. Fiber is fantastic for digestion and assists avoid constipation together with other digestive concerns. Fiber is not only good for the digestive system but likewise assists eliminate toxins from the body.
Smoothies can help you cleanse the body and enhance your metabolism. Smoothies can be quickly absorbed by the body, and depending upon the ingredients utilized, they can benefit a range of systems within the body. For example, ingredients in smoothies such as lemon, cruciferous vegetables, beetroot, turmeric, and dandelion greens might work to cleanse the liver.
Individuals that are looking to lose weight might also find that smoothies assist them stick to their diet because they are satisfied for much longer periods. Smoothies and diet smoothies can be filling, which is why they are a fantastic option when it comes to pleasing appetite cravings.
Individuals that drink smoothies take pleasure in a variety of tasty tastes through the combination of different ingredients. Unlike some treats that you might buy off a rack, smoothies include ingredients that are fantastic for the body and contain lower quantities of sugar and fat than you may expect.
Having stated that, it is always recommended to take into consideration the sugar content of the various ingredients, especially fruits that you put in your blender. Some fruits contain especially high levels of sugar, which suggests that you may easily exceed your RDA of sugar if you are not too mindful.
Fruits are clearly good for health and will go a long way towards making your smoothies taste even better. An easy Google search is all it takes to discover the precise amount of sugar in any piece of fruit. If a particular fruit is high in sugar, you can prevent putting too much of it in your smoothie at any one time. You ought to likewise moderate other fruits integrated into the smoothie.
Berries such as raspberries and blueberries are generally lower in sugar. It is an excellent concept, however, to always inspect how much of these sugars you add to your smoothie at any given time. You ought to also never add pure sugar to smoothies.
Smoothies delight in massive popularity amongst dieters given that they please yearnings in a scrumptious way without you feeling starving right after. If you only use healthy ingredients and monitor your consumption of sugar from the smoothies, then it might be possible to actually lose weight.
If you drink fresh veggie and fruit smoothies, you can more quickly meet your day-to-day recommended intake of minerals and vitamins. The number of calories in a smoothie, however, isn’t as high as what you can discover in a candy bar or cookie.
When individuals drink smoothies, they can often feel full for longer than they would after eating similar foods in other methods.
Use Healthy Ingredients Just
If you are aiming to make your smoothies healthy to promote total optimal health and to assist you with your weight-loss efforts, then you need to avoid adding some foods, such as cookies, ice cream, together with other processed, high-fat treats.
Utilize skimmed milk rather than whole milk or even cream if you are looking to lower the number of calories in your smoothies. While the difference in calories between whole and skimed milk may be small, it can affect your diet considerably in time. Better yet, instead of using regular milk, you can utilize coconut milk, almond milk, or just water.
Related Article: green smoothie diet pdf
Keto Diet Smoothies
Keto diet smoothies are an outstanding way to integrate the very best of 2 worlds. For instance, you can use a blender to consist of vegetables and fruits, which are good for your diet in a delicious way. Keto smoothies also contain healthy fats and that’s why they are so rewarding and filling when you consume them.
Healthy fats that can be consisted of in your smoothie include avocado, chia seeds, coconut milk, flax seeds, and numerous nut butters. These ingredients also include protein that can help you feel complete and offer you with energy for a workout. When choosing a whey protein powder, guarantee that it does not consist of sugar substitutes or artificial sweeteners.
Using Kefir in Your Smoothies
A healthy way to delight in a meal replacement is utilizing kefir in your smoothies. Kefir offers the body a healthy boost of calcium together with B vitamins, particularly B12. Kefir likewise includes high quantities of protein and probiotic lactobacilli.
Kefir smoothies can be made using yogurt, vegetables, or fruit. To keep the calorie count low as described above, you ought to use non-fat or low-fat milk.
Green smoothies are a healthy and popular liquid food made of fresh vegetables, fruits, herbs, nuts, or any combination thereof.
Among the various green smoothie recipes currently available, they vary from complex to very easy and can be made using varied ingredients. Green smoothies are likewise usually used a blender or a juicer.
Some examples of green smoothie ingredients include fresh fruits such as bananas, orange, lime, lemon, grape, blueberry, or apply. Non-starchy vegetables such as cucumber, celery, or carrots are likewise used routinely.
It is likewise always suggested to consist of an acidic ingredient such as lemon juice to avoid the extreme ripening of vegetables.
Nuts such as cashews, almonds, and walnuts are sometimes likewise used in a green smoothie. Whenever possible, it is always much better to use raw nuts because roasted nuts are more likely to ruin their taste and nutritional value. Prevent using salted nuts in the smoothies you make.
Herbs such as fresh spinach leaves or mint leaves are likewise typically added to green smoothies. Such herbs not only offer a fantastic flavor but also extra nutrients.
Smoothies are abundant in nutrients and can be fantastic for health. Smoothies are a rejuvenating drink that’s terrific for either lunch or breakfast and help you fell full for a lot longer.
If you would like to use a tested system that has been used successfully by numerous individuals throughout the world and be guided by a professional health coach such as Drew, you certainly require to provide the 21-Day Smoothie Diet a try today. In simply 3 weeks, you might find yourself entirely changed. Ten Day Smoothie Diet

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Since I don't have much experience with them (or remember very many from when I was little), I'm playing picture-book catchup in anticipation of my daughter being old enough for them. I'm mostly pulling ideas from the 1001 Children's Books You Must Read Before You Die book, but I'm going off on my own, or choosing other books by the same authors, on occasion.
Zen Shorts by Jon Muth
This lovely book has three children encountering a panda in their backyard. Each goes to visit him, and he tells them a story adapted from an ancient zen story that speaks to their particular needs at the time. The art is soft and inviting, and the stories each have good morals, even though they stem from a religious tradition not my own.
Are We There Yet? by Allison Lester
A family with three kids (our narrator is the middle one, named Grace) takes a three-month camping trip around Australia, seeing all the different types of places the country has to offer, and staying with friends all around, or camping by the beach or in the forest. I'm not big on camping, but this sounds like a wonderful thing to do in general. The book is a bit wordy, but very interesting. I'd love to see other books like this about traveling around different countries.
Llama Llama Red Pajama by Anna Dewdney
Karina hasn't had issues going to sleep since she was a tiny baby, so I'm not sure how much this will resonate with her as she gets older, but it's a pretty adorable story about a pretty adorable baby llama who's afraid of being alone before going to sleep. The cadence is very pleasing to read aloud, and the expressions on the llama baby's face are priceless.
George, the Dragon, and the Princess by Christopher Wormell
This is not, as you might expect, the story of Saint George and the Dragon. Nope, George here is a little mouse, timid and clumsy. But when a dragon roars onto the scene, George bravely runs against him, and thankfully, the dragon is afraid of mice! Told with sparing words (one line per page) and gorgeous painterly images, this is one I'll probably introduce to Karina relatively soon.
The Adventures of the Dish and the Spoon by Mini Grey
I was really looking forward to this one - a story about the adventures of the dish and spoon after they ran away, involving show business and bank robbery? Sign me up! I did like the story, but it was a bit busy, I thought. It was a bit of a challenge to keep up with the story and stay engaged because each page just had a bit too much happening on it.
Who's Afraid of the Big Bad Book? by Lauren Child
This one intrigued me immediately, because I love stories where people go into books. I did enjoy the story on this one, with the little boy going into his fairy tale book and realizing that the stories are all messed up because he's mistreated the book - drawing mustaches on princesses, cutting people out, pasting pages in upside down, etc. But the art style is really manic and even though I liked the idea of making it look like paper cut-outs (as per the theme of the book), it just wasn't pleasant for me to look at, verging on ugly at times.

domain: entertainment