Multifunctional acetyl-CoA carboxylase 1 is essential for very long chain fatty acid elongation and embryo development in Arabidopsis

Acetyl-CoA carboxylase (ACCase) catalyses the carboxylation of acetyl-CoA, forming malonyl-CoA, which is used in the plastid for fatty acid synthesis and in the cytosol in various biosynthetic pathways including fatty acid elongation. In Arabidopsis thaliana, ACC1 and ACC2, two genes located in a tandem repeat within a 25-kbp genomic region near the centromere of chromosome 1, encode two multifunctional ACCase isoforms. Both genes, ACC1 and ACC2, appear to be ubiquitously expressed, but little is known about their respective function and importance. Here, we report the isolation and characterisation of two allelic mutants disrupted in the ACC1 gene. Both acc1-1 and acc1-2 mutations are recessive and embryo lethal. Embryo morphogenesis is impaired and both alleles lead to cucumber-like structures lacking in cotyledons, while the shortened hypocotyl and root exhibit a normal radial pattern organisation of the body axis. In this abnormal embryo, the maturation process still occurs. Storage proteins accumulate normally, while triacylglycerides (TAG) are synthesised at a lower concentration than in the wild-type seed. However, these TAG are totally devoid of very long chain fatty acids (VLCFA) and consequently enriched in C18:1, like all lipid fractions analysed in the mutant seed. These data demonstrate, in planta, the role of ACCase 1 in VLCFA elongation. Furthermore, this multifunctional enzyme also plays an unexpected and central function in embryo morphogenesis, especially in apical meristem development.     

Cyclohexanedione herbicides are inhibitors of rat heart acetyl-CoA carboxylase

Acetyl CoA carboxylase (ACC) catalyzes the carboxylation of acetyl CoA to form malonyl CoA. In skeletal muscle and heart, malonyl CoA functions to regulate lipid oxidation by inhibition of carnitine palmitoyltransferase-1, an enzyme which controls the entry of long chain fatty acids into mitochondria. We have found that several members of the cyclohexanedione class of herbicides are competitive inhibitors of rat heart ACC. These compounds constitute valuable reagents for drug development and the study of ACCbeta, a validated anti-obesity target.     

Arabidopsis late-flowering fve mutants are affected in both vegetative and reproductive development

The development of wild-type Arabidopsis thaliana (L.) Heyhn and two late-flowering fve mutants has been analysed under different environmental conditions. In wild-type plants, short-day photoperiods delay the floral transition as a consequence of lengthening all the developmental phases of the plant. Moreover, short days also alter the inflorescence structure by reducing the internode elongation and delaying the establishment of the floral developmental programme in the lateral meristems of the inflorescence and co-florescences. Mutations at the FVE locus cause a delay in flowering time, and a change in the inflorescence structure, similar to the effect of short photoperiods on wild-type plants. However, the effect of the fve mutations is additive to the effect of short days, and all the aspects of the Fve phenotype are corrected by vernalization. These results seem to indicate that FVE is not simply involved in timing the transition from vegetative to reproductive growth, but that it could play a role during all stages of plant development.     

Multiple vacuoles in impaired tonoplast trafficking3 mutants are independent organelles

Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol 3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol 3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells.     

Potent biphenyl- and 3-phenyl pyridine-based inhibitors of acetyl-CoA carboxylase

We report the synthesis and enzymatic evaluation of potent inhibitors of acetyl-CoA carboxylases (ACCs) containing biphenyl or 3-phenyl pyridine cores. These compounds inhibit both ACC1 and ACC2, or are moderately selective for either enzyme, depending on side chain substitution. Typical activities of the most potent compounds in this class are in the low double-digit to single-digit nanomolar range in in vitro assays using human ACC1 and ACC2 enzymes.     

Knockdown of the plastid-encoded acetyl-CoA carboxylase gene uncovers functions in metabolism and development

De novo fatty acid biosynthesis in plants relies on a prokaryotic-type acetyl-CoA carboxylase (ACCase) that resides in the plastid compartment. The enzyme is composed of four subunits, one of which is encoded in the plastid genome, whereas the other three subunits are encoded by nuclear genes. The plastid gene (accD) encodes the β-carboxyltransferase subunit of ACCase and is essential for cell viability. To facilitate the functional analysis of accD, we pursued a transplastomic knockdown strategy in tobacco (Nicotiana tabacum). By introducing point mutations into the translational start codon of accD, we obtained stable transplastomic lines with altered ACCase activity. Replacement of the standard initiator codon AUG with UUG strongly reduced AccD expression, whereas replacement with GUG had no detectable effects. AccD knockdown mutants displayed reduced ACCase activity, which resulted in changes in the levels of many but not all species of cellular lipids. Limiting fatty acid availability caused a wide range of macroscopic, microscopic, and biochemical phenotypes, including impaired chloroplast division, reduced seed set, and altered storage metabolism. Finally, while the mutants displayed reduced growth under photoautotrophic conditions, they showed exaggerated growth under heterotrophic conditions, thus uncovering an unexpected antagonistic role of AccD activity in autotrophic and heterotrophic growth.

Analysis of the only plastid genome-encoded fatty acid biosynthesis gene reveals functions in plastid division and seed development, and antagonistic roles in autotrophic and heterotrophic growth.     

Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.     

Purification and properties of acetyl-CoA carboxylase phosphatase

Acetyl-CoA carboxylase phosphatase has been purified from the rat epididymal fat pad. The phosphatase occurs in a complex with the carboxylase. In the purification of the phosphatase, the high molecular weight complex was initially separated by sucrose gradient centrifugation, and the phosphatase was isolated from the complex by adjusting to 80% saturation with ethanol and by chromatography on Sephadex G-75. The molecular weight of the phosphatase is 71,000 as determined by sodium dodecyl sulfate gel electrophoresis and gel chromatography on Sephacryl-200 in the presence of 6 M urea. The Km for acetyl-CoA carboxylase and glycogen phosphorylase a are 1.5 microM and 37 microM, respectively. The phosphatase has a broad substrate specificity, being active toward glycogen synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, phosphorylase a, phosphoprotamine, and p-nitrophenyl phosphate, in addition to acetyl-CoA carboxylase from fat tissue and liver. Acetyl-CoA carboxylase inhibits the dephosphorylation of phosphoprotamine, indicating that the same activity is responsible for dephosphorylating both substrates. The phosphatase requires no metal ion for activity and is not inhibited by the rat liver phosphorylase phosphatase inhibitor protein. The significance of these findings is discussed in relation to the regulation of acetyl-CoA carboxylase, and the phosphatase is compared to other phosphoprotein phosphatases.     

Methylammonium-resistant mutants ofNicotiana plumbaginifolia are affected in nitrate transport

This work reports the isolation and preliminary characterization ofNicotiana plumbaginifolia mutants resistant to methylammonium.Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up byNicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.     

Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes

An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: The hepatocytes are made permeable by digitonin. 64 micrograms of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on mitochondrial permeability. Enzyme activity is measured by coupling the carboxylase reaction to the fatty acid synthase reaction. The advantages offered by this procedure over existing assays are: rapidity, no need to prepare cell extracts, absence of product inhibition, no interference by mitochondrial enzymes, useful in systems with bicarbonate buffers, and simple separation of radioactive substrate from labelled products. Using this coupled enzyme assay a good correlation was observed between changes in the activity of acetyl-CoA carboxylase and changes in the rate of fatty acid synthesis in hepatocytes as effected by short-term modulators.     

Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajan

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.     

Physiological regulation of acetyl-CoA carboxylase gene expression: effects of diet, diabetes, and lactation on acetyl-CoA carboxylase mRNA

We measured acetyl-CoA carboxylase mRNA levels in various tissues of the rat under different nutritional and hormonal states using a cDNA probe. We surveyed physiological conditions which are known to alter carboxylase activity, and thus fatty acid synthesis, to determine whether changes in the levels of carboxylase mRNA are involved. The present studies include the effects of fasting and refeeding, diabetes and insulin, and lactation on carboxylase mRNA levels. Northern blot analysis of liver RNA revealed that fasting followed by refeeding animals a fat-free (high carbohydrate) diet dramatically increased the amount of carboxylase mRNA compared to the fasted condition. These changes in the level of mRNA correspond to changes in the activity and amount of acetyl-CoA carboxylase. Acetyl-CoA carboxylase mRNA levels in epididymal fat tissue decreased upon fasting and increased to virtually normal levels after 72 h of refeeding, closely resembling the liver response. The amount of acetyl-CoA carboxylase mRNA decreased markedly in epididymal fat tissue of diabetic rats as compared to nondiabetic animals. However, 6 h after injection of insulin the mRNA level returned to that of the nondiabetic animals. Gestation and lactation also affected the levels of carboxylase mRNA in both liver and mammary gland. Maximum induction in both tissues occurred 5 days postpartum. These studies suggest that these diverse physiological conditions affect fatty acid synthesis in part by altering acetyl-CoA carboxylase gene expression.     

Ferritin mutants of Escherichia coli are iron deficient and growth impaired, and fur mutants are iron deficient

Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by M?ssbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of "reactive ferrous iron." It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear.     

Dephosphorylation and activation of acetyl-CoA carboxylase by phosphorylase phosphatase

Acetyl-CoA carboxylase from rat epididymal fat tissue is activated by phosphorylase phosphatase, a reaction which is inhibited by phosphatase inhibitor-1. This activation is accompanied by a corresponding loss of ³²P from the labelled enzyme. These results establish that dephosphorylation of the enzyme causes its activation.     

A biotin analog inhibits acetyl-CoA carboxylase activity and adipogenesis

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of long chain fatty acids. In this study, we observed that treatment of 3T3-L1 cells with biotin chloroacetylated at the 1' nitrogen reduced the enzymatic activity of cytosolic acetyl-CoA carboxylase and concomitantly inhibited the differentiation of 3T3-L1 cells in a dose-dependent manner. Treatment with chloroacetylated biotin blocked the induction of PPARgamma, STAT1, and STAT5A expression that normally occurs with adipogenesis. Moreover, addition of chloroacetylated biotin inhibited lipid accumulation, as judged by Oil Red O staining. Our results support recent studies that indicate that acetyl-CoA carboxylase may be a suitable target for an anti-obesity therapeutic.