Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose

The peroxisomal ABC transporter, Comatose (CTS), a full length transporter from Arabidopsis has intrinsic acyl-CoA thioesterase (ACOT) activity, important for physiological function. We used molecular modelling, mutagenesis and biochemical analysis to identify amino acid residues important for ACOT activity. D863, Q864 and T867 lie within transmembrane helix 9. These residues are orientated such that they might plausibly contribute to a catalytic triad similar to type II Hotdog fold thioesterases. When expressed in Saccharomyces cerevisiae, mutation of these residues to alanine resulted in defective of β-oxidation. All CTS mutants were expressed and targeted to peroxisomes and retained substrate-stimulated ATPase activity. When expressed in insect cell membranes, Q864A and S810N had similar ATPase activity to wild type but greatly reduced ACOT activity, whereas the Walker A mutant K487A had greatly reduced ATPase and no ATP-dependent ACOT activity. In wild type CTS, ATPase but not ACOT was stimulated by non-cleavable C14 ether-CoA. ACOT activity was stimulated by ATP but not by non-hydrolysable AMPPNP. Thus, ACOT activity depends on functional ATPase activity but not vice versa, and these two activities can be separated by mutagenesis. Whether D863, Q864 and T867 have a catalytic role or play a more indirect role in NBD-TMD communication is discussed

The COMATOSE ATP-binding cassette transporter is required for full fertility in arabidopsis

COMATOSE (CTS) encodes a peroxisomal ATP-binding cassette transporter required not only for beta-oxidation of storage lipids during germination and establishment, but also for biosynthesis of jasmonic acid and conversion of indole butyric acid to indole acetic acid. cts mutants exhibited reduced fertilization, which was rescued by genetic complementation, but not by exogenous application of jasmonic acid or indole acetic acid. Reduced fertilization was also observed in thiolase (kat2-1) and peroxisomal acyl-Coenzyme A synthetase mutants (lacs6-1, lacs7-1), indicating a general role for beta-oxidation in fertility. Genetic analysis revealed reduced male transmission of cts alleles and both cts pollen germination and tube growth in vitro were impaired in the absence of an exogenous carbon source. Aniline blue staining of pollinated pistils demonstrated that pollen tube growth was affected only when both parents bore the cts mutation, indicating that expression of CTS in either male or female tissues was sufficient to support pollen tube growth in vivo. Accordingly, abundant peroxisomes were detected in a range of maternal tissues. Although gamma-aminobutyric acid levels were reduced in flowers of cts mutants, they were unchanged in kat2-1, suggesting that alterations in gamma-aminobutyric acid catabolism do not contribute to the reduced fertility phenotype through altered pollen tube targeting. Taken together, our data support an important role for beta-oxidation in fertility in Arabidopsis (Arabidopsis thaliana) and suggest that this pathway could play a role in the mobilization of lipids in both pollen and female tissues

The NBDs that wouldn't die. A cautionary tale in the use of isolated nucleotide binding domains of ABC transporters

COMATOSE (CTS), the plant homologue of Adrenoleukodystrophy protein, is a full length ABC transporter localised in peroxisomes. In a recent article, we reported that the two nucleotide binding domains of CTS are not functionally equivalent in vivo. Mutations in conserved residues in the Walker A (K487A) and B (D606N) motifs of NBD1 resulted in a null phenotype, whereas identical mutations in the equivalent residues in NBD2 (K1136A and D1276N) had no detectable effect.1 In order to study the effect of these mutations on the ATPase activity of the nucleotide binding domains, we cloned and expressed the isolated NBDs as maltose binding protein (MBP) fusion proteins. We show that ATPase activity is associated with the isolated MBP-NBDs. However, mutations of amino acids located in conserved motifs did not result in striking reduction in activity despite well characterized roles in ATP binding and hydrolysis. We urge caution in the interpretation of results obtained from the study of isolated NBD fusions and their extrapolation to the mechanism of ATP hydrolysis in ABC transporter proteins

BIG regulates dynamic adjustment of circadian period in Arabidopsis thaliana

Circadian clocks drive rhythms with a period near 24 h, though the molecular basis of the regulation of circadian period is little understood. We have demonstrated previously that metabolites affect the free-running period of the circadian oscillator of Arabidopsis thaliana, with endogenous sugars acting as an accelerator and exogenous nicotinamide acting as a brake. Changes in circadian oscillator period are thought to adjust the timing of biological activities through the process of entrainment, in which the circadian oscillator becomes synchronised to rhythmic signals such as light and dark cycles, and changes in internal metabolism. To identify molecular components associated with the dynamic adjustment of circadian period we performed a forward genetic screen. We identified mutants that were either period insensitive to nicotinamide (sin) or period oversensitive to nicotinamide (son). We mapped son1 to BIG, a gene of unknown molecular function that has previously been reported to have a role in light signalling. We found that son1 has an early entrained phase suggesting that the dynamic alteration of circadian period contributes to the correct timing of biological events. Our data provide an insight in to how dynamic period adjustment of circadian oscillators contributes to establishing a correct phase relationship with the environment and identifies that BIG is involved in this process

A glutathione s-transferase confers herbicide tolerance in rice

Plant glutathione S-transferases (GSTs) have been a focus of attention due to their role in herbicide detoxification. OsGSTL2 is a glutathione S-transferase, lambda class gene from rice (Oryza sativa L.). Transgenic rice plants over-expressing OsGSTL2 were generated from rice calli by the use of an Agrobacterium transformation system, and were screened by a combination of hygromycin resistance, PCR and Southern blot analysis. In the vegetative tissues of transgenic rice plants, the over-expression of OsGSTL2 not only increased levels of OsGSTL2 transcripts, but also GST and GPX expression, while reduced superoxide. Transgenic rice plants also showed higher tolerance to glyphosate and chlorsulfuron, which often contaminate agricultural fields. The findings demonstrate the detoxification role of OsGSTL2 in the growth and development of rice plants. It should be possible to apply the present results to crops for developing herbicide tolerance and for limiting herbicide contamination in the food chain

The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas vaginalis

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters

Natural Variation in an ABC Transporter Gene Associated with Seed Size Evolution in Tomato Species

Seed size is a key determinant of evolutionary fitness in plants and is a trait that often undergoes tremendous changes during crop domestication. Seed size is most often quantitatively inherited, and it has been shown that Sw4.1 is one of the most significant quantitative trait loci (QTLs) underlying the evolution of seed size in the genus Solanum—especially in species related to the cultivated tomato. Using a combination of genetic, developmental, molecular, and transgenic techniques, we have pinpointed the cause of the Sw4.1 QTL to a gene encoding an ABC transporter gene. This gene exerts its control on seed size, not through the maternal plant, but rather via gene expression in the developing zygote. Phenotypic effects of allelic variation at Sw4.1 are manifested early in seed development at stages corresponding to the rapid deposition of starch and lipids into the endospermic cells. Through synteny, we have identified the Arabidopsis Sw4.1 ortholog. Mutagenesis has revealed that this ortholog is associated with seed length variation and fatty acid deposition in seeds, raising the possibility that the ABC transporter may modulate seed size variation in other species. Transcription studies show that the ABC transporter gene is expressed not only in seeds, but also in other tissues (leaves and roots) and, thus, may perform functions in parts of the plants other than developing seeds. Cloning and characterization of the Sw4.1 QTL gives new insight into how plants change seed during evolution and may open future opportunities for modulating seed size in crop plants for human purposes

Identification of genes differentially expressed in a resistant reaction to Mycosphaerella pinodes in pea using microarray technology

<p>Abstract</p> <p>Background</p> <p>Ascochyta blight, caused by <it>Mycosphaerella pinodes </it>is one of the most important pea pathogens. However, little is known about the genes and mechanisms of resistance acting against <it>M. pinodes </it>in pea. Resistance identified so far to this pathogen is incomplete, polygenic and scarce in pea, being most common in <it>Pisum </it>relatives. The identification of the genes underlying resistance would increase our knowledge about <it>M. pinodes-</it>pea interaction and would facilitate the introgression of resistance into pea varieties. In the present study differentially expressed genes in the resistant <it>P. sativum </it>ssp. <it>syriacum </it>accession P665 comparing to the susceptible pea cv. Messire after inoculation with <it>M. pinodes </it>have been identified using a <it>M. truncatula </it>microarray.</p> <p>Results</p> <p>Of the 16,470 sequences analysed, 346 were differentially regulated. Differentially regulated genes belonged to almost all functional categories and included genes involved in defense such as genes involved in cell wall reinforcement, phenylpropanoid and phytoalexins metabolism, pathogenesis- related (PR) proteins and detoxification processes. Genes associated with jasmonic acid (JA) and ethylene signal transduction pathways were induced suggesting that the response to <it>M. pinodes </it>in pea is regulated via JA and ET pathways. Expression levels of ten differentially regulated genes were validated in inoculated and control plants using qRT-PCR showing that the P665 accession shows constitutively an increased expression of the defense related genes as peroxidases, disease resistance response protein 39 (DRR230-b), glutathione S-transferase (GST) and 6a-hydroxymaackiain methyltransferase.</p> <p>Conclusions</p> <p>Through this study a global view of genes expressed during resistance to <it>M. pinodes </it>has been obtained, giving relevant information about the mechanisms and pathways conferring resistance to this important disease. In addition, the <it>M. truncatula </it>microarray represents an efficient tool to identify candidate genes controlling resistance to <it>M. pinodes </it>in pea.</p