Browsing by Subject "Apyrase"

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Characterization of apyrases in pea, arabidopsis, and cotton
(2010-05) Kays, Julia Elizabeth; Roux, Stanley J.; Clark, Gregory B.
Apyrases are proteins that bind to and hydrolyze most nucleoside di- and triphosphates, but not nucleoside monophosphates. Some function inside the cell; others hydrolyze extracellular nucleotides in the extracellular matrix. In Arabidopsis, apyrases and eATP have been implicated in diverse responses, ranging from phosphate mobilization to herbicide and toxin resistance to overall plant growth. Isolated garden pea nuclei exhibit phytochrome-dependent, red/far red-reversible changes in apyrase activity and in protein phosphorylation. O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification that functions in a manner analogous but often antagonistic to O-phosphorylation. Given the known red light effects in pea nuclei and the link between O-GlcNAc and O-phosphorylation, we tested whether O-GlcNAc modification in pea nuclei might change in response to red light (Rc). Western blots showed that anti-O-GlcNAc antibody could sometimes recognize modifications in pea nuclei proteins, but not consistently. Experiments testing red light irradiation, O-GlcNAc transferase inhibitors, and GlcNAcase inhibitors all yielded similarly inconsistent results. As the project progressed we learned that the O-GlcNAc modification is very labile, and that this lability was the probable basis of the inconsistent results. Methods to overcome this technical problem were not readily available and so this project was not pursued further. A second project focused on identifying the members of the protein complexes associated with apyrase in partially purified nuclear preparations. The apyrase complex was isolated from etiolated seedlings of both Arabidopsis and pea by co-immunoprecipitation using antibodies raised against purified protein from each system. Apyrase activity was also compared in etiolated and red-light irradiated nuclei in both systems. For both experiments and in both systems, the complex of proteins associated with apyrase in etiolated and Rc-irradiated nuclei were not consistently different. The discovery of a cotton EST with a sequence similar to other plant apyrases opened a new line of investigation in a model system with fibers that are suited for the study of growth kinetics of single cells. This putative apyrase is expressed at high levels in elongating fibers but not in fibers that are not elongating or in ovules, suggesting that apyrase may influence cotton fiber elongation. Studies of fiber growth kinetics in the presence of inhibitors that block apyrase activity, chemical inhibitors of animal P2X-type purinoceptors, and anti-apyrase antibodies also favor this hypothesis. I cloned the full length cDNAs of GhApy1 and GhApy2. Antibodies raised against a suspected antigenic and variable sequence of GhApy1 were produced and confirmed to recognize native cotton apyrase. Crude protein isolated from pollen exhibited apyrase activity.
Functional analyses of Arabidopsis apyrases 3 through 7
(2011-05) Yang, Jian, 1981-; Roux, Stanley J.; Huq, Enamul; Mauseth, James; Mehdy, Mona; Richardson, Richard
Apyrases (NTP-diphosphohydrolases, EC 3.6.1.5) are a family of enzymes that catalyze the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di- phosphates in the presence of divalent cations. In Arabidopsis, AtAPY1 and AtAPY2 function in part as ectoapyrases and have been shown to play important roles in controlling the concentration of extracellular nucleotides, which, in turn, can regulate pollen germination and growth, and cell expansion in diverse plant tissues. We used an NCBI nucleotide blast keyed to Apyrase Conserved Regions (ACRs) to identify five other AtAPYs (3-7). To assess the biological function of each of these five AtAPY genes, the phenotypes of their T-DNA insertion mutants were analyzed. We did not observe any obvious phenotypes from the T-DNA insertion single knockout of any of these genes. However, double knockout mutants of AtAPY6 and 7 exhibited late anther dehiscence and low male fertility. Transmission and scanning electron microscopy revealed that the pollen grains of double knockout mutant of AtAPY6 and 7 are largely deformed in shape and in most cases, the cell walls of the pollen grains are interconnected. Using an AtAPY6-YFP fusion protein in transgenic Arabidopsis, AtAPY6 was localized to intracellular vesicles. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays and promoter:GUS fusion analysis demonstrated that the transcripts of both AtAPY6 and 7 are expressed in mature pollen grains, AtAPY6 is also expressed in the veins and hydathode regions of rosette leaves, and AtAPY7 is expressed in more diverse tissues such as roots, leaves, stems, pistils and sepals. The tissue specificity of AtAPYs 3, 4 and 5 expression was also determined using qRT-PCR assays and promoter:GUS fusion analysis. AtAPY3 and AtAPY4 were primarily expressed in roots but not in rosette leaves. AtAPY5 was expressed primarily in rosette leaves but only weakly in roots. AtAPY5 and AtAPY7 were upregulated when the rosette leaves are wounded or exposed to drought stress. RNA interference (RNAi) was performed to simultaneously suppress the gene expression of AtAPYs 3, 4, 5 to around 10% of that of the wild type. However, we did not observe any obviously altered phenotypes in the RNAi lines. The suppression of AtAPYs 3, 4, 5 by RNAi in the background of AtAPY6 single knockout did not cause any phenotype either. A possible explanation for this lack of phenotype in the RNAi lines is that functional redundancy exists between AtAPYs 3-5 and AtAPYs 1-2 and/or AtAPYs 6-7.
Isolation and characterization of Pisum sativum apyrases, PsNTP9 and PsNTP9-DM, cloned and expressed in Escherichia coli
(2019-02-06) Wallen, Michael Andrew, Jr; Roux, Stanley J.
Adenosine triphosphate (ATP) is widely known as a fuel source for many biochemical processes, and to a lesser degree also as a signaling molecule in plants and animals. When plants are subjected to biotic or abiotic stress or undergoing exocytosis, they release ATP into the extracellular matrix (ECM). The release of ATP sets off a signal transduction pathway, first rapidly increasing the concentrations of cytosolic calcium, reactive oxygen species, and nitric oxide. How these changes specifically influence physiology is the object of much research in both plants and animals. Some of the changes that are affected influence growth and development, stomatal function, and gravitropism. Apyrases and other phosphatases control the concentration of the released nucleotides by breaking phosphate bonds from nucleoside triphosphates and diphosphates. Research aimed at the discovery of receptors, signaling pathway components, and processes has been successful to some extent. There are now known purinergic receptors in both plants and animal cells. We have cloned a truncated version of Pisum sativum (ps) NTP9. We used a pET-22B vector to add a histidine tag and transformed the vector into the BL21 Escherichia coli with a T7 promoter to enable IPTG induction of the LAC operon and expression of the enzyme. The pET-22B vector was incubated in separate samples with BL21 cells. Cells were propagated, and the expression of recombinant proteins PsNTP9, and separately, a double mutant PsNTP9-DM with a second calmodulin-binding domain, were induced ectopically. Cells were broken open by shaking them and mixing them with lysis buffer. Centrifugation was performed to separate the supernatant containing the released apyrases from the particulate wall fraction. The enzymes were purified by affinity chromatography, then their purity was evaluated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Western blots were performed to verify presence of the apyrases using a commercial anti-histidine antibody to detect PsNTP9 and PsNTP9-DM. Once suitable amounts of our proteins of interest were harvested, we performed Bradford assays to determine the protein concentration of the samples and carried out an apyrase activity assay to determine the specific activity of the purified enzymes and compare it to that of other known phosphatases.
A radiolabeling approach to purinoceptor-like receptor identification in plants and evidence for apyrase (APY1 and APY2) regulation of stomatal aperture in arabidopsis
(2010-12) Fraley, Devin Scott; Roux, Stanley J.; Clark, Gregory B.
Adenosine triphosphate (ATP) is well recognized for its role as the primary cellular energy currency. However, studies dating back to 1929 have reshaped our understanding of ATP as not only an energy source, but also as a signaling agent. Among the most important of these discoveries are animal purinergic receptors (P2X and P2Y receptors) that perceive extracellular ATP (eATP), primarily in the nervous system. Though eATP is an established receptor agonist in animals and applied poorly hydrolyzable ATP analogs have numerous effects on growth in plants, eATP is not widely accepted as a signal in plants where no purinoceptor has been identified. Here, enriched outside-out plasma membrane vesicles were isolated and proteins labeled with a radioactive ATP analog (8N₃ATP[α²³P]) to identify a putative purinoceptor-like receptor. We used etiolated seedlings to capture proteins from plant tissue that was actively growing and used sodium carbonate washes to separate peripheral and integral membrane proteins. With this method, we have generated lists of plasma membrane ATP binding proteins, and therefore possible eATP receptors. Ectoapyrases are phosphohydrolases thought to regulate eATP in both animals and plants. Here, we also investigated the expression and role of the candidate ectoapyrases AtAPY1 and AtAPY2 in guard cells and stomatal responses. AtAPY1 and AtAPY2 transcript and protein expression was confirmed in guard cells. Early genetic studies using an apy2 knock out with induced RNAi-silencing of APY1 suggest a role for these apyrases in stomatal regulation. In response to treatment with five hours light, the apyrase-suppressed line features wider stomatal aperture when compared to WS wild-type.