NTPDases (Apyrases) (EC 126.96.36.199) hydrolyze di- and triphosphate nucleotides, but not monophosphate nucleotides. They are categorized as E-type ATPases, have a broad divalent cation (Mg(2+), Ca(2+)) requirement for activation, and are insensitive to inhibitors of F-type, P-type, and V-type ATPases. Among the seven NTPDases identified in Arabidopsis, only APYRASE 1 (AtAPY1) and APYRASE 2 (AtAPY2) have been previously characterized. In this work, either AtAPY1 or AtAPY2 tagged with C-terminal green fluorescence protein (GFP) driven by their respective native promoter can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, and confocal microscopy reveals that these two Arabidopsis apyrases reside in the Golgi apparatus. In Saccharomyces cerevisiae, both AtAPY1 and AtAPY2 can complement the Golgi-localized GDA1 mutant rescuing its aberrant protein glycosylation phenotype. In Arabidopsis, microsomes of wildtype show higher substrate preferences toward UDP compared to other NDP substrates. Loss-of-function Arabidopsis AtAPY1 mutants exhibit reduced microsomal UDPase activity, and this activity is even more significantly reduced in the loss-of-function AtAPY2 mutant and in the AtAPY1/AtAPY2 RNAi technology repressor lines. Microsomes from wildtype plants also have detectable GDPase activity, which is significantly reduced in apy2 but not apy1 mutants. The GFP tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity confirming that they both also have functional competency. The cell walls of apy1, apy2 and the RNAi silenced lines all have increased composition of galactose, but the transport efficiency of UDP-galactose across microsomal membranes was not altered. Taken together these results reveal that AtAPY1 and AtAPY2 are Golgi localized nucleotide diphosphatases and are likely to have roles in regulating UDP/GDP concentrations in the Golgi lumen.
"In plants, this enzyme has been characterized from potato (Solanum tuberosum; Kalckar 1944; Molnar & Lorand 1961; Kettlun et al. 1982), and various legumes, such as Glycine max (soya; Day et al. 2000), Medicago truncatula (barrel medic; Cohn et al. 2001), and Pisum sativum (pea; Hsieh et al. 2000). It has been identified from various plant tissues and has multiple cellular locations, such as the cytoskeleton (Shibata et al. 1999), filamentous structures associated with ribosomes, nuclei (Shibata et al. 2002), chromatin (Matsumoto et al. 1984), nuclear membrane (Tong et al. 1993), the outer surface of the cytoplasmic membrane, the cell wall (Thomas et al. 1999) and in the Golgi (Dunkley et al. 2004; Chiu et al. 2012; Parsons et al. 2012; Schiller et al. 2012). These findings suggest that apyrase in plants may be involved in diverse functions and signalling inside and outside the cells. "
[Show abstract][Hide abstract] ABSTRACT: Apyrase (ATP diphosphohydrolase, EC 188.8.131.52) catalyzes hydrolysis of nucleoside tri- and di-phosphates to nucleoside monophosphates and orthophosphates. In the present study, the spatio-temporal expression of an apyrase gene (PsAPY1) in pea (Pisum sativum L. var. Alaska), was investigated during early stages of apical hook development using nonradioactive mRNA in-situ hybridization. During the formation of apical hook; at 45 hours after sowing (HAS), expression of PsAPY1 was obvious in epidermis and vascular bundle. By 60 HAS, the apical hook was completely formed. At this stage, transcript accumulation became higher than at the previous stage and expression was also visible in the cortex tissues of the developing hook. However, at 78 HAS, the curvature of the hook was reduced and hook was in the process of opening. At this time, expression of PsAPY1 was visible in all the above-mentioned tissues although the level of expression was slightly lower than at the previous stage (60 HAS). Apical hook formation provides a unique mechanism of protection for delicate shoot meristem in dicot plants. Its establishment is orchestrated by differential elongation rates of cells within the structure. The expression pattern of a gene provides essential information concerning the likely appearance and localization of its encoded protein and this helps to understand the mechanism of development of plant cells and tissues. Higher expression of PsAPY1 during the process of hook development indicates its essential role in the process of formation and maintenance of hook curvature and thus aids in protection of delicate shoot meristem.
"Two apyrases (APYs) in Arabidopsis (Arabidopsis thaliana) with high sequence similarity, APY1 and APY2, play a major role in growth regulation (Wu et al., 2007), in part because their expression is required for normal auxin transport (Liu et al., 2012). Recent evidence indicates that they have a Golgi localization (Chiu et al., 2012; Schiller et al., 2012), where they could regulate growth by controlling glycoprotein, glycolipid, and wall polysaccharide synthesis. One source of eATP is secretory vesicles that have a high ATP concentration in their lumen (Rudnick, 2008; Geigenberger et al., 2010). "
[Show abstract][Hide abstract] ABSTRACT: Plant cells release ATP into their extracellular matrix as they grow, and extracellular ATP (eATP) can modulate the rate of cell growth in diverse tissues. Two closely related apyrases in Arabidopsis thaliana, APY1 and APY2, function in part to control the concentration of eATP. The expression of APY1/APY2 can be inhibited by RNAi, and this suppression leads to an increase in the concentration of eATP in the growth medium and severely reduces growth. To clarify how the suppression of APY1 and APY2 is linked to growth inhibition the gene expression changes that occur in seedlings when apyrase expression is suppressed were assayed by microarray and qRT-PCR analyses. The most significant gene expression changes induced by apyrase suppression were in genes involved in biotic stress responses, which include those regulating wall composition and extensibility. These expression changes predicted specific chemical changes in the walls of mutant seedlings, and two of these, wall lignification and decreased methyl ester bonds, were verified by direct analyses. Taken together the results are consistent with the hypothesis that APY1 and 2 and eATP play important roles in the signaling steps that link biotic stresses to plant defense responses and growth changes.
"Apyrase proteins serve to regulate extracellular ATP concentration in animal cells (Plesner, 1995; Gaddie and Kirley, 2010), and a similar role may exist for these proteins in plant cells. The Arabidopsis nucleoside triphosphate–diphosphohydrolases termed apyrase 1 and 2 have been implicated in e-ATP signaling (Clark et al., 2011; Liu et al., 2012), although they may do so from a Golgi locale (Chiu et al., 2012; Schiller et al., 2012) rather than from a plasma membrane site. When ecto-apyrase activity is inhibited by antibodies raised to APY1 and APY2, the [eATP] of media in which pollen tubes are growing rises several fold and pollen tube growth is inhibited (Wu et al., 2007). "
[Show abstract][Hide abstract] ABSTRACT: Recent data indicate that nucleotides are released into the extracellular matrix during plant cell growth, and that these extracellular nucleotides induce signaling changes that can, in a dose-dependent manner, increase or decrease the cell growth. After activation of a presumed receptor, the earliest signaling change induced by extracellular nucleotides is an increase in the concentration of cytosolic Ca(2+), but rapidly following this change is an increase in the cellular level of nitric oxide (NO). In Arabidopsis, mutants deficient in nitrate reductase activity (nia1nia2) have drastically reduced nitric oxide production and cannot transduce the effects of applied nucleotides into growth changes. Both increased levels of extracellular nucleotides and increased NO production inhibit auxin transport and inhibit growth, and these effects are potentially due to disruption of the localization and/or function of auxin transport facilitators. However, because NO- and auxin-induced signaling pathways can intersect at multiple points, there may be diverse ways by which the induction of NO by extracellular ATP could modulate auxin signaling and thus influence growth. This review will discuss these optional mechanisms and suggest possible regulatory routes based on current experimental data and predictive computational analyses.
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